Screening assays, modulators and modulation of progressive glycation end product receptor (RAGE) activation

Abstract

A method of screening for a candidate substance according to the ability of the candidate substance to modulate RAGE activity wherein such RAGE activity is induced by a co-localized active-type GPCR, the method comprising the steps of contacting a RAGE polypeptide with a GPCR polypeptide in the presence of a candidate substance, wherein the GPCR polypeptide is constitutively active and / or activated by the addition of an agonist, a partial agonist or an allosteric modulator of the GPCR; and detecting whether the candidate substance is a modulator of RAGE ligand-independent RAGE activation by co-localized activation-type GPCR by detecting an effect indicative of RAGE activation modulated by the presence of the candidate substance and / or by detecting RAGE-dependent signaling modulated by the presence of the candidate substance.

Description

This application is a divisional application of Chinese patent application No. 201880068213.4. The filing date of the original application is August 21, 2018. The name of the invention is “Screening assays, regulators and regulation of activation of the receptor for advanced glycation end products (RAGE)”. Field of the Invention

[0001] The present invention generally relates to screening assays for identifying modulators of receptor activation associated with certain diseases and / or conditions, to such modulators, and to treatment methods comprising administering such modulators. More specifically, the present invention relates to modulators that activate the receptor for advanced glycation end products (RAGE) via certain co-localized activated G protein-coupled receptors (GPCRs), including activated angiotensin II receptor type 1 (AT1R) and activated CC chemokine receptor 2 (CCR2), by means of a RAGE ligand-independent mechanism (also referred to as RAGE ligand-independent RAGE transactivation), and modulate or not modulate RAGE activation by RAGE ligands (including S100A8 / A9, advanced glycation end products (AGEs), and HMGB1. The present invention also relates to screening assays for identifying such modulators and methods of using the modulators to treat RAGE-related disorders. Background of the Invention

[0002] The receptor for advanced glycation end products (RAGE) is a multivalent type I transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily (Neeper et al., 1992). The 50-55 kDa glycosylated RAGE protein is constitutively expressed in a limited range of cells (e.g., vascular endothelium, type II pneumocytes, leukocytes), although RAGE expression can be induced in most cell types and tissues after injury and inflammation (Ballinger et al., 2005). RAGE expression is significantly upregulated in important inflammatory and metabolic diseases, including but not limited to cardiovascular disease (CVD), cancer, diabetes, chronic kidney disease (CKD), ischemic injury and Alzheimer's disease (Yan et al., 2010).

[0003] It has been previously shown that genetic deletion of the AGER gene encoding RAGE in mice results in protection from numerous diseases and disease processes, including certain cancers (Malik et al., 2015) and inflammatory diseases (Chuah et al., 2013), the latter including atherosclerosis and diabetic complications. For example, in apolipoprotein E (apoE) knockout (KO) mice, deletion of RAGE results in less plaque accumulation with age and attenuates diabetes-accelerated atherosclerosis (Soro-Paavonen et al., 2008). Similarly, deletion of AGER can attenuate renal damage in diabetic mice without affecting glucose control (Thomas et al., 2005).

[0004] Polymorphisms in AGER genes have been associated with numerous diseases and disease processes in humans, including but not limited to arthritis, atherosclerotic and diabetic complications, cancer risk, obesity, epilepsy, and cognitive impairment, including Alzheimer's disease.

[0005] Binding of advanced glycation end products (AGEs) and non-AGE ligands (including members of the S100 calgranulin family, HMGB1, amyloid beta-1, and Mac-1) to the extracellular domain of RAGE activates a series of signaling cascades involved in inflammation, injury, and dysfunction (including nuclear factor κB (NFκB) and the renin-angiotensin aldosterone system (RAAS)).

[0006] In experimental models, inhibition of ligand-mediated RAGE activation using soluble decoy receptors attenuated atherogenesis and vascular injury (Schmidt et al. 1999), suggesting that the pathological effects of RAGE in these settings are mediated in part by ligand-mediated RAGE activation.

[0007] The precise molecular mechanisms by which RAGE is activated and its full biological effects are manifested are poorly understood, and therefore, the ability to target these clinically important signaling pathways in the clinical setting has not yet emerged.

[0008] Renin-angiotensin aldosterone system (RAAS) is a key homeostatic pathway, which is also involved in the formation and progression of many common diseases and disease processes. The inhibition of renin-angiotensin aldosterone system (RAAS) with angiotensin converting enzyme (ACE) inhibitors or angiotensin II receptor type 1 (AT1R) blockers (inhibitors) is widely used to manage many diseases and / or conditions, including hypertension, cardiovascular disease (CVD), heart failure, chronic kidney disease (CKD) and diabetic complications. RAAS inhibition has also been shown to be beneficial in preventing diabetes (Tikellis et al., 2004), neuroprotection (Thoene-Reineke et al., 2011), regulating the growth of certain cancers (Shen et al., 2016) and even in aging, while genetic deletion of AT1R brings longevity in mice (Benigni et al., 2009).

[0009] These effects of RAAS blockade are additive and independent of the blood pressure reduction achieved with RAAS blockade, as comparable blood pressure reduction with other drugs has not resulted in the same benefit (Lee et al., 1993). Specifically, AT1R activation by angiotensin II (Ang II) triggers the induction of oxidative stress, activation of nuclear factor kappa B (NFκB), and inflammation through pathways distinct from those that cause vasoconstriction.

[0010] Activation of the renin-angiotensin aldosterone system (RAAS) is known to be an important mediator of atherosclerosis (Lee et al., 1993; and Jacoby et al., 2003). Atherosclerosis is increased after angiotensin (Ang) II infusion and is associated with physiological RAAS activation in experimental models, including a low-salt diet (Tikellis et al., 2012), diabetes (Goldin et al., 2006; and Soro-Paavonen et al., 2008), and genetic deletion of angiotensin-converting enzyme 2 (Ace2) (Thomas et al., 2010), regardless of its effects on blood pressure homeostasis. Similarly, inhibition of the RAAS has additional anti-atherosclerotic effects that are independent of systemic blood pressure reduction (Candido et al., 2002; Candido et al., 2004; and Knowles et al., 2000). Ang II has numerous direct pro-atherogenic effects (Daugherty et al., 2000; Ferrario et al., 2006; and Ekholm et al., 2009), including induction of oxidative stress (Rajagopalan et al., 1996), vascular adhesion (Grafe et al., 1997), and inflammation (Marvar et al., 2010).

[0011] These pro-atherogenic effects are thought to be primarily mediated by activation of the angiotensin type 1 receptor (AT1R) and the subsequent induction of reactive oxygen species (ROS) and activation of NFκB signaling (Li et al., 2008). However, the signaling mechanisms underlying these effects are poorly understood, including their relative independence from conventional vasoconstrictor signaling via the AT1R.

[0012] It has also been shown that the pathogenesis of atherosclerosis involves certain chemokine signaling pathways, and that macrophage infiltration into arterial lesions directly contributes to this abnormal inflammatory disease (Boisvert et al., 2004). In fact, all known CC and CXC chemokine receptors, as well as CX3CR1 and XCR1, have been involved in inflammation (Murphy et al. 2000; Zlotnik and Yoshie 2000). The main physiological function of chemokine ligands (CCLs) is to "regulate cell migration during conventional immune surveillance, inflammation and development" (Allen et al., 2007). CCLs are released in response to proinflammatory cytokines and selectively bind to a large family of G protein-coupled receptors that mediate the physiological response to chemokines. Chemokines were originally called chemotactic cytokines.

[0013] Studies in animal models of chronic inflammatory diseases have shown that inhibition of the binding between MCP-1 (monocyte chemoattractant protein-1, also known as monocyte chemoattractant protein-1, monocyte chemoattractant and activating factor (MCAF), and chemokine (CC motif) ligand 2 (CCL2)) and CCR2 (chemokine (CC motif) receptor 2) by antagonists suppresses the inflammatory response. The interaction between MCP-1 and its corresponding receptor CCR2 has been implicated (Rollins, 1996; Dawson et al., 2003) in the pathology of inflammatory diseases such as uveitis, atherosclerosis, rheumatoid arthritis, multiple sclerosis, Crohn's disease, nephritis, organ allograft rejection, fibrotic lung, renal insufficiency, diabetes and diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic retinitis, diabetic microangiopathy, tuberculosis, sarcoidosis, Invasive staphylococcal infections, inflammation following cataract surgery, allergic rhinitis, allergic conjunctivitis, chronic urticaria, allergic asthma, periodontal disease, periodontitis, gingivitis, gum disease, dilated cardiomyopathy, myocardial infarction, myocarditis, chronic heart failure, vascular stenosis, restenosis, reperfusion disorders, glomerulonephritis, solid tumors and cancers, chronic lymphocytic leukemia, chronic myeloid leukemia, multiple myeloma, malignant myeloma, Hodgkin's disease, and bladder, breast, cervical, colon, lung, prostate, or stomach cancer.

[0014] MCP-1 and CCR2 KO mice have all been shown to significantly reduce the infiltration of mononuclear cells to inflammatory lesions in the absence of these signal transduction pathways. In addition, this type of KO mice resists the formation of asthma, atherosclerosis and uveitis induced by experimental allergic encephalomyelitis (EAE, human multiple sclerosis model), cockroach allergen. During treatment with TNFα antagonists (e.g., monoclonal antibodies and soluble receptors) at dosage levels relevant to reducing MCP-1 expression and infiltrating macrophage number, rheumatoid arthritis patients and Crohn's disease patients improve.

[0015] MCP-1 has been implicated in the pathogenesis of seasonal and chronic allergic rhinitis and has been found in the nasal mucosa of a large proportion of patients with dust mite allergy. MCP-1 has also been found to induce histamine release from basophils in vitro. During allergic disease, allergens and histamine have been shown to trigger (i.e., upregulate) the expression of MCP-1 and other chemokines in the nasal mucosa of patients with allergic rhinitis, suggesting the existence of a positive feedback loop in such patients.

[0016] Kidney disease is relevant to the chronic inflammation that is characterized as kidney macrophage gathering.It has been determined that diabetic kidney produces monocyte chemoattractant protein-1 (MCP-1 / CCL2) and is the leading factor (Tesch et al., 2008) that affects macrophage gathering in the kidney disease derived from diabetic nephropathy.In multiple animal models, suppress CCR2 and / or suppress specific CCR2 approach and / or suppress CCR2 part MCP-1 and show and reduce kidney damage (Tesch et al., 2008; Rao V et al., 2006; Kang et al., 2010; Kitagawa et al., 2004; Park J et al., 2008).

[0017] Tesch (2008) noted that selectively targeting MCP-1 has been shown to be an effective treatment in animal models of kidney disease, including diabetic nephropathy. Treatments involving small molecule antagonists of CCR2 (INCB3344, propagermanium, and RS-504393) have been shown to inhibit inflammation in mouse models of multiple sclerosis, renal ischemia-reperfusion injury, ureteral obstruction, and diabetic nephropathy, as well as in rat models of arthritis. Engineered biological antagonists of CCR2 have also been shown to be effective. Subcutaneous infusion of cells transfected with a vector expressing a truncated, inactive form of MCP-1 has been found to inhibit renal inflammation in a mouse model of lupus nephritis. Similarly, muscle transfection with 7ND (a mutant of MCP-1) reduced renal inflammation in mouse models of renal ischemia-reperfusion injury, lupus nephritis, and diabetic nephropathy. To date, human trials of chemokine monotherapy for inflammatory diseases have not resulted in drug approval. Anders HJ et al. considered why single chemokine antagonist therapy has not been effective in treating the disease and discussed possible explanations, including the redundancy of single chemokine mediators and the variable expression patterns of chemokine receptors (Anders HJ et al. 2010). Therefore, there is a need in the art for effective treatments for diseases caused by activation of the CCR2 pathway.

[0018] It is important to point out that the concept that RAGE can be activated in a RAGE ligand-independent manner via the co-localized activating GPCR that is the subject of the present invention has implications for a wide range of GPCRs, particularly those involved in inflammation and cell proliferation.

[0019] It is in this context that a novel functional interaction between certain co-localized activating GPCRs, including AT1R and CCR2, and RAGE in a RAGE ligand-independent manner was described. SUMMARY OF THE INVENTION

[0020] It is known that RAGE signaling, the renin angiotensin aldosterone system (RAAS) and certain chemokine signaling pathways functionally interact in pathways involved in the formation and progression of vascular complications. For example, RAGE ligands bind to RAGE and can induce proinflammatory signaling, which can be reduced by antagonists (inhibitors) of AT1R (e.g., Fukami et al. 2004). Equally, AT1R receptors are activated by Ang II to increase the formation and release of RAGE ligands, and inhibiting the binding of RAGE ligands to RAGE or reducing the intervention of RAGE ligands can attenuate the damage caused by Ang II-AT1R (e.g., Thomas et al. 2005). Some downstream signaling pathways and mediators induced after RAGE ligand activation of RAGE, especially those that lead to inflammation, are also similar to those signaling pathways and mediators induced after Ang II activation of AT1R (e.g., NFκB activation).

[0021] This prior art does not suggest or disclose any evidence of complexation between RAGE and GPCRs, such as angiotensin receptors (e.g., AT1R) or certain chemokine receptors (e.g., CCR2). In the absence of any RAGE ligand, or indeed in the presence of a RAGE ligand-binding extracellular domain that does not require RAGE, it is not foreseeable that activation of a co-localized GPCR by the corresponding ligand of such a GPCR, such as activation of the angiotensin receptor by Ang II or activation of CCR2 by MCP-1, would directly lead to activation of RAGE, particularly the RAGE cytoplasmic tail; subsequent induction of signaling via RAGE is not foreseeable. Therefore, it is not foreseeable that modulation of RAGE ligand-independent activation of the RAGE cytoplasmic tail would involve modulation of signaling induced following activation of a co-localized GPCR, such as by binding of Ang II to AT1R or binding of MCP-1 to CCR2.

[0022] One of the prominent features of RAGE is that it is activated by multiple ligands at multiple sites on its extracellular domain, rather than a single ligand and a single binding site that can be used for inhibition. RAGE can be activated by advanced glycation end products (AGEs) and other non-AGE ligands, including high-mobility group box-1 (HMGB-1), S-100 / calgranulin, SAA, Aβ, C3a, heat shock protein 70 (HSP70), acidic and cysteine-rich stromal cell damage-associated glycoprotein secreted protein (SPARC), β2-integrin Mac-1 (CD11b), phosphatidylserine (PS), double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), lipopolysaccharide (LPS), and advanced oxidation protein products.

[0023] Activation of the RAGE extracellular domain by RAGE ligands triggers NFκB activation and subsequent NFκB-driven gene expression, leading to inflammation, oxidative stress, fibrosis, and cell proliferation (Bierhaus et al., 2001).

[0024] RAGE ligand-induced signaling also triggers a positive feedback loop in which RAGE ligand-receptor interactions increase RAGE expression via NFκB activation, thereby enhancing subsequent RAGE-induced cellular activation. In fact, the only means known to the inventors to strongly downregulate RAGE expression is to reduce RAGE activation. This is in contrast to other receptors, such as the low-density lipoprotein (LDL) receptor, in which elevated levels of ligand reduce receptor expression.

[0025] Importantly, the inventors have shown that upon activation of certain co-localized GPCRs (such as AT1R activation by Ang II or CCR2 activation by MCP-1), the RAGE cytoplasmic tail is activated, independent of any RAGE ligand or RAGE extracellular domain, thereby initiating downstream signaling that leads to activation of NFκB, a key transcription factor involved in inflammation, oxidative stress, fibrosis, cell proliferation, and cell survival. The lack of RAGE expression, and in particular the lack of expression of key domains in the RAGE cytoplasmic tail, independent of expression of the RAGE extracellular domain, prevents the induction of NFκB activation upon activation of co-localized GPCRs (such as AT1R activation by Ang II or CCR2 activation by MCP-1). Without wishing to be bound by theory, the inventors believe that activation of the RAGE cytoplasmic RAGE tail by certain co-localized activating GPCRs (including AT1R and CCR2) is the dominant pathway by which RAGE is activated. Additionally, and again not wishing to be bound by theory, the inventors believe that de novo expression of RAGE in cells subjected to, for example, injury, stress or hypoxia provides a pathway for pro-inflammatory signaling via activation of established GPCR signaling.

[0026] The inventors have shown that RAGE ligand-independent activation of the RAGE cytoplasmic tail, following activation of certain co-localized GPCRs (such as AT1R by Ang II or CCR2 by MCP-1), also triggers signaling that increases RAGE expression.

[0027] RAGE has been implicated in many aspects of tumor biology, including tumor cell growth, migration, and invasion (Malik et al., 2015; Abe et al., 2008). Many cancers have elevated levels of RAGE (illustrative examples are breast, colon, kidney, and stomach cancers; Taguchi et al., 2000). Lung cancer is an exception, in which RAGE expression is reduced because RAGE is a normal part of lung function and is lost as lung cells differentiate and become more malignant (Marinakis et al., 2014). In C6 glioma cells, tumor volume was significantly reduced in tumors containing cells in which RAGE was blocked. In contrast, tumors that overexpressed RAGE grew rapidly and invaded surrounding tissues very efficiently (Taguchi et al., 2000). Therapeutics that block RAGE signaling have been pursued as cancer therapeutics for many common cancers, including but not limited to: glioblastoma multiforme / medulloblastoma (Taguchi et al., 2000); pancreatic cancer (Malik et al., 2015; Leclerc et al., 2015); melanoma (Malik et al., 2015); prostate cancer (Malik et al., 2015); breast cancer (Malik et al., 2015); liver cancer / hepatocellular carcinoma (Logsdon et al., 2007; Volz et al., 2010); and colon cancer (Sparvero et al., 2009).

[0028] RAGE has been implicated in a range of brain diseases for which preclinical and clinical studies have supported the potential use of RAGE inhibitors in their treatment, including but not limited to Alzheimer's disease (Cai et al., 2016). Other brain diseases in which RAGE signaling is implicated include but are not limited to amyotrophic lateral sclerosis (Ray et al., 2016); Huntington's disease (Ray et al., 2016); Creutzfeld-Jakob disease (Ray et al., 2016); neurodegenerative diseases such as diabetic neuropathy, familial amyloid polyneuropathy, Charcot neuroarthropathy, and vasculitic neuropathy (Ray et al., 2016); neuropathic pain (Wan et al., 2016); glioma formation and progression (Angelopoulou et al., 2016); and ischemic brain injury / stroke (Xia et al., 2010).

[0029] Under healthy conditions, lung RAGE expression is the highest among all tissues. However, RAGE expression in the lung is normally found only in type 1 pneumocytes. Upregulation of RAGE signaling in other cells and other parts of the lung has been implicated in a range of lung conditions, including but not limited to: chronic obstructive pulmonary disease (COPD) / emphysema (Sukkar et al., 2012); asthma (Sukkar et al., 2012); damage due to smoking / pollution; acute lung injury / acute respiratory distress syndrome (ARDS) (Guo et al., 2012); and pulmonary fibrosis.

[0030] RAGE is critically involved in numerous inflammatory conditions and is therefore a potential therapeutic target for their treatment. Such conditions include, but are not limited to: inflammatory arthritis (Sparvero et al., 2009; Chuah et al., 2013); osteoarthritis (Xie et al., 2013); diseases of the retina (Barile et al., 2007); atherosclerosis (Soro-Paavonen et al., 2008; Schmidt et al., 1999; Park et al., 1998; Zhou et al., 2003; Yan et al., 2010); vascular calcification (Ott et al., 2014); cardiomyopathy (Volt et al., 2010; Russo et al., 2016); ischemic heart disease / cardiac remodeling / fibrosis (Yan et al., 2010; Rama et al., 2010). Ramasamy et al., 2012); heart failure (Ramasamy et al., 2012); diabetic and nondiabetic kidney disease (Fukami et al., 2015; Gugliucci et al., 2014); inflammatory bowel disease (Ott et al., 2014); preeclampsia (Daffu et al., 2013); polycystic ovary syndrome (Garg et al., 2015); hepatic steatosis, fibrosis, ischemic and nonischemic liver injury (Yamagishi et al., 2015); spinal cord injury (Yamagishi et al., 2015); skin inflammation and aging (Tong et al., 2014); and keratitis (Tong et al., 2014).

[0031] The present invention arises, in part, from the inventors' determination that RAGE forms receptor heteromeric complexes in the cell membrane with certain co-localized GPCRs, including AT1R and CCR2.

[0032] Furthermore, the present invention arises in part from the inventors' recognition that activation of certain co-localized GPCRs, such as the angiotensin receptor in the form of the AT1R, in this case with Ang II, or certain chemokine receptors such as CCR2, in this case with MCP-1, triggers RAGE ligand-independent activation of the RAGE cytoplasmic tail.

[0033] The inventors have shown that activation of certain co-localized GPCRs (such as AT1R activation by Ang II or CCR2 activation by MCP-1) leads to activation of domains of the RAGE cytoplasmic tail through a common mechanism. This transactivation pathway does not require the release of RAGE ligands or their binding to the RAGE extracellular domain (i.e., it is RAGE ligand-independent RAGE activation).

[0034] Even though there are published data suggesting that the RAGE cytoplasmic tail is phosphorylated (Sakaguchi et al., 2011), the inventors have shown that RAGE ligand-independent signaling induced upon activation of certain co-localized GPCRs (such as activation of the AT1R receptor by Ang II) does not require phosphorylation of the RAGE cytoplasmic tail at serine 391 or any other site in the RAGE cytoplasmic tail. In addition, the inventors have shown that RAGE ligand-dependent signaling induced upon binding of RAGE ligands (e.g., S100A8 / A9) to the RAGE extracellular domain does not necessarily require phosphorylation of the RAGE cytoplasmic tail at serine 391 or any other site in the cytoplasmic tail, as RAGE homologs from other mammals and RAGE mutants lacking any residue capable of sustaining phosphorylation are still able to be activated and induce signaling in response to both RAGE ligand-dependent and RAGE ligand-independent RAGE activation. In addition, in the absence of the target for RAGE phosphorylation, N-truncated constructs of RAGE (e.g., S391A-RAGE 362-404 ), demonstrating that the regulatory effects of the RAGE constructs described by the inventors are independent of RAGE phosphorylation.

[0035] Prior art shows that inhibitors of PKCζ inhibit RAGE ligand-dependent (e.g., s100-induced) signaling through RAGE, as well as many other PKCζ-dependent pathways. In humans and animals, severe diseases are caused by genetic deletions of PKCζ. Researchers have shown that inhibitors of PKCζ also inhibit RAGE ligand-independent (i.e., transactivation-induced) signaling through full-length RAGE. However, N-truncated constructs of RAGE (e.g., RAGE 362-404 ) is not affected by inhibition of PKCζ, confirming that the regulatory effects of the RAGE constructs described by the inventors are independent of PKCζ.

[0036] Inhibitors of common pathways induced upon RAGE activation (e.g., myD88, TIRAP, interleukin-1 receptor-associated kinase 4 (IRAK4), or NFκB) nonspecifically block both RAGE ligand-dependent (e.g., s100-induced) and RAGE ligand-independent (i.e., transactivation-induced) signaling via RAGE. Since other receptors (e.g., TLRs) also use these signaling molecules / pathways, inhibition of any of these mediators would be nonspecific for RAGE signaling and would affect many other functions of these signaling mediators, which could be detrimental to human health (e.g., unlike RAGE deletion, genetic deletion of myD88, TIRAP, IRAK4, or NFκB is harmful to humans and animals).

[0037] The inventors have further shown that selective modulation (e.g., inhibition) of RAGE ligand-independent signaling can be achieved by selectively targeting signaling mediated through the RAGE cytoplasmic tail, and that the inventors' assays and modulators identified therefrom act on this transactivation (RAGE ligand-independent RAGE activation) process.

[0038] The inventors have further shown that dual inhibition of RAGE ligand-dependent RAGE activation and RAGE ligand-independent RAGE signaling transactivation can also be achieved by selectively targeting signaling mediated through the RAGE cytoplasmic tail, and that the inventors' assays and modulators identified therefrom are able to act simultaneously on both modes of RAGE activation due to a common mediator. This is completely different from soluble RAGE. 22-331 , RAGE neutralizing antibodies and small molecules that selectively bind to the extracellular domain of RAGE and may potentially inhibit only RAGE ligand-dependent RAGE activation.

[0039] The inventors have further shown that modulation of RAGE ligand-dependent signaling and / or RAGE ligand-independent RAGE signaling transactivation by selectively targeting signaling mediated through the RAGE cytoplasmic tail can be achieved without modulating the interaction between RAGE and Diaphran-1 (Diaph1), where prior art suggests that Diaphran-1 (Diaph1) may be a regulator of ligand-dependent RAGE activation (Manigrasso, MB et al. 2016). In addition, in the absence of Diaph1, N-truncated constructs of RAGE (e.g., RAGE 362-404 ), confirming that the regulatory effects of the RAGE constructs described by the inventors are independent of Diaph1.

[0040] Sakaguchi and collaborators found that when HEK293 cells were treated with RAGE ligands S100A11, S100A12, HMGB1, or AGE, resulting in RAGE ligand-dependent RAGE activation, the common proinflammatory adaptor proteins TIRAP, MyD88, and IRAK were predominantly coprecipitated with RAGE overexpressed in the cells. These interactions were not specific for RAGE, as TIRAP, MyD88, and IRAK also function as adaptor proteins for all toll-like receptors (TLRs) except TLR-3 to activate NFKB transcription.

[0041] Following this work, the same group published findings suggesting that S391E-RAGE mimics the phosphorylation state of RAGE and masks the adaptor protein TIRAP, thereby preventing endogenous RAGE signaling. 387-395 (RAGE(E)-I) acts as an inhibitor of specific aspects of RAGE ligand-dependent signaling (i.e., inhibition of apoptosis, cell migration, and invasion) (Putranto et al., 2013). However, the inventors have shown that RAGE activation does not require phosphorylation. In addition, masking these common adaptor proteins will also affect signaling through TLRs (such as TLR-2 and TLR-4), some of which can also be activated by RAGE ligands (such as s100 proteins), which may explain the findings of Puranto et al. In the same experiments, they also argued that S391A-RAGE 387-395 is not a suitable inhibitor as it did not show any appreciable TIRAP binding and did not attenuate apoptosis induced by the RAGE ligand S100B (Putranto et al., 2013). They also noted that S391E-RAGE 387-395 The entire RAGE ligand-induced signaling pathway was not inhibited, as the growth of U-87MG cells was not significantly affected as assessed by measuring intracellular ATP levels (Putranto et al., 2013). Therefore, Putran and co-workers postulated that the RAGE ligand-dependent pathway that was inhibited and the RAGE cytoplasmic tail fragment they used was clearly distinct from the RAGE ligand-independent RAGE activation via the co-localized activated GPCR and modulator that is the subject of the present invention. In fact, they used S391A-RAGE 387-395 The negative results obtained are in contrast to the teaching of the present invention.In this publication, Putranto et al. never contemplated RAGE ligand-independent activation of the RAGE cytoplasmic tail by means of a co-localized GPCR.

[0042] EP 1 415 997-A1 details the identification and use of polypeptides that bind directly or indirectly to the RAGE cytoplasmic tail and thereby inhibit or enhance signal transduction resulting from ligand binding to RAGE and subsequent NFκB activation, as well as downstream pathways resulting from such activation. The present invention differs from this teaching in several ways. First, this teaching does not contemplate RAGE ligand-independent signaling via RAGE or dual inhibition of RAGE ligand-dependent and RAGE ligand-independent signaling via RAGE. Second, the claims in EP 1 415 997-A1 relate to the use of polypeptides that bind to unidentified elements in the RAGE cytoplasmic tail. In contrast, the inventors have demonstrated that polypeptides encoding the RAGE cytoplasmic tail and mutant forms thereof can be used to selectively bind to signaling molecules involved in RAGE ligand-independent signaling via RAGE or both RAGE ligand-dependent and RAGE ligand-independent signaling via RAGE, resulting in modulation of subsequent NFκB activation and downstream pathways resulting from such activation. Third, the inventors have demonstrated the ability to modulate RAGE ligand-independent signaling via RAGE using selectively modified polypeptides containing key elements of the RAGE cytoplasmic tail. Fourth, EP 1 415 997-A1 does not demonstrate modulation of RAGE ligand-dependent signaling via RAGE. Furthermore, the only polypeptide specifically identified in EP 1 415 997-A1 as capable of modulating RAGE ligand-dependent signaling is PKCζ, a well-known binding partner and signaling mediator of full-length RAGE. The inventors have shown that PKCζ is not required for their regulators to function.

[0043] After binding to CpG-DNA, RAGE is monoubiquitinated by the F-box protein (FBXO10) at cytoplasmic residue K374, triggering its endocytosis and lysosome-mediated degradation (Evankovich et al. 2017). No other proinflammatory RAGE ligands have been observed to undergo endocytosis and / or RAGE ubiquitination.

[0044] RAGE ubiquitination is partially dependent on S391, such that upon overexpression of FBXO10, the S391A-RAGE mutant is partially resistant to ubiquitination and subsequent degradation.

[0045] These data suggest that under certain circumstances K374R and S391A-RAGE mutants can be resistant to ubiquitination, which may allow these mutants to accumulate at higher levels than wild-type RAGE. However, this potential increased stability / resistance to degradation cannot explain the rapid modulation of RAGE ligand-independent RAGE activation following activation of a colocalized GPCR, nor the inhibition of RAGE ligand-independent RAGE activation signaling by the S391A-RAGE mutant even in the presence of a 1000-fold excess of wild-type RAGE delivered, as detailed below, nor the fact that this modulation occurs similarly in the presence and absence of K374.

[0046] Without limiting the generality of the following detailed description, the inventors have demonstrated that activation of certain co-localized GPCRs (e.g., activation of AT1R by, for example, Ang II or activation of CCR2 by, for example, MCP-1) triggers activation of the co-localized RAGE cytoplasmic tail. This activation can occur in the absence of the RAGE extracellular domain and is therefore completely unrelated to RAGE ligands or their interactions with the RAGE extracellular domain. Without wishing to be bound by theory, the inventors believe that this transactivation of RAGE by certain co-localized GPCRs represents an important RAGE activation mechanism. Consistent with this premise, the inventors demonstrate that selective restoration of RAGE ligand-independent RAGE signaling in AGER apoE double KO (DKO) mice restores atherosclerosis to levels not significantly different from those observed in RAGE-rich apoE KO mice, even though RAGE ligand-dependent signaling remains completely absent.

[0047] Many of the adverse signaling events induced by AT1R activation are attenuated when RAGE expression is absent (eg, genetically deleted or silenced, or in healthy cells that do not express RAGE, or when RAGE ligand-independent RAGE activation via activated AT1R is prevented or inhibited).

[0048] Meanwhile, RAGE-independent AT1R signaling pathways inhibited by AT1R antagonists, such as the Gq signaling pathway induced by AT1R activation leading to the induction of phosphoinositide and calcium influx, were not affected by RAGE deletion, silencing of RAGE expression, or inhibition of RAGE function.

[0049] Thus, modulation, and in particular inhibition, of RAGE ligand-independent RAGE activation by certain co-localized activating GPCRs (such as AT1R or CCR2) provides additional advantages for therapeutic intervention targeting pathogenic signaling induced by RAGE after activation of co-localized GPCRs. For example, in specific embodiments, such modulators allow for aggressive targeting of the adverse effects of AT1R without disrupting blood pressure regulation or causing feedback "escape" from AT1R inhibition (as occurs after AT1R inhibition) that limits the use of RAAS inhibitors. Such modulators will also only affect cells and tissues in which this transactivation pathway is constitutively active (e.g., leukocytes, endothelial cells) or induced (e.g., sites of inflammation and injury), leaving RAAS and other GPCR-mediated signaling unaffected in cells that also do not express RAGE (e.g., healthy smooth muscle cells).

[0050] Activation of AT1R has hemodynamic and non-hemodynamic effects. Hemodynamic effects are those that cause changes in blood flow and include changes in blood volume, blood pressure, flow or velocity, resistance, cardiac output, turbulence, and wall tension. AT1R blockers (inhibitors) can show hemodynamic effects (e.g., lower blood pressure, changes in resistance and cardiac output) as well as non-hemodynamic effects (e.g., triggering oxidative stress and inflammation). In states where RAAS is activated (e.g., heart disease, kidney disease, hypertension), both hemodynamic and non-hemodynamic pathways are activated.

[0051] RAGE ligand-independent RAGE activation via the activated AT1R is the only mediator of the non-hemodynamic (non-blood flow) effects of AT1R activation. The inventors have observed that complete genetic deletion of RAGE has no direct hemodynamic effects (e.g., no effect on blood pressure, vascular resistance or flow, blood volume) and does not modulate the hemodynamic effects of AT1R activation or inhibition. A major advantage of targeting RAGE ligand-independent RAGE activation via the activated AT1R is that it is therefore not subject to the constraints of blood pressure regulation, which limit how much blood pressure can be lowered before adverse hemodynamic effects render the treatment unsafe.

[0052] Furthermore, changes in blood flow automatically trigger feedback (homeostatic) responses that maintain a constant level of blood flow. These feedback responses serve to bias or circumvent the hemodynamic effects of RAAS inhibition by AT1R inhibition or inhibition of angiotensin-converting enzyme (ACE). In contrast, selective inhibition of non-hemodynamic pathways induced by RAAS activation, achieved through inhibition of RAGE ligand-independent RAGE activation via activated angiotensin receptors (such as AT1R), is independent of feedback / escape responses. The absence of such feedback responses supports the durability and efficacy of this inhibitory effect. Regulators of RAGE ligand-independent RAGE activation via colocalized activated GPCRs

[0053] In one form, the present invention comprises modulators of RAGE activity, wherein such RAGE activity is induced by certain co-localized active GPCRs.

[0054] In one form, the invention comprises modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs.

[0055] In one form, the invention comprises a modulator of RAGE-dependent signaling induced by certain co-localized activated GPCRs.

[0056] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs function in the absence of any RAGE ligand.

[0057] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs function in the presence of a truncated RAGE extracellular domain.

[0058] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs function in the presence of a truncated RAGE extracellular domain of no more than 40, no more than 20, no more than 10, or no more than 5 amino acids in length.

[0059] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs comprise the complete extracellular domain of RAGE conjugated to a RAGE transmembrane domain analog, fragment, or derivative that is greater than 5, greater than 10, or greater than 20 amino acids in length.

[0060] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs function in the absence of the RAGE ligand-binding RAGE extracellular domain.

[0061] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs do not contain the extracellular domain of RAGE.

[0062] In one form of the invention, the modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs do not contain RAGE extracellular domain analogs, fragments or derivatives.

[0063] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs comprise fragments of the extracellular domain of RAGE.

[0064] In one form of the invention, a modulator of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs comprises a fragment of the extracellular domain of RAGE that is no more than 40, no more than 20, no more than 10, or no more than 5 amino acids in length.

[0065] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs does not bind to the extracellular domain of RAGE.

[0066] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs inhibit or enhance signaling induced by the co-localized activating GPCR via the C-terminal cytoplasmic tail of RAGE.

[0067] In one form of the invention, modulators of RAGE ligand-independent RAGE activation inhibit binding that occurs at the C-terminal cytoplasmic tail of RAGE via certain co-localized activating GPCRs.

[0068] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs inhibit or promote the interaction between the RAGE transmembrane domain and certain GPCRs.

[0069] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activated GPCRs inhibit the interaction between the RAGE transmembrane domain and certain GPCRs.

[0070] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activated GPCRs inhibit or enhance the ability of activated GPCRs to modulate RAGE-dependent signaling that is dependent on the proximity of the RAGE transmembrane domain and certain GPCRs.

[0071] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activated GPCRs inhibit the ability of activated GPCRs to modulate RAGE-dependent signaling that is dependent on the proximity of the RAGE transmembrane domain and certain GPCRs.

[0072] In one form of the invention, modulators of RAGE ligand-independent RAGE activation by certain co-localized activating GPCRs inhibit or promote the ability of the activating GPCR to modulate RAGE-dependent signaling that is dependent on the proximity of the RAGE transmembrane domain and certain GPCRs, and inhibit or promote signaling induced by the co-localized activating GPCR that occurs through the C-terminal cytoplasmic tail of RAGE.

[0073] In one form of the invention, modulators of RAGE ligand-independent RAGE activation by certain co-localized activated GPCRs inhibit the ability of activated GPCRs to modulate RAGE-dependent signaling that is dependent on the proximity of the RAGE transmembrane domain and certain GPCRs, and inhibit signaling induced by the co-localized activated GPCRs through the C-terminal cytoplasmic tail of RAGE.

[0074] Throughout this specification, unless the context requires otherwise, a co-localized GPCR refers to a member of the G protein-coupled receptor superfamily (GPCR; also known as seven transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptors, and G protein-linked receptors; certain other 7TM proteins have been classified as members of the G protein-coupled receptor superfamily, including GPR107, GPR137, OR51E1, TPRA1, GPR143, and GPR157) that is co-expressed endogenously or by transfection in the same cell as RAGE. Note that not all members of this superfamily are coupled to G proteins, and in this context, the term GPCR includes superfamily members that are not coupled to G proteins. Co-expression in the same cell can be demonstrated by a variety of techniques known to those skilled in the art, including co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), and microscopy. A co-localized GPCR is preferably one that is sufficiently proximal to RAGE to allow for a functional interaction between the GPCR and RAGE. Even more preferably, the co-located GPCR is such a GPCR that is sufficiently adjacent to RAGE so that a suitable proximity assay can detect this proximity. Examples of suitable proximity assays are BRET, FRET, enzyme fragment complementation, split luciferase complementation, split fluorophore complementation, TANGO assay, NanoLuc Binary Technology (NanoBIT) assay, proximity ligation assay (PLA) or any other proximity assay capable of detecting the proximity of two proteins, regardless of whether one or more of these proteins are labeled or tagged to facilitate the use of the assay. Such proximity assays can be configured in different ways, and the receptor-heteromeric inspection technology (receptor-HIT) layout and its derivatives are preferred layouts for such proximity assays (WO2008 / 055313; Jaeger et al., 2014).

[0075] Throughout this specification, unless the context requires otherwise, an activated GPCR is intended to mean a GPCR that is in an active state, which may result from binding of an agonist, partial agonist and / or allosteric modulator, and / or from constitutive activity that does not require ligand binding.

[0076] Throughout this specification, unless the context requires otherwise, certain co-localized activating GPCRs of the present invention are GPCRs that are expressed in the same cells as RAGE and for which, upon activation by the corresponding ligand of the certain co-localized GPCR or when the GPCR is constitutively active, an effect on RAGE is detected that is shown to modulate RAGE activation and / or modulate the induction of RAGE-dependent signaling.

[0077] In one embodiment, the effect on RAGE that is indicative of modulation of RAGE activation is a change in intracellular trafficking, such as that detected by a change in the proximity of luciferase-conjugated RAGE (e.g., RAGE / Rluc8) to an intracellular compartment marker (e.g., a fluorophore-labeled Rab class, such as Rabl, Rab4, Rab5, Rab6, Rab7, Rab8, Rab9 and / or Rabl 1 (e.g., Venus-Rab4, Venus-Rab5, Venus-Rab6, Venus-Rab7, Venus-Rab8, Venus-Rab9 and / or Venus-Rab11)) and / or a plasma membrane marker (e.g., a fluorophore-conjugated K-ras fragment (e.g., Venus-K-ras)) using bioluminescence resonance energy transfer (BRET) upon addition of a corresponding ligand for a co-localized GPCR (Tiulpakov et al., 2016).

[0078] In another embodiment, the effect on RAGE is a change in RAGE-dependent signaling, such as by luciferase-conjugated RAGE (e.g., RAGE-Rluc8) and a RAGE-interacting group (e.g., a fluorophore-labeled protein that interacts with the cytoplasmic tail of RAGE, such as IQGAP-1, protein kinase Cζ (PKCζ), Dock7, MyD88, TIRAP, ERK1 / 2, (Jules et al., 2013; Ramasamy et al., 2016), olfactory receptor 2T2, ADP / ATP translocase 2, protein phosphatase 1G, intercellular adhesion molecule 1, protein DJ-1 (PARK7), calponin-3, brain development regulatory protein, filamin B, Ras-related protein Rab-13, Radixin / Ezrin / Mosin, proteolipid protein 2, corona protein, S100 The presence of α-secreting proteins was detected by changes in the proximity of A11, succinyl-CoA ligase (GDP-forming) subunit α, Hsc70-interacting protein, inhibitor of apoptosis 5, neuropilin, splicing stimulatory factor, growth factor receptor binding protein 2, sec61 β subunit, or Nck1.

[0079] In another embodiment, the effect on RAGE is a change in RAGE-dependent signaling as detected by a change in the canonical activation of NFκB when certain co-localized GPCRs are activated by their corresponding ligands, as measured by one or more of: Detecting IkB kinase (IKK) activity by monitoring in vitro phosphorylation of substrates (e.g., GST-IκBα); Detection of IκB degradation kinetics, including phosphorylation / ubiquitination and / or degradation of IκB and / or IκB-α; Detection of p65 (Rel-A) phosphorylation / ubiquitination, such as by use of antibodies, gel shift, EMSA, and / or mass spectrometry; Detection of cytoplasmic to nuclear shuttling / translocation of NFκB components / subunits (e.g., p65 / phospho-p65); Detection of NFκB subunit dimerization / complexation; Detection of active NFκB components / subunits by binding to immobilized DNA sequences / oligonucleotides containing NFκB response elements / NFκB consensus binding motifs, such as by using electrophoretic mobility shift assays or gel shift assays, SELEX, protein-binding microarrays, or sequencing-based methods; Chromatin immunoprecipitation (ChIP) analysis to detect in situ binding of NFκB to DNA at promoters and enhancers of specific genes; In vitro kinase assay for NFκB kinase activity; NFκB transcriptional activity can be measured using NFκB reporter assays via transgenic expression of reporter constructs (e.g., LacZ Fluc, eGFP SEAP, and NF-gluc) using methods such as plasmid transfection, reporter cell lines, minicircles, retroviruses, or lentiviruses; Changes in expression of NFκB downstream targets (e.g., cytokines, growth factors, adhesion molecules, and mitochondrial anti-apoptotic genes) measured by real-time PCR assays, protein assays, or functional assays (Note: The pleiotropic nature of NFκB is reflected in its current list of approximately 500 transcriptional targets (see http: / / www.bu.edu / nf-kb / gene-resources / target- genes / Deadline: August 2, 2017); and • Measurement of functional or structural changes induced by NFκB-dependent signaling, such as POLKADOTS in T cells, adhesion in endothelial cells, activation in leukocytes, or tumorigenicity.

[0080] In another embodiment, the effect on RAGE is a change in RAGE signaling as detected by a change in NFκB atypical activation by measuring one or more of the following: Detection of NIK (NFκB-inducible kinase); Detect IKKα activation / phosphorylation; NIK kinase activity is measured by performing a kinase assay based on the ability to autophosphorylate or phosphorylate a substrate; Produces NFκB dimers containing p52, such as p52 / RelB; Detection of phospho-NFκB2 p100 (Ser866 / 870); Detects the partial degradation (called processing) of the precursor p100 into p52; Detecting the translocation of p52 / RelB to the nucleus; Detection of p52 / RelB binding to NFκB sites; Measuring NFκB transcriptional activity using NFκB reporter assays with transgenic expression of reporter constructs (e.g., LacZ Fluc, eGFP SEAP, NF-gluc) using such methods as plasmid transfection, reporter cell lines, minicircles, retroviruses, or lentiviruses; and • Measure changes in expression of downstream targets of NFκB non-canonical signaling, such as CXCL12, by real-time PCR, protein expression, or by functional assays. Colocalized GPCRs

[0081] In one embodiment, certain co-localized activating GPCRs of the present invention are those GPCRs that are expressed in the same cells as RAGE and are associated with RAGE-related disorders.

[0082] In one embodiment, certain co-localized activating GPCRs of the present invention are those GPCRs that are expressed in the same cells as RAGE, are associated with a RAGE-associated disorder, and when removed or inhibited result in a reduction or alleviation of the RAGE-associated disorder.

[0083] In one embodiment, certain co-localized activated GPCRs of the invention are those involved in inflammation.

[0084] In one embodiment, certain co-localized activating GPCRs of the present invention are those GPCRs that are involved in inflammation and that when removed or inhibited result in a decrease or reduction in inflammation.

[0085] In one embodiment, certain co-localized activated GPCRs of the invention are those involved in cell proliferation.

[0086] In one embodiment, certain co-localized activating GPCRs of the present invention are those GPCRs that are involved in cell proliferation and that when removed or inhibited result in a decrease or reduction in cell proliferation.

[0087] In fact, there is evidence that many GPCRs are involved in inflammation to some extent and these levels can be distinguished according to the level of evidence: 1-No evidence has been found so far; 2- The receptor structure or motifs within the receptor are similar to known inflammatory / immune receptors or motifs involved in inflammatory / immune processes; 3-receptor binding to ligands that mediate inflammatory / immune processes; 4-Receptors are associated with / involved in inflammatory / immune diseases; 5- At least one paper describes the direct involvement of the receptor in inflammatory / immune processes; 6-receptor is expressed in inflammatory / immune cells; and 7- Well characterized receptors involved in inflammatory / immune processes (e.g. http: / / www.guidetopharmacology.org database).

[0088] Family A GPCRs (other than olfactory, vomeronasal, opsins) and the current level of evidence for their involvement in inflammation (see key points above):

[0089] Family A olfactory GPCRs and the current level of evidence for their involvement in inflammation (see key points above):

[0090] Family A vomeronasal and opsin GPCRs and the current level of evidence for their involvement in inflammation (see key points above):

[0091] Family B GPCRs and the current level of evidence for their involvement in inflammation (see key points above):

[0092] Family C GPCRs and the current level of evidence for their involvement in inflammation (see key points above):

[0093] Frizzled Family GPCRs and the current level of evidence for their involvement in inflammation (see key points above): Subtype Level of evidence References FZD1 6 (Neumann et al., 2010) FZD2 6 (Zhao et al., 1995) FZD3 6 (Lu et al., 2004) FZD4 5 (You et al., 2008) FZD5 5 (You et al., 2008) FZD6 7 (Wu et al., 2009) FZD7 5 (Wada et al., 2013) FZD8 5 (Gregory et al., 2010) FZD9 5 (Wada et al., 2013) FZD10 1 (Dijksterhuis et al., 2014) SMO 1 (Dijksterhuis et al., 2014)

[0094] Other 7TM proteins that have been classified as members of the GPCR superfamily and the current level of evidence for their involvement in inflammation (see key points above):

[0095] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of ADGRA2, ADGRB2, ADGRB3, ADGRF3, ADGRG4, ADGRV 1, CELSR1, CELSR2, CELSR3, OX1 receptor, OX2 receptor, PTH1 receptor, PTH2 receptor, AMY1 receptor, AMY2 receptor, AMY3 receptor, AM1 receptor, AM2 receptor, GPR63, GPR75, NMU2 receptor, OPN5, V1B receptor, y6 receptor, 5-HT4 receptor, GPR101, GPR119, GPR135, GPR137, GPR141, GPR149, GPR150, GPR151, GPR152, GPR157, GPR19, GPR25, GPR37, GPR37L1, GPR50, GPR62, LGR5, MRGPRE, MRGPRF, NTS2 receptor, OPN4, OPN4, OR10A7, OR10AG1, OR10Q1, OR10W1, OR12D3, OR13C2, OR13C3, OR13C4, OR13C5, OR13C8, OR13F1, OR13G1, OR1A2, OR1L1, OR1S1, OR1S2, OR2AK2, OR2D2, OR2D3, OR4A1 5. OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K15, OR4K17, OR4N5, OR5AC2, OR5AK2, OR5AP2, OR5AR1, OR5AS1, OR5B12, OR5B17, OR5B2, OR5 B21, OR5B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5J2, OR5K3, OR5L1, OR5L2, OR5M1, OR5M10, OR5M11, OR5M3, OR5M8, OR5M9, OR5R1, OR5T1, OR5T2, OR5T3, OR5W2, OR6C74, OR6K6, OR6M1, OR6Q1, OR6X1, OR8H1, OR8H2, OR8H3, OR8J1, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR8U8, OR9A4, OR9G1, OR9G4, OR9G9, OR9Q2, TAAR3, TPRA1, Y4 receptor, 5-HT1D receptor, 5-HT1E receptor, ADGRB1, AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor,GABAB1, GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER, GPR107, GPR139, GPR156, GPR158, GPR161, GPR171, GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor, HCA1 receptor, LPA1 receptor, LPA3 receptor, L PA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1, MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1D2, OR2AG1, OT receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1A receptor, V2 receptor, α2A-adrenergic receptor, α2B-adrenergic receptor adrenergic receptor, α2C-adrenergic receptor, β1-adrenergic receptor, β3-adrenergic receptor, 5-HT1B receptor, 5-HT1F receptor, 5-HT2B receptor, 5-HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR 182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR11H1, OR2A1,OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1, OR2C3, OR2J3, OR2L13, OR2T11, OR2T34, OR2 W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51 D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1, O R51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D 1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H 1, OR52I 1, OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2, OR9A2, ketoglutarate receptor, P2RY10, P2RY8, P2RY9 Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sst1 receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R3 9, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenergic receptor, α1B-adrenergic receptor, α1D-adrenergic receptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4,ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemokine receptor, CX3CR1, C XCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLT1 receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2 / ALX, FPR2 / ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, G PR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlu1 receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, O XE receptor, P2Y1 receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PAR1, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2-adrenergic receptor, κ receptor, μ receptor.

[0096] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding CCR4.

[0097] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding CCR5.

[0098] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding CCR4 and CCR5.

[0099] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding CCR4, CCR5, CCR10, and CXCR3.

[0100] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0101] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0102] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding adrenergic α1A receptor, CCR3, M2 receptor, and OX1 receptor.

[0103] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding adrenergic α1A receptor, CCR3, CCR4, M2 receptor, and OX1 receptor.

[0104] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding adrenergic α1A receptor, CCR3, CCR5, M2 receptor, and OX1 receptor.

[0105] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, M2 receptor, and OX1 receptor.

[0106] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, OX1 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0107] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from paragraph

[00095] , excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, M2 receptor, and OX1 receptor.

[0108] In one embodiment, certain co-localized activating GPCRs of the invention are GPCRs selected from paragraph

[00095] , except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, OX1 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor, and somatostatin 3 receptor.

[0109] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of OX1 receptor, OX2 receptor, PTH1 receptor, PTH2 receptor, AMY1 receptor, AMY2 receptor, AMY3 receptor, AM1 receptor, AM2 receptor, GPR63, GPR75, NMU2 receptor, OPN5, V1B receptor, y6 receptor, 5-HT4 receptor, GPR101, GPR119, GPR135, GPR137, GPR141, GPR149, GPR150, GPR151, GPR152, GPR157, GPR19, GPR25, GPR37, GPR37L1, GPR50, GPR62, LG R5, MRGPRE, MRGPRF, NTS2 receptor, OPN4, OPN4, OR10A7, OR10AG1, OR10Q1, OR10W1, OR12D3, OR13C2, OR13C3, OR13C4, OR13C5, OR13C8, OR13F1, OR13G1, OR1A2, OR1L1, OR1S1, OR1S2, OR2AK2, OR2D2, OR2D3, OR4A15, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K15, OR4K17, OR4N5, OR5AC2, OR5AK 2. OR5AP2, OR5AR1, OR5AS1, OR5B12, OR5B17, OR5B2, OR5B21, OR5B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5J2, OR5K3, OR5L1, OR5L2, OR5M 1. OR5M10, OR5M11, OR5M3, OR5M8, OR5M9, OR5R1, OR5T1, OR5T2, OR5T3, OR5W2, OR6C74, OR6K6, OR6M1, OR6Q1, OR6X1, OR8H1, OR8H2, OR8H3, OR8J1, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR8U8, OR9A4, OR9G1, OR9G4, OR9G9, OR9Q2, TAAR3, TPRA1, Y4 receptor, 5-HT1D receptor, 5-HT1E receptor, ADGRB1, AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor, GABAB1, GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER,GPR107, GPR139, GPR156, GPR158, GPR161, GPR171, GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor, HCA1 receptor, LPA1 receptor, LPA3 receptor, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1, MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1D2, OR2AG1, OT Receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1A receptor, V2 receptor, α2A-adrenergic receptor, α2B-adrenergic receptor, α2C-adrenergic receptor, β1-adrenergic receptor, β3-adrenergic receptor, 5-HT1B receptor, 5-HT1F receptor, 5-HT2B receptor, 5-HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, C CK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, G PR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR1 1H1, OR2A1, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1, OR2C3, OR2J3, OR2 L13,OR2 T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51 B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR 51J1, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J3, OR52K 1. OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2, OR9A2, ketoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrR P receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sst1 receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R4 1. TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenergic receptor, α1B-adrenergic receptor, α1D-adrenergic receptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor,BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemokine receptor, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLT1 receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2 / ALX, FPR2 / ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlu1 receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PAR1, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2-adrenergic receptor, κ receptor, μ receptor.

[0110] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding CCR4.

[0111] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding CCR5.

[0112] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding CCR4 and CCR5.

[0113] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding CCR4, CCR5, CCR10, and CXCR3.

[0114] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0115] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0116] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of OX1 receptor, OX2 receptor, PTH1 receptor, PTH2 receptor, AMY1 receptor, AMY2 receptor, AMY3 receptor, AM1 receptor, AM2 receptor.

[0117] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding the adrenergic α1A receptor, CCR3, M2 receptor, and OX1 receptor.

[0118] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding adrenergic α1A receptor, CCR3, CCR4, M2 receptor, and OX1 receptor.

[0119] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding adrenergic α1A receptor, CCR3, CCR5, M2 receptor, and OX1 receptor.

[0120] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, M2 receptor, and OX1 receptor.

[0121] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, OX1 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0122] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, M2 receptor, and OX1 receptor.

[0123] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000109], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, OX1 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0124] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of 5-HT4 receptor, GPR101, GPR119, GPR135, GPR137, GPR141, GPR149, GPR150, GPR151, GPR152, GPR157, GPR19, GPR25, GPR37, GPR37L1, GPR50, GPR62, LGR5, MRGPRE, MRGPRF, NTS2 receptor, OPN4, OPN4, OR10A7, OR10AG1, OR10Q1, OR10W1, OR12D3, OR13C2, OR13C3, OR13C4, OR1 3C5, OR13C8, OR13F1, OR13G1, OR1A2, OR1L1, OR1S1, OR1S2, OR2AK2, OR2D2, OR2D3, OR4A15, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K15, OR4K17, OR4N5, OR5AC2, OR5AK2, OR5AP2, OR5AR1, OR5AS1, OR5B12, OR5B17, OR5B2, OR5B21, OR5B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5 I1, OR5J2, OR5K3, OR5L1, OR5L2, OR5M1, OR5M10, OR5M11, OR5M3, OR5M8, OR5M9, OR5R1, OR5T1, OR5T2, OR5T3, OR5W2, OR6C74, OR6K6, OR6M1, OR6Q1, OR6X1, OR8H1, OR8H2, OR8H3, OR8J1, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR8U8, OR9A4, OR9G1, OR9G4, OR9G9, OR9Q2, TAAR3, TPRA1, Y4 receptor, 5-HT1D receptor, 5-HT1E receptor body, ADGRB1, AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor, GABAB1, GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER, GPR107, GPR139, GPR156, GPR158, GPR161, GPR171, GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor,HCA1 receptor, LPA1 receptor, LPA3 receptor, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1, MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1D2, OR2AG1, OT receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1A receptor, V2 receptor, α2A-adrenergic receptor, α2B-adrenergic receptor, α2C-adrenergic receptor, β1-adrenergic receptor, β3-adrenergic receptor, 5-HT1B receptor, 5-HT1F receptor, 5-HT2B receptor, 5-HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 Receptors, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, G PR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, O R11H1, OR2Al, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1, OR2C3, OR2J3, OR2 L13, OR2T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6,OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1 , OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D 1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2, OR9A2, ketoglutarate receptor, P2RY1 0, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sst1 receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenergic receptor, α1B-adrenergic receptor, α1D-adrenergic receptor, δ receptor, 5-HT1A receptor, 5-HT2 A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, Bl receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2,Chemokine receptors, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLT1 receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2 / ALX, FPR2 / ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor Body, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlu1 receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PAR1, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2-adrenergic receptor, κ receptor, μ receptor.

[0125] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding CCR4.

[0126] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding CCR5.

[0127] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding CCR4 and CCR5.

[0128] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding CCR4, CCR5, CCR10, and CXCR3.

[0129] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0130] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0131] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding the adrenergic α1A receptor, CCR3, and M2 receptor.

[0132] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding adrenergic α1A receptor, CCR3, CCR4, and M2 receptor.

[0133] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding adrenergic α1A receptor, CCR3, CCR5, and M2 receptor.

[0134] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, and M2 receptor.

[0135] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0136] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, and M2 receptor.

[0137] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000124], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0138] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of 5-HT1D receptor, 5-HT1E receptor, ADGRB1, AT2 receptor, BB1 receptor, BB3 receptor, CGRP receptor, CRF1 receptor, CRF2 receptor, ETA receptor, ETB receptor, FZD4, FZD5, FZD7, FZD8, FZD9, GABAB receptor, GABAB1, GABAB2, GAL1 receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, GnRH2 receptor, GPER, GPR107, GPR139, GPR156, GPR158, GPR161, GPR17 1. GPR179, GPR39, GPR45, GPR88, GPRC5A, GPRC5B, GPRC5C, H3 receptor, HCA1 receptor, LPA1 receptor, LPA3 receptor, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1, MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor, NTS1 receptor, OR1D2, OR2AG1, OT receptor, PAC1 receptor, RXFP1 receptor, secretin receptor, TSH receptor, UT receptor, V1A receptor, V2 receptor, α2A-adrenergic receptor, α2B -Adrenergic receptor, α2C-adrenergic receptor, β1-adrenergic receptor, β3-adrenergic receptor, 5-HT1B receptor, 5-HT1F receptor, 5-HT2B receptor, 5-HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnR H1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR182, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6,LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor, M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR11H1, OR2A1, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C1, OR2C3, OR2J3, OR2L13, OR2T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR 51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, O R51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2, OR9A2, ketoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sstl receptor, sst2 receptor, sst3 receptor, sst4 receptor , sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8,TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenergic receptor, α1B-adrenergic receptor, α1D-adrenergic receptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 Receptors, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemokine receptors, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLT1 receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP Receptor, FPR1, FPR2 / ALX, FPR2 / ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlu1 receptor, mGlu5 receptor, MRGPRX 2. MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PAR1, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2-adrenergic receptor, κ receptor, μ receptor.

[0139] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding CCR4.

[0140] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding CCR5.

[0141] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding CCR4 and CCR5.

[0142] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding CCR4, CCR5, CCR10, and CXCR3.

[0143] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0144] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0145] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding the adrenergic α1A receptor, CCR3, and M2 receptor.

[0146] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding adrenergic α1A receptor, CCR3, CCR4, and M2 receptor.

[0147] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding adrenergic α1A receptor, CCR3, CCR5, and M2 receptor.

[0148] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, and M2 receptor.

[0149] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0150] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, and M2 receptor.

[0151] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000138], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0152] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of 5-HT1B receptor, 5-HT1F receptor, 5-HT2B receptor, 5-HT2C receptor, 5-HT5A receptor, 5-HT6 receptor, 5-HT7 receptor, ADGRE4P, ADGRF1, ADGRG1, ADGRG3, ADGRG5, calcitonin receptor-like receptor, CB1 receptor, CB2 receptor, CCK1 receptor, CCK2 receptor, CT receptor, D1 receptor, body, D2 receptor, D3 receptor, D4 receptor, D5 receptor, FFA1 receptor, FFA3 receptor, FSH receptor, FZD1, FZD2, FZD3, GHRH receptor, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR148, GPR153, GPR160, GPR162, GPR17, GPR173, GPR174, GPR176, GPR18, GPR1 82, GPR20, GPR22, GPR26, GPR27, GPR3, GPR33, GPR35, GPR6, GPR61, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kisspeptin receptor, LGR4, LGR6, LH receptor, LPA2 receptor, LPA6 receptor, M1 receptor , M2 receptor, M3 receptor, M4 receptor, M5 receptor, MAS1L, MC3 receptor, MC5 receptor, MCH2 receptor, mGlu4 receptor, mGlu7 receptor, mGlu8 receptor, MRGPRG, NOP receptor, NPBW1 receptor, NPBW2 receptor, OPN3, OR11H1, OR2A1, OR2A2, OR2A4, OR2A42, OR2A7, OR2B11, OR2B6, OR2C 1. OR2C3, OR2J3, OR2L13, OR2T11, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4 , OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F2, OR 51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52 A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8,OR52H1, OR52I1, OR52I2, OR52J3, OR52K1, OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2, OR9A2, ketoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sst1 receptor, sst2 receptor, sst3 receptor, sst4 receptor body, sst5 receptor, TA1 receptor, TAAR2, TAAR5, TAAR6, TAAR8, TAAR9, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2 R8, TAS2R9, TRH1 receptor, Y1 receptor, Y2 receptor, Y5 receptor, α1A-adrenergic receptor, α1B-adrenergic receptor, α1D-adrenergic receptor, δ receptor, 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, C CR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemokine receptor, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLT1 receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2 / ALX, FPR2 / ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183,GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlu1 receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 Receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PAR1, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2-adrenergic receptor, κ receptor, μ receptor.

[0153] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding CCR4.

[0154] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding CCR5.

[0155] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding CCR4 and CCR5.

[0156] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding CCR4, CCR5, CCR10, and CXCR3.

[0157] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0158] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0159] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding the adrenergic α1A receptor, CCR3, and M2 receptor.

[0160] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding adrenergic α1A receptor, CCR3, CCR4, and M2 receptor.

[0161] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding adrenergic α1A receptor, CCR3, CCR5, and M2 receptor.

[0162] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, and M2 receptor.

[0163] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0164] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, and M2 receptor.

[0165] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000152], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α1A receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, M2 receptor, neurotensin NTS2 receptor, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0166] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of 5-HT1A receptor, 5-HT2A receptor, A1 receptor, A2A receptor, A2B receptor, A3 receptor, ACKR1, ACKR2, ACKR3, ACKR4, ADGRE1, ADGRE2, ADGRE3, ADGRE5, apelin receptor, AT1 receptor, B1 receptor, B2 receptor, BB2 (GRP) receptor, BLT1 receptor, BLT2 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, C aS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemokine receptor, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CysLT1 receptor, CysLT2 receptor, DP1 receptor, DP2 receptor, EP1 receptor, EP2 receptor, EP3 receptor, EP4 receptor, FFA2 receptor, FFA4 receptor, FP receptor, FPR1, FPR2 / ALX , FPR2 / ALX, FPR3, FZD6, GAL2 receptor, GAL3 receptor, ghrelin receptor, GPR132, GPR15, GPR18, GPR183, GPR21, GPR31, GPR32, GPR34, GPR4, GPR55, GPR55, GPR65, GPR68, H1 receptor, H2 receptor, H4 receptor, IP receptor, LPA5 receptor, MAS1, MC1 receptor, MCH1 receptor, mGlu1 receptor, mGlu5 receptor, MRGPRX2, MT1 receptor, MT2 receptor, NK1 receptor, NK3 receptor, NMU1 receptor, OXE receptor, P2Y1 receptor, P2Y11 receptor, P2Y13 receptor, P2Y14 receptor, P2Y2 receptor, P2Y6 receptor, PAF receptor, PAR1, PAR2, PAR3, PAR4, PKR1, PKR2, S1P1 receptor, S1P2 receptor, S1P3 receptor, S1P4 receptor, S1P5 receptor, succinate receptor, TP receptor, VPAC1 receptor, VPAC2 receptor, XCR1, β2-adrenergic receptor, κ receptor, μ receptor.

[0167] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR4.

[0168] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR5.

[0169] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR4 and CCR5.

[0170] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR4, CCR5, CCR10, and CXCR3.

[0171] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, and prostaglandin E4 receptor.

[0172] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic β2 receptor, apelin receptor, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, platelet activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, and prostaglandin E4 receptor.

[0173] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR3.

[0174] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR3 and CCR4.

[0175] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR3 and CCR5.

[0176] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR3, CCR4, and CCR5.

[0177] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0178] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], excluding CCR3, CCR4, CCR5, CCR10, and CXCR3.

[0179] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000166], except adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenergic α2A receptor, adrenergic α2C receptor, adrenergic β1 receptor, adrenergic β2 receptor, adrenergic β3 receptor, apelin receptor, CCR3, CCR4, CCR5, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, dopamine D2 receptor, glucagon-like peptide receptor 1, neurotensin NTS2 receptor, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, prostaglandin E4 receptor, somatostatin 1 receptor and somatostatin 3 receptor.

[0180] In one embodiment, certain co-localized activating GPCRs of the invention are GPCRs selected from the group consisting of angiotensin receptors, including AT1R, and certain chemokine receptors, including CCR1, CCR2, CCR6, CCR7, CXCR2, CXCR4, CXCR6, and CXCR7.

[0181] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of angiotensin receptors, including AT1R, and certain chemokine receptors, including CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2, CXCR4, CXCR6, and CXCR7.

[0182] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of angiotensin receptors, including AT1R, and certain chemokine receptors, including CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2, CXCR4, and CXCR6.

[0183] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of angiotensin receptors, including AT1R, and certain chemokine receptors, including CCR1, CCR2, CCR6, CCR7, CXCR2, CXCR4, and CXCR6.

[0184] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of angiotensin receptors, including AT1R, and certain chemokine receptors, including CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2, and CXCR6.

[0185] In one embodiment, certain co-localized activating GPCRs of the present invention are GPCRs selected from the group consisting of angiotensin receptors, including AT1R, and certain chemokine receptors, including CCR1, CCR2, CCR6, CCR7, CXCR2, and CXCR6.

[0186] In one embodiment, the co-localized activating GPCR of the present invention is vasopressin receptor 2.

[0187] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE.

[0188] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0189] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0190] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0191] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0192] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0193] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0194] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0195] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0196] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0197] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0198] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0199] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0200] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0201] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0202] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0203] In one embodiment, the certain chemokine receptors are chemokine receptors that are co-expressed in the same cells as RAGE, are involved in inflammation and are selected from the group consisting of: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0204] In one form of the invention, certain co-localized activating GPCRs of the invention are GPCRs selected from the group consisting of adenosine 1A receptor, adrenergic α1A receptor, adrenergic α1B receptor, adrenergic α2B receptor, angiotensin receptor AT1R, bradykinin receptor B2, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR2, CXCR4, CXCR5, dopamine D1 receptor, endothelin receptor type A, endothelin receptor type B, histamine H3 receptor, muscarinic M1 receptor, muscarinic M2 receptor, muscarinic M3 receptor, neuropeptide Y1 receptor, neurotensin 1 receptor, orexin receptor 1, orexin receptor 2, prostaglandin E1 receptor, serotonin 5-HT1a receptor, serotonin 5-HT2a receptor, serotonin 5-HT2b receptor, serotonin 5-HT2c receptor, serotonin 5-HT4b receptor, somatostatin 2 receptor, sphingosine 1-phosphate receptor S1P1, sphingosine 1-phosphate receptor S1P3, thyrotropin-releasing hormone receptor 1, vasopressin receptor 1A, vasopressin receptor 1B, or vasopressin receptor 2.

[0205] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding CCR4.

[0206] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding CCR5.

[0207] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding CCR4 and CCR5.

[0208] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding CCR4, CCR5, and CXCR4.

[0209] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding adrenergic α1A receptor, CCR3, muscarinic M2 receptor, and orexin receptor 1.

[0210] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding adrenergic α1A receptor, CCR3, CCR4, muscarinic M2 receptor, and orexin receptor 1.

[0211] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding adrenergic α1A receptor, CCR3, CCR5, muscarinic M2 receptor, and orexin receptor 1.

[0212] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding adrenergic α1A receptor, CCR3, CCR4, CCR5, muscarinic M2 receptor, and orexin receptor 1.

[0213] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000204], excluding adrenergic α1A receptor, CCR3, CXCR4, muscarinic M2 receptor, and orexin receptor 1.

[0214] In preferred forms of the invention, certain co-localized activating GPCRs of the invention are GPCRs selected from the group consisting of: adrenergic α1A receptor, adrenergic α1B receptor, angiotensin receptor AT1R, bradykinin receptor B2, CCR2, CCR3, CCR4, CCR6, CCR9, CXCR4, CXCR5, dopamine D1 receptor, endothelin receptor type B, histamine H3 receptor, muscarinic M2 receptor, neuropeptide Y1 receptor, neurotensin 1 receptor, orexin receptor 1, orexin receptor 2, prostaglandin E1 receptor, serotonin 5-HT2b receptor, serotonin 5-HT2c receptor, serotonin 5-HT4b receptor, somatostatin 2 receptor, sphingosine 1-phosphate receptor S1P3, vasopressin receptor 1A, or vasopressin receptor 1B.

[0215] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000214], excluding CCR4.

[0216] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000214], excluding CCR4 and CXCR4.

[0217] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000214], excluding adrenergic α1A receptor, CCR3, muscarinic M2 receptor, and orexin receptor 1.

[0218] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000214], excluding adrenergic α1A receptor, CCR3, CCR4, muscarinic M2 receptor, and orexin receptor 1.

[0219] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000214], excluding adrenergic α1A receptor, CCR3, CCR4, CXCR4, muscarinic M2 receptor, and orexin receptor 1.

[0220] In a particularly preferred form of the invention, certain co-localized activating GPCRs of the invention are GPCRs selected from the group consisting of: adrenergic α1A receptor, adrenergic α1B receptor, angiotensin receptor AT1R, bradykinin receptor B2, CCR2, CCR6, CCR9, CXCR4, CXCR5, dopamine D1 receptor, endothelin receptor type B, histamine H3 receptor, muscarinic M2 receptor, neuropeptide Y1 receptor, orexin receptor 1, orexin receptor 2, prostaglandin E1 receptor, serotonin 5-HT2c receptor, serotonin 5-HT4b receptor, somatostatin 2 receptor, sphingosine 1-phosphate receptor S1P3, vasopressin receptor 1A, or vasopressin receptor 1B.

[0221] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000220], excluding CXCR4.

[0222] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000220], excluding adrenergic α1A receptor, CCR3, muscarinic M2 receptor, and orexin receptor 1.

[0223] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000220], excluding adrenergic α1A receptor, CCR3, CXCR4, muscarinic M2 receptor, and orexin receptor 1.

[0224] In one form of the invention, certain co-localized activating GPCRs of the invention are GPCRs selected from the group consisting of adenosine A1 receptor (ADORA1), adrenergic α2B receptor, angiotensin receptor AT1 (AT1R), bradykinin receptor 2 (B2R), CCR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR9, CXCR2, CXCR4, CXCR5, neuropeptide Y1 receptor (NPY1R), orexin receptor 2, sphingosine 1-phosphate receptor 1 (S1PR1), thyrotropin-releasing hormone receptor 1 (TRHR1), vasopressin receptor 1A (V1aR), vasopressin receptor 1B (V1bR), and vasopressin receptor 2 (V2R).

[0225] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000224], excluding CCR4.

[0226] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000224], excluding CCR5.

[0227] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000224], excluding CCR4 and CCR5.

[0228] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000224], excluding CXCR4.

[0229] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000224], excluding CCR4, CCR5, and CXCR4.

[0230] In preferred forms of the invention, certain co-localized activating GPCRs of the invention are GPCRs selected from the group consisting of adrenergic α2B receptor, angiotensin receptor AT1 (AT1 R), bradykinin receptor 2 (B2R), CCR1, CCR2, CCR4, CCR5, CCR6, CCR9, CXCR2, CXCR4, neuropeptide Y1 receptor (NPY1 R), orexin receptor 2, sphingosine 1-phosphate receptor 1 (S1PR1), thyrotropin-releasing hormone receptor 1 (TRHR1), vasopressin receptor 1A (V1aR), vasopressin receptor 1B (V1bR) and vasopressin receptor 2 (V2R).

[0231] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000230], excluding CCR4.

[0232] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000230], excluding CCR5.

[0233] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000230], excluding CCR4 and CCR5.

[0234] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000230], excluding CXCR4.

[0235] In one embodiment, certain co-localized activating GPCRs of the present invention are selected from the GPCRs of paragraph [000230], excluding CCR4, CCR5, and CXCR4.

[0236] In a specific form of the invention, the co-localized activating GPCR of the invention is an angiotensin receptor.

[0237] In a specific form of the invention, the co-localized activating GPCR of the invention is AT1R.

[0238] In a specific form of the invention, the co-localized activating GPCR of the invention is a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR6, CCR7, CXCR2, CXCR4, CXCR6, and CXCR7.

[0239] In a specific form of the invention, the co-localized activating GPCR of the invention is a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2 and CXCR6.

[0240] In a specific form of the invention, the co-localized activating GPCR of the invention is a chemokine receptor selected from the group consisting of CCR1, CCR2, CCR6, CCR7, CXCR2 and CXCR6.

[0241] In a specific form of the invention, the co-localized activating GPCR of the invention is a chemokine receptor selected from CCR2 and CCR6.

[0242] In a specific form of the invention, the co-localized activating GPCR of the invention is CCR2.

[0243] In a specific form of the invention, the co-localized activating GPCR of the invention is CXCR4.

[0244] In one form of the invention, a RAGE ligand is a ligand that interacts with the extracellular domain of RAGE to modulate RAGE activation. Thus, in this form of the invention, RAGE ligand-independent RAGE activation refers to RAGE activation that occurs without interaction of a ligand with the extracellular domain of RAGE.

[0245] Preferably, the RAGE ligand is one that interacts with the extracellular domain of RAGE to modulate RAGE activation and does not interact with the transmembrane domain or cytoplasmic tail of RAGE or motifs contained therein. Thus, in this preferred form of the invention, RAGE ligand-independent RAGE activation refers to RAGE activation that does not occur by way of a ligand interacting with the extracellular domain of RAGE unless the ligand also interacts with the transmembrane domain or cytoplasmic tail of RAGE or motifs contained therein.

[0246] The extracellular domain (also known as the extracellular domain) of RAGE consists of three immunoglobulin-like regions: an N-terminal V-domain followed by two C-domains (referred to as C and C' or alternatively, C1 and C2). The primary ligand-binding portion is the V-domain, however, RAGE activation can also be mediated by ligand binding to the C-domain. Most ligands tend to bind to the V domain and / or the C1 domain because ligands tend to be negatively charged, however, there is at least one example of ligand binding to the C2 domain (S100A6; Leclerc et al., 2007). Although the C1-domain and C2-domain may not typically bind directly to ligands, they may play an important role in stabilizing the V-domain to mediate its interaction with ligands. RAGE has a single transmembrane domain and a cytoplasmic tail. In humans, the RAGE cytoplasmic tail is 43 amino acids long (residues 362 to 404). This cytoplasmic tail contains motifs that are critical for RAGE-dependent cell activation.

[0247] In one form of the invention, a RAGE ligand is a ligand that interacts with the extracellular V, C1 and / or C2 domains of the RAGE extracellular domain to activate RAGE. In this form of the invention, RAGE ligand-independent RAGE activation refers to RAGE activation that occurs without interaction of the ligand with the extracellular V, C1 or C2 domains of the RAGE extracellular domain.

[0248] Preferably, the RAGE ligand does not interact with the RAGE transmembrane domain or cytoplasmic tail or motifs contained therein. Thus, in this form of the invention, RAGE ligand-independent RAGE activation refers to RAGE activation that does not occur by way of a ligand interacting with the extracellular V, C1 or C2 domains of the RAGE extracellular domain unless the ligand also interacts with the RAGE transmembrane domain or cytoplasmic tail or motifs contained therein.

[0249] In one form of the invention, a modulator that modulates RAGE ligand-independent RAGE activation via a co-localized activating GPCR (eg, an activating angiotensin receptor such as AT1R or CCR2) also modulates RAGE ligand-dependent RAGE activation.

[0250] In a preferred embodiment of the invention, the modulators of the invention do not modulate, or differentially modulate, or modulate to varying degrees, a RAGE-independent signaling pathway associated with a certain co-localized activated GPCR.

[0251] In a preferred embodiment, the modulators of the invention do not inhibit or inhibit to a lesser extent one or more RAGE-independent certain co-localized GPCR signaling pathways.

[0252] In one form of the invention, the RAGE-independent co-localized GPCR signaling pathway is the Gq signaling pathway. In one form of the invention, the RAGE-independent co-localized GPCR signaling pathway is the Gi / o signaling pathway. In one form of the invention, the RAGE-independent co-localized GPCR signaling pathway is the Gs signaling pathway. In one form of the invention, the RAGE-independent co-localized GPCR signaling pathway is the calcium signaling pathway. In one form of the invention, the RAGE-independent co-localized GPCR signaling pathway is the phospholipase C signaling pathway. In another form of the invention, the RAGE-independent co-localized GPCR signaling pathway is beta-arrestin-mediated extracellular regulated kinase (ERK) signaling.

[0253] In a particularly preferred embodiment, wherein the co-localized activated GPCR is activated AT1R, the modulators of the invention do not modulate, or to a lesser extent modulate, one or more RAGE-independent AT1R signaling pathways.

[0254] In a particularly preferred embodiment, wherein the co-localized activated GPCR is activated AT1R, the modulators of the invention do not inhibit or inhibit to a lesser extent one or more RAGE-independent AT1R signaling pathways.

[0255] In one form of the invention, the RAGE-independent AT1R signaling pathway is the Gq signaling pathway. In another form of the invention, the RAGE-independent AT1R signaling pathway is beta-arrestin-mediated extracellular regulated kinase (ERK) signaling.

[0256] In another particularly preferred embodiment, wherein the co-localized activated GPCR is activated CCR2, the modulators of the invention do not modulate or modulate to a lesser extent one or more RAGE-independent CCR2 signaling pathways.

[0257] In another particularly preferred embodiment, wherein the co-localized activated GPCR is activated CCR2, the modulators of the invention do not inhibit or inhibit to a lesser extent one or more RAGE-independent CCR2 signaling pathways.

[0258] In one form of the invention, the RAGE-independent AT1R signaling pathway is the Gi / o signaling pathway. In another form of the invention, the RAGE-independent CCR2 signaling pathway is β-arrestin-mediated extracellular regulated kinase (ERK) signaling. In another form of the invention, the RAGE-independent CCR2 signaling pathway is the phospholipase C signaling pathway. regulator

[0259] In one form of the invention, the modulator of the present invention is an activator, inhibitor, allosteric modulator or a functional or non-functional substitute of the RAGE cytoplasmic tail. A functional substitute is a modulator that replaces the RAGE cytoplasmic tail in the presence of certain co-located GPCRs and can be activated by them to induce downstream RAGE-dependent signal transduction in the presence or absence of wild-type RAGE expression. A non-functional substitute is a modulator that replaces the RAGE cytoplasmic tail in the presence of certain co-located GPCRs and cannot be activated or induce downstream RAGE-dependent signal transduction by them, and inhibits the signal transduction that normally occurs by activating the RAGE cytoplasmic tail and the RAGE-dependent signal transduction resulting therefrom.

[0260] In one form of the invention, a modulator of the invention is an activator, inhibitor, allosteric regulator or non-functional replacement of the transmembrane domain of RAGE or a portion thereof.

[0261] Non-functional surrogates are regulators that take over the RAGE transmembrane domain in the presence of certain co-localized GPCRs and are unable to be activated by them or to induce downstream RAGE-dependent signaling, and inhibit signaling that normally occurs through activation of the RAGE cytoplasmic tail and the resulting RAGE-dependent signaling therefrom.

[0262] In one form of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or a portion thereof and the RAGE extracellular domain.

[0263] In one form of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or portion thereof and the RAGE cytoplasmic tail.

[0264] In one form of the invention, the modulator comprises the RAGE transmembrane domain or portion thereof and a fragment of the RAGE extracellular domain and a fragment of the RAGE cytoplasmic tail.

[0265] In one form of the invention, a modulator of the invention comprises a fragment of the extracellular domain of RAGE that is no more than 40, no more than 20, no more than 10, or no more than 5 amino acids in length.

[0266] By way of example, the inventors have shown that RAGE 362-404 It is a functional substitute for RAGE and is capable of being activated by certain co-localized GPCRs (such as AT1R and CCR2) and inducing downstream RAGE-dependent signaling due to RAGE ligand-independent RAGE activation in the presence or absence of wild-type RAGE expression. 362-404When fused with cell-penetrating peptide (TAT) and marker protein (mCherry), AT1R is activated by Ang II and then expressed with TAT-mCherry-RAGE. 362-404 Oligopeptide treatment restored Ang II-dependent inflammation and atherosclerosis in AgerApoe DKO mice in the absence of wild-type RAGE expression.

[0267] RAGE 362-404 The sequence is SEQ ID NO: 1: [L 362 WQRRQRRGEERKAPENQEEEEERAELNQSEEPEAGESSTGGP 404 ]

[0268] By way of additional modulator examples, the inventors have shown that S391A-RAGE 362-404 It is a nonfunctional surrogate for RAGE that is not activated by certain colocalized GPCRs and inhibits RAGE-dependent signaling. 362-404 Expression of S391A-RAGE inhibited RAGE ligand-independent wild-type RAGE activation via activated AT1R and RAGE ligand-dependent wild-type RAGE activation via RAGE ligand S100A8 / A9. 362-404 When fused with a cell-penetrating peptide (TAT) and a marker protein (mCherry), use TAT-mCherry-S391A-RAGE 362-404 Oligopeptide treatment inhibits RAGE ligand-independent RAGE activation via activated AT1R to attenuate Ang II-dependent inflammation and atherosclerosis in apolipoprotein E knockout mice. Additional examples are provided below.

[0269] S391A-RAGE 362-404 The sequence is SEQ ID NO: 2: [L 362 WQRRQRRGEERKAPENQEEEEERAELNQ A EEPEAGESSTGGP 404 ]

[0270] By way of another example of a modulator, the inventors have shown that RAGE 338-361 Inhibits RAGE ligand-independent wild-type RAGE activation via activated AT1R. 362-404 Overcome this inhibition.

[0271] RAGE 338-361The sequence is SEQ ID: 3.

[0272] [L 338 GTLALALGILGGLGTAALLIGVI 361 ]

[0273] In one form, the invention comprises modulators of RAGE ligand-independent RAGE activation by certain co-localized activating GPCRs, which modulate transactivation of the RAGE cytoplasmic tail triggered by activation of such certain co-localized activating GPCRs, such as angiotensin receptors (e.g., AT1R) or chemokine receptors (e.g., CCR2).

[0274] In one form, the invention includes modulators of RAGE ligand-independent activation of the cytoplasmic tail of RAGE by colocalizing activated GPCRs that bind to Ras GTPase-activating-like protein (IQGAP1) or other RAGE-associated proteins, including protein kinase Cζ (PKCζ), Dock7, MyD88, TIRAP, IRAK4, ERK1 / 2, olfactory receptor 2T2, ADP / ATP translocase 2, protein phosphatase 1G, intercellular adhesion molecule 1, protein DJ-1 (PARK7), calponin-3, brain development regulatory protein, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipid protein 2, coronarin, S100 A11, succinyl CoA ligase [GDP-forming] subunit alpha, Hsc70-interacting protein, inhibitor of apoptosis 5, neuropilin, splicing stimulating factor, growth factor receptor binding protein 2, sec61 β subunit or Nck1, or disrupt the binding of these elements to RAGE, to regulate RAGE transactivation via certain co-localized activating GPCRs, such as angiotensin receptors (such as AT1R) or chemokine receptors (such as CCR2).

[0275] In one form of the invention, the modulators of the invention bind to certain co-localized activated GPCRs, cytoplasmic elements of RAGE, and / or elements in complex with either of the former two, including IQGAP-1, PKCζ, Dock7, MyD88, TIRAP, IRAK4, ERK1 / 2, olfactory receptor 2T2, ADP / ATP translocase 2, protein phosphatase 1G, intercellular adhesion molecule 1, protein DJ-1 (PARK7), calponin-3, brain development regulatory protein, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipid protein 2, coronarin, S100 A1 1, succinyl CoA ligase [GDP-forming] subunit alpha, Hsc70-interacting protein, inhibitor of apoptosis 5, neuropilin, splicing stimulating factor, growth factor receptor binding protein 2, sec61 The β subunit or Nck1 regulates RAGE ligand-independent signaling through the RAGE cytoplasmic tail by modulating these signaling elements required for RAGE transactivation through certain co-localized activating GPCRs, such as angiotensin receptors (such as AT1R) or chemokine receptors (such as CCR2).

[0276] In one form of the invention, modulators of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs also activate RAGE by binding to cytoplasmic elements of RAGE and / or elements complexed with RAGE in the cytosol (e.g., IQGAP-1, PKCζ, Dock7, MyD88, IRAK4, TIRAP, ERK1 / 2, olfactory receptor 2T2, ADP / ATP translocase 2, protein phosphatase 1G, intercellular adhesion molecule 1, protein DJ-1 (PARK7), calponin-3, brain development regulatory protein, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipid protein 2, coronarin, S100 A11, succinyl CoA ligase [GDP-forming] subunit alpha, Hsc70-interacting protein, inhibitor of apoptosis 5, neuropilin, splicing stimulating factor, growth factor receptor binding protein 2, sec61 β subunit or Nck1), regulates RAGE ligand-dependent activation of the RAGE cytoplasmic tail to inhibit RAGE ligand-mediated signaling through these elements.

[0277] In a specific embodiment, the modulator comprises, consists of, or consists essentially of the amino acid sequence as shown in SEQ ID NO: 1, or an analog, fragment or derivative thereof.

[0278] In some embodiments, the modulator is introduced by gene delivery (e.g., by using viruses or artificial non-viral gene delivery such as electroporation, microinjection, gene guns, impalefection, hydrostatic pressure, continuous infusion, sonication, lipofection, liposomes, nanobubbles, and polymer gene carriers) and as a result of the transcription and translation processes, the peptide fragment, biologically active analog, or derivative is produced by the cell.

[0279] In some embodiments of this aspect, the modulator has an improved ability to form a complex with certain co-localized GPCRs (e.g., AT1R or CCR2) or elements complexed therewith. For example, a RAGE analog or derivative can be distinguished from the wild-type RAGE polypeptide or fragment sequence by substitution, addition, or deletion of at least one amino acid residue, or by addition or substitution of an unusual or unconventional amino acid or non-amino acid residue.

[0280] In some embodiments, the modulator lacks or has a modification of serine 391 that is normally present in the wild-type human RAGE polypeptide. In an illustrative example of this type, a fragment, analog, or derivative of the RAGE cytoplasmic tail lacks the serine at position 391 of the wild-type RAGE sequence (e.g., RAGE 370-390 The construct is truncated at Glu390). Suitably, the serine at position 391 is deleted or substituted with another amino acid residue, analog, or derivative to disrupt or eliminate the signaling conferred by the serine at this site upon activation of the co-localized GPCR. In one embodiment, the serine at position 391 is deleted or substituted with another amino acid residue selected from the group consisting of alanine, aspartic acid, phenylalanine, histidine, lysine, arginine, tyrosine, asparagine, valine, glycine, cysteine, or glutamic acid.

[0281] In some embodiments, the modulator retains serine 391 that is normally present in the wild-type human RAGE polypeptide or is replaced by another amino acid, or an analog or derivative thereof, that retains the same function of the serine at position 391. In an illustrative example of this type, a fragment of the RAGE cytoplasmic tail retains serine at position 391 of the wild-type RAGE sequence (e.g., RAGE 370-404 Constructs). Suitably, the serine at position 391 is substituted with another amino acid residue, or an analog or derivative thereof, to replicate the signaling conferred by the RAGE construct containing serine at this position upon activation of a co-localized GPCR. In one embodiment, the serine at position 391 is substituted with another amino acid residue selected from the group consisting of proline, glutamine, threonine, leucine, isoleucine, methionine, or tryptophan.

[0282] In some embodiments, the modulator lacks or has impaired binding to Diaphragmatic 1 (Diaph1) relative to human wild-type RAGE. In illustrative examples of this type, the peptide or its analog, fragment or derivative lacks the RAGE-Diaph1 binding site (e.g., RAGE 370-390 、RAGE 374-390 or RAGE 379-390 ) or have an altered Diaph1 binding site (such as 366A / 367A) to eliminate or disrupt this site. Suitably, the residues at 366 / 367 are deleted or substituted with other residues (such as alanine) to disrupt or eliminate this site, and in doing so, improve the affinity for binding to other targets by reducing the constraints caused by wild-type binding to Diaph1.

[0283] In one aspect of the invention, the modulators of the invention include isolated or purified peptides comprising, consisting of, or consisting essentially of an amino acid sequence represented by Formula I: Z1 M Z2 (I) in Z1 is absent or is selected from at least one of a proteinaceous portion comprising about 1 to about 50 amino acid residues; and M is an amino acid sequence as shown in SEQ ID NO: 1, or an analog, fragment or derivative thereof; and Z2 is absent or is a proteinaceous portion comprising about 1 to about 50 amino acid residues.

[0284] In some embodiments of the invention described above, the modulator (e.g., a fragment of the RAGE cytoplasmic tail, an analog thereof, or a derivative thereof, as described above and elsewhere herein) is capable of penetrating cell membranes. In a non-limiting example of this type, the RAGE modulator is conjugated, fused, or otherwise linked to a cell membrane penetrating molecule (e.g., the HIV TAT motif, as described below in SEQ ID NO: 4).

[0285] SEQ ID NO: 4: [YGRKKRRQRRR].

[0286] In some forms of the invention, the modulator is a non-peptide molecule that shares with the peptide modulators described above the ability to bind to and / or interfere with elements associated with RAGE ligand-independent RAGE activation via certain co-localized activated GPCRs. These non-peptide modulators may or may not contain structural similarities to functionally important domains contained in the peptide modulators.

[0287] In a preferred form, the non-peptide modulator contains structural similarity to functionally important domains contained in the peptide modulator, as represented by the pharmacophore described in paragraph [000318].

[0288] In a preferred form of the invention, the modulator is an inhibitor.

[0289] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by a colocalized activated GPCR, the modulator is also an inhibitor of a colocalized GPCR and / or an inhibitor of a colocalized GPCR signaling pathway.

[0290] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by a co-localized activated GPCR, the modulator is also an inhibitor of RAGE ligand-dependent activation of RAGE and / or an inhibitor of constitutively active RAGE and / or an inhibitor of the RAGE signaling pathway.

[0291] In certain forms of the invention, wherein a co-localized GPCR is AT1R, the modulator, in addition to being an inhibitor of RAGE ligand-independent RAGE activation, is also an AT1R inhibitor and / or an inhibitor of the AT1R signaling pathway.

[0292] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation via an activated angiotensin receptor, preferably activated AT1R, the modulator is also an inhibitor of RAGE ligand-dependent RAGE activation and / or an inhibitor of constitutively active RAGE and / or an inhibitor of the RAGE signaling pathway.

[0293] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by a co-localized activated GPCR, the modulator is also an inhibitor of a co-localized GPCR and / or an inhibitor of a co-localized GPCR signaling pathway and an inhibitor of RAGE ligand-dependent RAGE activation and / or an inhibitor of constitutively active RAGE and / or an inhibitor of the RAGE signaling pathway.

[0294] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by an activated angiotensin receptor, preferably activated AT1R, the modulator is also an inhibitor of AT1R and / or an inhibitor of the AT1R signaling pathway and an inhibitor of RAGE ligand-dependent RAGE activation and / or an inhibitor of constitutively active RAGE and / or an inhibitor of the RAGE signaling pathway.

[0295] In certain forms of the invention, wherein a co-localized GPCR is a chemokine receptor, preferably CCR2, the modulator, in addition to being an inhibitor of RAGE ligand-independent RAGE activation, is also a chemokine receptor inhibitor, preferably a CCR2 inhibitor, and / or an inhibitor of a chemokine signaling pathway, preferably a CCR2 signaling pathway.

[0296] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by activated forms of certain chemokine receptors, preferably activated CCR2, the modulator is also an inhibitor of RAGE ligand-dependent RAGE activation and / or an inhibitor of constitutively active RAGE and / or an inhibitor of the RAGE signaling pathway.

[0297] In certain forms of the invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by an activated chemokine receptor, preferably activated CCR2, the modulator is also an inhibitor of a chemokine receptor, preferably a CCR2 inhibitor and / or an inhibitor of a chemokine signaling pathway, preferably a CCR2 signaling pathway and an inhibitor of RAGE ligand-dependent RAGE activation and / or an inhibitor of constitutively active RAGE and / or an inhibitor of the RAGE signaling pathway.

[0298] In certain forms of the invention, the modulator is a functional replacement for the RAGE cytoplasmic tail or portion thereof and is capable of being activated by certain co-localized GPCRs (e.g., activated AT1R and activated CCR2) and inducing downstream RAGE-dependent signaling in the presence or absence of wild-type RAGE expression.

[0299] In certain forms of the invention, the modulator is a non-functional surrogate of the RAGE cytoplasmic tail or a portion thereof that is incapable of being activated by a co-localized GPCR or promoting downstream RAGE-dependent signaling and that inhibits signaling through the RAGE cytoplasmic tail and RAGE-dependent signaling.

[0300] In certain forms of the invention, the modulator is a non-functional replacement for the RAGE transmembrane domain or portion thereof that is incapable of being activated by a co-localized GPCR or promoting downstream RAGE-dependent signaling and inhibits signaling through the RAGE cytoplasmic tail and RAGE-dependent signaling.

[0301] In certain forms of the invention, the modulator comprises a RAGE transmembrane domain or a portion thereof and a fragment of the RAGE extracellular domain. In certain forms of the invention, the modulator comprises a RAGE transmembrane domain or a portion thereof and a fragment of the RAGE cytoplasmic tail.

[0302] In certain forms of the invention, the modulator comprises the RAGE transmembrane domain or portion thereof and a fragment of the RAGE extracellular domain and a fragment of the RAGE cytoplasmic tail.

[0303] In certain forms of the invention, the modulator of RAGE ligand-independent RAGE activation by certain co-localized activating GPCRs comprises a fragment of the ligand-binding extracellular domain of human wild-type RAGE, said fragment being no more than 40, no more than 20, no more than 10, or no more than 5 amino acids in length.

[0304] The inventors have further discovered that a peptide comprising residues 370-390 of the RAGE cytoplasmic tail (see SEQ ID NO: 5) is an inhibitory peptide that inhibits both RAGE ligand-independent and RAGE ligand-dependent wild-type RAGE activation.

[0305] SEQ ID NO: 5: [G 370 EERKAPENQEEEEERAELNQ 390 ].

[0306] RAGE exists 363-404 The solution NMR structure of (Rai V et al., 2012) showed that the N-terminus of this peptide (residues 363-376) is well ordered. 362-404 A Rosetta-derived model (model 4) was obtained, which is consistent with the NMR structure ( http: / / www.rcsb.org / pdb / explore / explore.do?structureId=2LMB, Accessed August 25, 2016)), and this also suggests that the remainder of the peptide forms an alpha helix.

[0307] RAGE was constructed by truncating model 4 370-390 The initial model (Model 4_ 370-390 ). Model 4 is a theoretical model of the RAGE cytoplasmic tail, generated by inputting the sequence into the I-Tasser web server (http: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ). See also Yang et al. (2015), Roy et al. (2010), and Y Zhang (2008). All five models displayed by the I-Tasser server predict that region 370-390 forms a helix. These models and the NMR structure are aligned by the C-alpha carbon of the peptide sequence backbone. Model 4 was selected as the preferred model because the predicted structural region corresponding to the Diaphanous 1 binding site in Model 4 is closest to the NMR structure recorded for this region.

[0308] Using GROMACS, a 20 nanosecond molecular dynamics simulation of model 4 in water was performed (Hess et al., 2008). The molecular dynamics simulation showed that model 4_ 370-390 Strong interactions were observed between numerous charged side chains, suggesting that these interactions stabilize the folded structure and suggesting that any conservation of these residues may be due to their role in stabilizing the peptide structure.

[0309] Blast search was used to identify RAGE 370-390 The sequences are aligned as follows: CLUSTAL 2.0.10 multiple sequence alignment Model 4_ 370-390 .pdb ---GEERKAPENQ--EEEEERAELNQ-- gi|505855911|ref|XP_004621364.RRRRGEERKVPENQ--EEEEERAELKQSGE gi|836716008|ref|XP_012791097.RRRRGEERKVPENQ--EEEEERAELKQSGE gi|830242517|ref|XP_012589882.RRR-GEQRKAPENR--EEEEERAELNQSEE gi|830242520|ref|XP_012589883.RRR-GEQRKAPENR--EEEEERAELNQSEE gi|830242532|ref|XP_012589884.RRR-GEQRKAPENR--EEEEERAELNQSEE gi|859958468|ref|XP_012905636.RPR-REERKAPENQ--EEEEERAELNQSEE gi|505855913|ref|XP_004621365.RRRRGEERKVPENQ--EEEEERAELKQSGE gi|859958474|ref|XP_012905637.RPR-REERKAPENQ--EEEEERAELNQSEE gi|674092933|ref|XP_008819684.QHR-GEERKTPENQ--EDEEERAELNQSEE gi|852803202|ref|XP_012890437.QHR-GEERKAPENQ--EEEEERAELNQSEE gi|586986169|ref|XP_006931651.RRQ-GEERKAPENQEEEEEEEREELNQSGE gi|752437365|ref|XP_011235981.RHR-REERKAPENQ--EEEEERAELNQSEE gi|671038558|ref|XP_008710071.RHR-REERKAPENQ--EEEEERAELNQSVE gi|859958450|ref|XP_012905633.RPR-REERKAPENQ--EEEEERAELNQSEE gi|1040099494|gb|OBS60144.1|QPR-GEERKTPENQ--EDEEERAELNQSED gi|674092931|ref|XP_008819683.QHR-GEERKTPENQ--EDEEERAELNQSEE gi|641730582|ref|XP_008155542.RHR-GEERKAPENQA-EEEEERAELNQSQE gi|641730580|ref|XP_008155541.RHR-GEERKAPENQA-EEEEERAELNQSQE gi|946738855|ref|XP_014389946.RRR-GEERKAPENQ--EEEEERAELHQSQE gi|940771956|ref|XP_006104444.RRR-GEERKAPENQ--EEEEERAELHQSQE gi|355748446|gb|EHH52929.1|RRQ-GEERKASENQ--EEEEERAELNQSEE gi|355561569|gb|EHH18201.1|RRQ-GEERKASENQ--EEEEERAELNQSEE gi|544428837|ref|XP_005553456.RRQ-GEERKASENQ--EEEEERAELNQSEE gi|635095937|ref|XP_007971201.RRQ-GEERKASENQ--EEEERAELNQSEE gi|402866556|ref|XP_003897445.RRQ-REERKASENQ--EEEERAELNQSEE gi|795466133|ref|XP_011890032.RRQ-GEERKASENQ--EEEERAELNQSEE gi|795466129|ref|XP_011890031.RRQ-GEERKASENQ--EEEERAELNQSEE gi|795317622|ref|XP_011824818.RRQ-GEERKASENQ--EEEERAELNQSEE gi|326693968|ref|NP_001192046.RRQ-GEERKASENQ--EEEERAELNQSEE gi|724802002|ref|XP_010376439.RRQ-GERKAPENQ--EEEERAELNQSEE gi|724801999|ref|XP_010376432.RRQ-GEERKAPENQ--EEEERAELNQSEE gi|795178216|ref|XP_011800170.RRQ-GEERKAPENQ--EEEERAELNQSEE gi|312182478|gb|ADQ42279.1|RRQ-GEERKASENQ--EEEERAELNQSEE gi|795178211|ref|XP_011800169.RRQ-GEERKAPENQ--EEEERAELNQSEE gi|332800965|ref|NP_001193858.QRR-GERKAPENQ--EEEERAELNQSEE gi|10835203|ref|NP_001127.1|QRR-GERKAPENQ--EEEERAELNQSEE gi|332800967|ref|NP_001193861.QRR-GERKAPENQ--EEEERAELNQSEE gi|823672830|gb|AKI71626.1|QRR-GERKAPENQ--EEEERAELNQSEE gi|190846|gb|AA03574.1|QRR-GERKAPENQ-EEEERAELNQSEE gi|194389738|dbj|BAG60385.1|QRR-GERKAPENQ--EEEERAELNQSEE gi|694915715|ref|XP_009449249.QRQ-GEERKAPENQ--EEEERAELNQSEE gi|694915717|ref|XP_009449250.QRQ-GEERKAPENQ--EEEERAELNQSEE gi|694915721|ref|XP_009449252.QRQ-GEERKAPENQ--EEEERAELNQSEE gi|397519329|ref|XP_003829814.QRQ-GEERKAPENQ--EEEERAELNQSEE gi|397519323|ref|XP_003829811.QRQ-GEERKAPENQ--EEEERAELNQSEE gi|820970747|ref|XP_012358508.RRQ-GEERKAPENQ--EEEERAELNQSEE gi|820970749|ref|XP_012358509.RRQ-GEERKAPENQ--EEEERAELNQSEE gi|817330292|ref|XP_012292176.RRR-GERKAPENQ--EEEEHAELNQSEE gi|817330294|ref|XP_012292177.RRR-GERKAPENQ--EEEEHAELNQSEE gi|725608250|ref|XP_010330526.RRR-GERKAPENQ--EEEEHAELNQSEE gi|725608252|ref|XP_010330527.RRR-GEERKAPENQ--EEEEEHAELNQSEE gi|296197788|ref|XP_002746422.RRRRGEERKAPENQ--EEEEEHAELNQSEE gi|826320184|ref|XP_012509111.RGQ-GEERKAPENQ--EEEEERAELNQSEE gi|826320169|ref|XP_012509105.RGQ-GEERKAPENQ--EEEEERAELNQSEE gi|826320175|ref|XP_012509107.RGQ-GEERKAPENQ--EEEEERAELNQSEE gi|826320172|ref|XP_012509106.RGQ-GEERKAPENQ--EEEEERAELNQSEE gi|829933710|ref|XP_012596554.RHQ-GEERKAPENQ--EEEEERAELNQSEE gi|829933718|ref|XP_012596557.RHQ-GEERKAPENQ--EEEEERAELNQSEE gi|829933722|ref|XP_012596558.RHQ-GEERKAPENQ--EEEEERAELNQSEE gi|743731194|ref|XP_010959751.QRR-GEERKAPENQ-EEEEEEERAELNQQEE gi|560905029|ref|XP_006178871.QRR-GEERKAPENQ-EEEEEEERAELNQQEE gi|593759840|ref|XP_007118666.QRR-GEERKAPENQ-EEEEEEERTELNQPEE gi|560986474|ref|XP_006215428.QRR-GEERKAPENQ-EEEEEEERAELNQQEE gi|927155182|ref|XP_013833109.QRR-GQERKAPENQ-EEDEEERAELNQPED gi|147225137|emb|CAN13265.1|QRR-GQERKAPENQ-EEDEEERAELNQPED gi|178056480|ref|NP_001116690.QRR-GQERKAPENQ-EEDEEERAELNQPED gi|162138238|gb|ABX82823.1|QRR-GQERKAPENQ-EEDEEERAELNQPED gi|471418692|ref|XP_004390841.KHR-GERKAPENQ--EEEEHAELNQSEE gi|471418700|ref|XP_004390845.KHR-GERKAPENQ--EEEEHAELNQSEE gi|471418694|ref|XP_004390842.KHR-GERKAPENQ-EEEEHAELNQSEE gi|829933714|ref|XP_012596556.RHQ-GEERKAPENQ--EEEERAELNQSEE gi|831224940|ref|XP_012660273.QCQ-GERKAPENQ--EEEERTELNQSEE gi|984103351|ref|XP_015342983.RRQ-GEERKAPENQ--EEEERAELNQSEE gi|532108558|ref|XP_005339001.RRQ-GEERKAPENQ--EEEERAELNQSEE gi|955504646|ref|XP_014638416.QHR-REERKAPENQ--EEEERAELNQSEE gi|478500097|ref|XP_004424372.QHR-REERKAPENQ--EEEERAELNQSEE gi|955504650|ref|XP_014638417.QHR-REERKAPENQ--EEEEERAELNQSEE gi|1048457071|ref|XP_017510394QCR-GEERKAPENQ--EEEEERAELSQSEE gi|589966171|ref|XP_006995615.QPR-REERKAPENQ--EDEEERAELNQSED gi|589966173|ref|XP_006995616.QPR-REERKAPENQ--EDEEERAELNQSED gi|532056239|ref|XP_005370828.QPR--EERKAPENE--EDEEERAELNQSED gi|532056241|ref|XP_005370829.QPR--EERKAPENE--EDEEERAELNQSED gi|532056245|ref|XP_005370831.QPR--EERKAPENE--EDEEERAELNQSED gi|641730578|ref|XP_008155540.RHR-GEERKAPENQA-EEEEERAELNQSQE

[0310] This analysis identified RAGE 370-390 Many strongly conserved residues are marked as follows: * (asterisk) indicates positions with completely conserved single residues. : (colon) indicates conservation between groups with strongly similar properties (score > 0.5 in the Gonnet PAM 250 matrix). . (dot) indicates conservation between groups with weakly similar properties (score = < 0.5 in the Gonnet PAM 250 matrix). ： ： ＊ ＊ · · ＊ ＊ · ＊ ： ＊ ＊ ＊ ： · ＊ ＊ · ＊ RAGE- G E E R K A P E N Q <![CDATA[ E ]]> E E E <![CDATA[ E ]]> R A <![CDATA[ E ]]> <![CDATA[ L ]]> N Q

[0311] Highly conserved residues may play a structural role. The underlined residues are located on one face of the helix and may represent a binding pharmacophore.

[0312] Test Model 4_RAGE 370-390 Structural and molecular dynamics simulations revealed numerous salt bridges in the structure. Molecular dynamics simulations suggest that these interactions are important structural features. Structural function is a likely explanation for the conserved nature of these amino acids.

[0313] Many strongly conserved amino acids do not participate in salt bridge formation. These amino acids are present in RAGE 370-390 On one face of the helix, it likely represents the binding interface. These amino acids are Glu380, Glu384, Glu387, and Leu388. Another highly conserved residue, Glu377, is also present on this face of the peptide and, in addition to forming an α-helix-stabilizing salt bridge to Lys374, may also participate in binding.

[0314] Replacement of the important hydrophobic residue L388 with alanine (e.g. L388A-RAGE 370-390 ) leads to the action of wild-type RAGE, which 370-390 The inhibitory effect achieved was lost. 385-390 and RAGE 385-404 ) resulted in loss of regulatory effects of these RAGE constructs. In contrast, N-truncation of RAGE eliminating 374 and 377 did not result in RAGE peptides acting as inhibitors (RAGE 379-390 ) or as a functional replacement for wild-type RAGE (e.g., RAGE 379-404 ) loss of function, suggesting that these conserved residues (374 and 377) are not essential for regulatory activity, even though they may play a role in stabilizing the α-helical tertiary structure of the RAGE cytoplasmic tail.

[0315] Consistent with the conserved nature of these four amino acids representing the binding interface, the inventors have further discovered that a peptide comprising only residues 379-390 of the RAGE cytoplasmic tail (i.e., RAGE 379-390 ) is an inhibitory peptide that inhibits both ligand-independent and ligand-dependent wild-type RAGE activation and found that RAGE 379-404 Can be activated by certain co-localized GPCRs in CHO cells.

[0316] In a preferred form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activated GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is peptide Q as set forth in SEQ ID NO: 6. 379 EEEEERAELNQ 390 or its derivatives.

[0317] SEQ ID NO: 6: [Q 379 EEEEERAELNQ 390 ]

[0318] The following shows the structure model derived from 4_RAGE 370-390 RAGE379-390 Peptide pharmacophore:

[0319] H4 is a hydrophobic residue and P1-P3 are polar residues, and the distances are shown in angstroms. The matrix of distances between site points is as follows, where P represents a polar site point (forming a hydrogen bond or being charged) and H represents a hydrophobic site point. Distances are in angstroms. Tolerances should be applied to the position of each site point.

[0320] Molecular dynamics simulations reveal RAGE 379-390 The interacting groups are mobile and tolerance should be applied to each group until , provided that the distances between the part points are positive in value.

[0321] As will be appreciated by those skilled in the art, additional smaller pharmacophores can be generated by taking subsets of the above, and the present invention encompasses such pharmacophores, methods of identifying compounds using such pharmacophores, and compounds so identified.

[0322] In one form, the invention further comprises a modulator of RAGE ligand-independent RAGE activation by a co-localized activating GPCR, said modulator comprising two or more features selected from the group consisting of a first charged or hydrogen-bonding group (A), a second charged or hydrogen-bonding group (B), a third charged or hydrogen-bonding group (C), and a hydrophobic group (D), wherein at up to Within the tolerance range of , the distances between the part points of the feature are as follows, provided that the distances between the part points are positive in value:

[0323] In a preferred form of the invention, tolerances up to The condition is that the distance between the location points is positive in value. In a preferred form of the invention, the tolerance is up to The condition is that the distance between the location points is positive in value. In a preferred form of the invention, the tolerance is up to The condition is that the distances between the part points are positive in value.

[0324] In a preferred form of the invention, the modulator comprises three or more features selected from the group specifically described above.

[0325] In a preferred form of the invention, the modulator comprises four or more features from the group specified above.

[0326] In one form of the invention, there is provided a modulator characterized in that the modulator comprises at least two features selected from one of the following groups: AB, AC, AD, BC, BD and CD.

[0327] In one form of the invention, a modulator is provided, characterized in that the modulator comprises at least three characteristics selected from one of the following groups: ABC, ABD, ACD and BCD.

[0328] In one form of the invention, a modulator is provided, characterized in that the modulator comprises at least four features selected from one of the following combinations: ABCD.

[0329] In one form of the invention, a modulator is provided, characterized in that the modulator comprises an additional charged or hydrogen-bonding group (P1) that binds to RAGE 370-390 The invention is consistent with the conservative stabilization effect of E377 in the present invention and thus comprises two or more features selected from the following: a first charged or hydrogen-bonding group (A), a second charged or hydrogen-bonding group (B), a third charged or hydrogen-bonding group (C), a fourth charged or hydrogen group (D) and a hydrophobic group (E), wherein the distances between the site points of the features are as follows, Within the tolerance range:

[0330] The modulator of RAGE ligand-independent RAGE activation may be a peptide or a non-peptidyl compound.

[0331] In one form of the invention, the hydrophobic group is an amino acid residue selected from the group consisting of: Ala, Val, Leu, He, Phe, Trp, Tyr.

[0332] In one form of the invention, the hydrophobic group is a chemical moiety selected from the group consisting of: C 1-8 Alkyl, C 1-8 Alkenyl, C 3-6 Cycloalkyl, aryl, substituted aryl, alkylaryl, heteroaryl, alkylheteroaryl.

[0333] "Alkyl" means an aliphatic hydrocarbon group which may be straight or branched and contains from about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups contain from about 1 to about 12 carbon atoms in the chain. More preferred alkyl groups contain from about 1 to about 6 carbon atoms in the chain. Branched means one or more lower alkyl groups, such as methyl, ethyl, or propyl, attached to a straight alkyl chain.

[0334] "Lower alkyl" means a group having from about 1 to about 6 carbon atoms in the chain, which group may be straight or branched. Alkyl may optionally be substituted with one or more substituents, which may be the same or different, each substituent being independently selected from halogen, alkyl, aryl, cycloalkyl, cyano, hydroxyl, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), -N(alkyl) 2, carboxyl, and -C(O)O-alkyl. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, and tert-butyl.

[0335] "Alkenyl" means an aliphatic hydrocarbon group which contains at least one carbon-carbon double bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably have about 2 to about 4 carbon atoms in the chain. Branching means one or more lower alkyl groups, such as methyl, ethyl or propyl, attached to a linear alkenyl chain.

[0336] "Lower alkenyl" means about 2 to about 6 carbon atoms in the chain, which may be straight or branched. Non-limiting examples of suitable alkenyl groups include ethenyl, propenyl, 2-butenyl, and 3-methylbutenyl. The term "substituted alkenyl" means that the alkenyl group may be substituted with one or more substituents, which may be the same or different, each substituent being independently selected from the group consisting of alkyl, aryl, and cycloalkyl.

[0337] "Alkynyl" means an aliphatic hydrocarbon group which contains at least one carbon-carbon triple bond and which may be straight or branched and comprises about 2 to about 15 carbon atoms in the chain. Preferred alkynyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably have about 2 to about 4 carbon atoms in the chain. Branching means one or more lower alkyl groups, such as methyl, ethyl, or propyl, attached to a linear alkynyl chain.

[0338] "Lower alkynyl" means about 2 to about 6 carbon atoms in the chain, which may be straight or branched. Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, 2-butynyl, and 3-methylbutynyl. The term "substituted alkynyl" means that the alkynyl group may be substituted with one or more substituents, which may be the same or different, each substituent being independently selected from the group consisting of alkyl, aryl, and cycloalkyl.

[0339] "Aliphatic" means and includes straight or branched chains of alkane, alkene, or alkyne carbon atoms. An aliphatic group may be optionally substituted with one or more substituents, which may be the same or different, each substituent being independently selected from the group consisting of H, halogen, halogen, alkyl, aryl, cycloalkyl, cycloalkylamino, alkenyl, heterocyclic, alkynyl, cycloalkylaminocarbonyl, hydroxy, thio, cyano, hydroxy, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), -N(alkyl)2)carboxyl, -C(O)O-alkyl, heteroaryl, aralkyl, alkylaryl, aralkenyl, heteroaralkyl, alkylheteroaryl, heteroaralkenyl, heteroalkyl, carbonyl, hydroxyalkyl, aryloxy, aralkyloxy, acyl, aroyl, nitro, amino, amido, Y1Y2N-alkyl-, Y1Y2N-alkyl-, Y1Y2NC(O)- and Y1Y2NSO2-, wherein Y1 and Y2 can be the same or different and are independently selected from hydrogen, alkyl, aryl and aralkyl.

[0340] "Heteroaliphatic" means an otherwise aliphatic group that contains at least one heteroatom (such as oxygen, nitrogen, or sulfur). The term "heteroaliphatic" includes substituted heteroaliphatics.

[0341] "Aryl" means an aromatic monocyclic or polycyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. An aryl group can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein. Non-limiting examples of suitable aryl groups include phenyl and naphthyl.

[0342] "Heteroalkyl" means an alkyl group as defined above wherein one or more hydrogen atoms are replaced by a heteroatom selected from N, S or O.

[0343] "Heteroaryl" means an aromatic monocyclic or polycyclic ring system comprising from about 5 to about 14 ring atoms, preferably from about 5 to about 10 ring atoms, wherein one or more of the ring atoms is an element other than carbon, such as nitrogen, oxygen or sulfur, alone or in combination. Preferred heteroaryl groups contain from about 5 to about 6 ring atoms. The "heteroaryl" group may be optionally substituted with one or more "ring system substituents," which may be the same or different and are as defined herein. The prefix aza, oxa or thio before the heteroaryl radical name means that at least a nitrogen, oxygen or sulfur atom, respectively, is present as a ring atom. The nitrogen atom of a heteroaryl group may be optionally oxidized to the corresponding N-oxide. Non-limiting examples of suitable heteroaryl groups include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyrido (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, oxazolidin ... yl), imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothiophenyl, quinolinyl, imidazolyl, thienopyridinyl, quinazolinyl, thienopyrimidinyl, pyrrolopyridinyl, imidazopyridinyl, isoquinolinyl, benzazaindolyl, 1,2,4-triazinyl, benzothiazolyl, and the like. The term "heteroaryl" also refers to partially saturated heteroaryl moieties, for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, and the like.

[0344] "Aralkyl" or "arylalkyl" means an aryl-alkyl group in which the aryl and alkyl groups are as previously described. Preferred aralkyl groups include lower alkyl groups. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenylethyl, and naphthylmethyl. The bond to the parent moiety is through the alkyl group.

[0345] "Alkylaryl" means an alkyl-aryl group in which the alkyl and aryl groups are as previously described. Preferred alkylaryl groups include lower alkyl groups. A non-limiting example of a suitable alkylaryl group is tolyl. The bond to the parent moiety is through the aryl group.

[0346] "Cycloalkyl" means a non-aromatic monocyclic or multicyclic ring system comprising about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 7 ring atoms. The cycloalkyl group can be optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined above. Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. Non-limiting examples of suitable multicyclic cycloalkyls include 1-decalinyl, norbornyl, adamantyl, and the like, as well as partially saturated species such as, for example, indanyl, tetrahydronaphthyl, and the like. "Halogen" means fluorine, chlorine, bromine, or iodine. Preferred are fluorine, chlorine, and bromine.

[0347] "Ring system substituent" means a substituent attached to an aromatic or non-aromatic ring system which, for example, replaces an available hydrogen on the ring system. The ring system substituents may be the same or different, each substituent being independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkyloxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio alkyl, -C(=N-CN)-NH, -C(=NH)-NH, -C(=NH)-NH(alkyl), Y1Y2N-, Y1Y2N-alkyl-, Y1Y2NC(O)-, Y1Y2NSO2-, and -SONY1Y2, wherein Y1 and Y2 can be the same or different and are independently selected from hydrogen, alkyl, aryl, cycloalkyl, and aralkyl. "Ring system substituent" may also mean a single moiety which simultaneously replaces two available hydrogens on two adjacent carbon atoms (one H on each carbon) of a ring system. Examples of such moieties are methylenedioxy, ethylenedioxy, -C(CH3)2-, and the like, which form moieties such as, for example:

[0348] It should be noted that in the ring systems of the present invention containing heteroatoms, there are no hydroxyl groups on carbon atoms adjacent to N, O, or S1 and no N or S groups on carbon adjacent to another heteroatom. Thus, for example, in the following ring: There is no -OH group directly attached to the carbons labeled 2 and 5.

[0349] It should also be noted that tautomeric forms, for example, of the following moieties: are considered equivalent in certain embodiments of the present invention.

[0350] "Alkynylalkyl" means an alkynyl-alkyl group in which the alkynyl and alkyl groups are as previously described. Preferred alkynylalkyl groups contain a lower alkynyl group and a lower alkyl group. The bond to the parent moiety is through the alkyl group. Non-limiting examples of suitable alkynyl groups include propargylmethyl.

[0351] "Heteroaralkyl" means a heteroaryl-alkyl group in which the heteroaryl and alkyl groups are as previously described. Preferred heteroaralkyl groups contain a lower alkyl group. Non-limiting examples of suitable aralkyl groups include pyridylmethyl and lin-3-ylmethyl. The bond to the parent moiety is through the alkyl group.

[0352] "Hydroxyalkyl" means a HO-alkyl group, wherein alkyl is as previously defined. Preferred hydroxyalkyl groups contain a lower alkyl group. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

[0353] "Acyl" means an HC(O)-, alkyl-C(O)-, or cycloalkyl-C(O)- group, wherein the various groups are as previously described. The bond to the parent moiety is through the carbonyl group. Preferred acyl groups contain lower alkyl groups. Non-limiting examples of suitable acyl groups include formyl, acetyl, and propionyl.

[0354] "Aroyl" means an aryl-C(O)- group in which the aryl group is as previously described. The bond to the parent moiety is through the carbonyl group. Non-limiting examples of suitable groups include benzoyl and 1-naphthoyl.

[0355] "Alkoxy" means an alkyl-O- group in which the alkyl group is as previously described. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond to the parent moiety is through the ether oxygen.

[0356] "Aryloxy" means an aryl-O- group in which the aryl group is as previously described. Non-limiting examples of suitable aryloxy groups include phenoxy and naphthoxy. The bond to the parent moiety is through the ether oxygen.

[0357] "Alkylthio" means an alkyl-S- group in which the alkyl group is as previously described. Non-limiting examples of suitable alkylthio groups include methylthio and ethylthio. The bond to the parent moiety is through the sulfur.

[0358] "Arylthio" means an aryl-S- group in which the aryl group is as previously described. Non-limiting examples of suitable arylthio groups include phenylthio and naphthylthio. The bond to the parent moiety is through the sulfur.

[0359] "Aralkylthio" means an aralkyl-S- group in which the aralkyl group is as previously described. Non-limiting example of a suitable aralkylthio group is benzylthio. The bond to the parent moiety is through the sulfur.

[0360] "Alkoxycarbonyl" means an alkyl-O-CO- group. Non-limiting examples of suitable alkoxycarbonyl groups include methoxycarbonyl and ethoxycarbonyl. The bond to the parent moiety is through the carbonyl group.

[0361] "Aralkyloxycarbonyl" means an aralkyl-OC(O)- group. Non-limiting example of a suitable aralkyloxycarbonyl group is benzyloxycarbonyl. The bond to the parent moiety is through the carbonyl group.

[0362] "Alkylsulfonyl" means an alkyl-S(O2)- group. Preferred groups are those wherein the alkyl group is a lower alkyl group. The bond to the parent moiety is through the sulfonyl group.

[0363] "Arylsulfonyl" means an aryl-S(O2)- group. The bond to the parent moiety is through the sulfonyl group.

[0364] The term "substituted" means that one or more hydrogen atoms on the designated atom are replaced with a radical selected from the indicated group, provided that the normal valence of the designated atom is not exceeded under the existing circumstances and that such substitution results in a stable compound. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0365] By "stable compound" or "stable structure" is meant a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0366] The term "optionally substituted" means optionally substituted with the specified group, radical or moiety.

[0367] When a functional group in a compound is referred to as "protected," this means that when the compound is subjected to a reaction, the group is in a modified form that precludes undesirable side reactions at the protected site. Suitable protecting groups will be recognized by one of ordinary skill in the art and by reference to standard texts such as, for example, Greene et al. (1991).

[0368] When any variable (eg, aryl, heterocycle, R2) occurs more than one time in any constituent or in the present invention, its definition on each occurrence is independent of its definition at every other occurrence.

[0369] In one form of the invention, each charged or hydrogen-bonding group is an amino acid residue independently selected from the group consisting of: Asp, Glu.

[0370] In one form of the invention, each charged or hydrogen-bonding group is an amino acid residue having a carboxylic acid moiety.

[0371] In one form of the invention, each charged or hydrogen-bonding group is independently selected from the following chemical moieties: carboxylic acids, hydroxamic acids, phosphonic and phosphinic acids, sulfonic and sulfinic acids, sulfonamides, acylsulfonamides and sulfonylureas, 2,2,2-trifluoroethan-1-ol and trifluoromethylketones, tetrazoles, 5-oxo-1,2,4-oxadiazoles and 5-oxo-1,2,4-thiadiazoles, thiazolidinediones, oxazolidinediones and oxadiazolidine-diones, 3-hydroxyisoxazoles and 3-hydroxyisothiazoles, substituted phenols, squaric acids, 3- and 4-hydroxyquinolin-2-ones, tetronic acid and tetramic acid. Acid), cyclopentane-1,3-diones and other cyclic and acyclic structures including boronic acids, mercaptoazoles and sulfonimidamides (Ballatore et al., 2013).

[0372] In one form, the present invention provides a method for identifying a modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), the method comprising the steps of: (1) comparing the three-dimensional structure of a compound to a pharmacophore comprising two or more features selected from the group consisting of a first charged or hydrogen-bonding group (A), a second charged or hydrogen-bonding group (B), a third charged or hydrogen-bonding group (C), and a hydrophobic group (D), wherein the distances between the features are, within a tolerance of ±10 Å, as follows: and (2) selecting compounds in which the hydrophobic and / or charged or hydrogen-bonding chemical moieties are so positioned.

[0373] The modulators of RAGE ligand-independent RAGE activation identified by the above methods including comparison with the pharmacophore can be peptides or non-peptidyl compounds.

[0374] In a preferred form of the invention, tolerances up to The condition is that the distance between the location points is positive in value. In a preferred form of the invention, the tolerance is up to The condition is that the distance between the location points is positive in value. In a preferred form of the invention, the tolerance is up to The condition is that the distances between the part points are positive in value.

[0375] In a preferred form of the invention, the modulator comprises three or more features selected from the group specifically described above. In a preferred form of the invention, the modulator comprises four features from the group specified above.

[0376] In one form of the invention, the comparison of the three-dimensional structure of the compound to the pharmacophore comprises comparing the minimum energy structure of the compound to the pharmacophore.

[0377] An efficient means of selecting compounds from a potentially large number of compounds involves screening one or more computerized databases of three-dimensional chemical structures of compounds using a computer program such as Catalyst (MS1) to compare the compounds against the pharmacophores of the present invention.

[0378] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is a peptide having the amino acid sequence as shown in SEQ ID NO: 1, or an analog, fragment or derivative thereof containing at least residues 379-390.

[0379] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is a peptide of formula SEQ ID NO: 1 or an analog or derivative thereof.

[0380] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is a peptide of formula SEQ ID NO: 2 or an analog or derivative thereof.

[0381] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is a peptide of formula SEQ ID NO: 5 or an analog or derivative thereof.

[0382] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is a peptide of formula SEQ ID NO: 6 or an analog or derivative thereof.

[0383] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is the S391A-E392X-RAGE peptide as depicted in SEQ ID NO: 7, or an analog or derivative thereof.

[0384] SEQ ID NO: 7: [L 362 WQRRQRRGEERKAPENQEEEEERAELNQA 391 ]

[0385] In one form of the invention, the modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), is the S391X-RAGE peptide as depicted in SEQ ID NO: 8, or an analog or derivative thereof.

[0386] SEQ ID NO: 8: [L 362 WQRRQRRGEERKAPENQEEEEERAELNQ 390 ]

[0387] Preferred specific derivatives include Q as described below in SEQ ID NO: 9 379 EEEEERAELNR 390 , Q as set forth in SEQ ID NO: 10 379 EEEEERAELNK 390 , K as described in SEQ ID NO: 11 379 EEEEERAELNQ 390 , K as described in SEQ ID NO: 12 379 EEEERAELNK 390 and K as set forth in SEQ ID NO: 13 379 EEEEERAELNR 390 .

[0388] SEQ ID NO: 9: [Q 379 EEEEERAELNR 390 ]

[0389] SEQ ID NO: 10: [Q 379 EEEEERAELNK 390 ]

[0390] SEQ ID NO: 11: [K 379 EEEEERAELNQ 390 ]

[0391] SEQ ID NO: 12: [K 379 EEEEERAELNK 390 ]

[0392] SEQ ID NO: 13: [K 379 EEEEERAELNR 390 ]

[0393] As used herein in relation to a modulator of the invention, such as SEQ ID NOs: 1, 2, 5 to 13, the term "derivative" refers to a modulator characterized in that its primary structure is taken from or derived from the C-terminal cytoplasmic tail of RAGE or a fragment thereof, but the modulator comprises amino acid additions, substitutions, truncations, chemical and / or biochemical modifications (acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, side chain methylation), labeling with radionucleotides or halogens, unusual or artificial amino acids (e.g., D-amino acids, N-methylated amino acids, tetra-substitutions, β-peptides, pyroglutamic acid; 2-aminoadipic acid; 3-aminoadipic acid; β-alanine; β-amino acid); 2-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid; pipecolic acid; 6-aminohexanoic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2″-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine; sarcosine; N-methylisoleucine; N-methylvaline; norvaline; norleucine; ornithine; statins), retroinverted sequences, cyclic peptides, peptoids, or linked to non-peptide drugs, non-peptide labels, non-peptide carriers, or non-peptide resins.

[0394] The inventors have further discovered that a peptide comprising residues 343-361 of wild-type RAGE (SEQ ID NO: 14) is an inhibitory peptide that inhibits both RAGE ligand-independent and RAGE ligand-dependent RAGE activation.

[0395] Substitutions encompass amino acid changes in which an amino acid is replaced with a different naturally occurring or unconventional amino acid residue. Such substitutions can be classified as "conservative," in which case the amino acid residue contained in the polypeptide is replaced with another naturally occurring amino acid having similar characteristics with respect to polarity, side chain functionality, or size, e.g. or It will be appreciated that certain unconventional amino acids may also be suitable replacements for naturally occurring amino acids. For example, ornithine, homoarginine, and dimethyllysine are related to His, Arg, and Lys.

[0396] Substitutions encompassed by the invention may also be "non-conservative," in which an amino acid residue present in a polypeptide is replaced with an amino acid having different properties, such as from a different group of naturally occurring amino acids (e.g., replacing a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally occurring amino acid is replaced with an unconventional amino acid.

[0397] Amino acid substitutions are generally single residue substitutions, but can be multiple residue substitutions, either clustered or dispersed. Preferably, amino acid substitutions are conservative.

[0398] Additions encompass additions of one or more naturally occurring or unconventional amino acid residues. Deletions encompass deletions of one or more amino acid residues.

[0399] As described above, the present invention includes peptides in which one or more amino acids have undergone side chain modifications. Examples of side chain modifications contemplated by the present invention include modification of amino groups, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxalation of lysine with pyridoxal 5-phosphate followed by reduction with NaBH4.

[0400] The guanidino group of arginine residues can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, phenylacetaldehyde, and glyoxal.

[0401] Carboxyl groups can be modified by carbodiimide activation with the formation of O-acylisoureas, followed by derivatization (e.g., to the corresponding amides). Thiol groups can be modified by various methods, such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation with performic acid to oxidize cysteine; formation of mixed disulfide bonds with other sulfhydryl compounds; reaction with maleimide, maleic anhydride, or other substituted maleimides; formation of mercury derivatives using 4-chloromercuric benzoate, 4-chloromercuric benzenesulfonic acid, phenylmercuric chloride, 2-chloromercuric-4-nitrophenol, and other mercurials; and carbamylation with cyanate esters at alkaline pH. In preferred forms of the invention, any modification of cysteine ​​residues must not affect the ability of the peptide to form the necessary disulfide bonds. It is also possible to replace the sulfhydryl group of a cysteine ​​with a selenium equivalent, such that the peptide forms a diselenide bond in place of one or more disulfide bonds.

[0402] Tryptophan residues can be modified, for example, by oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfhydryl halides. On the other hand, tyrosine residues can be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0403] Modification of the imidazole ring of a histidine residue can be achieved by alkylation with an iodoacetic acid derivative or N-ethoxycarbonylation with diethylpyrocarbonate.Proline residues can be modified, for example, by hydroxylation at the 4-position.

[0404] A list of some amino acids and other unnatural amino acids with modified side chains is shown in the table below:

[0405] These types of modifications may be important to stabilize the peptide if it is to be administered to an individual or used as a diagnostic reagent.

[0406] As used herein, conservative amino acid substitutions can include amino acid residues within a group having sufficiently similar physicochemical properties such that substitutions between members of the group retain the biological activity of the molecule (see, e.g., Grantham, R., 1974). In particular, conservative amino acid substitutions are preferably substitutions in which the amino acids are derived from the same class of amino acids (e.g., basic amino acids, acidic amino acids, polar amino acids, amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chains, amino acids with side chains that can form hydrogen bridges, e.g., amino acids with side chains having hydroxyl functional groups). Conservative substitutions in this case are, for example, substitutions of a basic amino acid residue (Lys, Arg, His) with another basic amino acid residue (Lys, Arg, His), substitutions of an aliphatic amino acid residue (Gly, Ala, Val, Leu, Ile) with another aliphatic amino acid residue, substitutions of an aromatic amino acid residue (Phe, Tyr, Trp) with another aromatic amino acid residue, substitutions of threonine with serine, or substitutions of leucine with isoleucine. Other conservative amino acid exchanges will be known to those skilled in the art. Isomer forms should preferably be maintained, for example, preferably replacing R or H with K, while preferably replacing r and h with k.

[0407] When considering replacement amino acids, preferred substitutions of the present invention are those described in Grantham, R. (1974) as having a D of less than 100, the contents of which are incorporated by reference. Most preferred substitutions are those described as having a D of less than 50.

[0408] Peptide modulators of the present invention include retro inverso isomers of SEQ ID NO: 1, 2, 5, 6, 7, 8, 9, 10, 11, 12 or 13 or modified or substituted variants thereof or peptides formed by addition or deletion (Li et al., 2010). Methods for treating, preventing or managing RAGE-related disorders

[0409] In another related aspect, the invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, comprising administering an effective amount of a modulator of RAGE ligand-independent RAGE activation by a colocalized activating GPCR of the invention.

[0410] In another aspect, the invention encompasses the use of a modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR for the manufacture of a medicament for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment.

[0411] In another aspect, the invention encompasses the use of a modulator of RAGE ligand-independent RAGE activation via a co-localized activating GPCR for treating, preventing, or managing a RAGE-related disorder in a patient in need of such treatment.

[0412] In a preferred form of the invention, certain co-localized GPCRs are angiotensin receptors. In a preferred form of the invention, certain co-localized GPCRs are AT1R.

[0413] In a preferred form of the invention, the certain co-localized GPCR is a certain chemokine receptor. In a preferred form of the invention, the certain co-localized GPCR is CCR2.

[0414] Additionally, the present invention provides methods for treating, preventing, or managing a RAGE-associated disorder in a patient in need of such treatment, comprising administering an effective amount of a modulator of RAGE ligand-independent RAGE activation by a certain colocalized activating GPCR of the invention in combination with a modulator of a certain colocalized GPCR and / or a modulator of a certain colocalized GPCR signaling pathway.

[0415] In a preferred form of the invention, certain co-localized GPCRs are angiotensin receptors. In a preferred form of the invention, certain co-localized GPCRs are AT1R.

[0416] In a preferred form of the invention, the certain co-localized GPCR is a certain chemokine receptor. In a preferred form of the invention, the certain co-localized GPCR is CCR2.

[0417] The method can comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation by a colocalized activated GPCR of the invention in combination with a modulator of a colocalized GPCR and / or a modulator of a colocalized GPCR signaling pathway, wherein the modulator of a colocalized GPCR and / or a modulator of a colocalized GPCR signaling pathway is administered at a dose lower than the dose typically administered for treating a condition associated with the colocalized GPCR.

[0418] The method can comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation by a colocalized activating GPCR of the invention in combination with a modulator of a colocalized GPCR and / or a modulator of a colocalized GPCR signaling pathway, wherein the modulator of a colocalized GPCR and / or a modulator of a colocalized GPCR signaling pathway is administered at a dose lower than the dose typically administered for the treatment of a disorder associated with RAGE.

[0419] In a particularly preferred form of the invention, the method may comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation via an activated angiotensin receptor of the invention in combination with a modulator of AT1R and / or a modulator of the AT1R signaling pathway, wherein the modulator of AT1R and / or the modulator of the AT1R signaling pathway is administered at a dose lower than the dose typically administered for the treatment of AT1R-related disorders.

[0420] In another particularly preferred form of the invention, the method may comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation of a certain chemokine receptor activated by the invention in combination with a modulator of CCR2 and / or a modulator of the CCR2 signaling pathway, wherein the modulator of CCR2 and / or the modulator of the CCR2 signaling pathway is administered at a dose lower than the dose typically administered for the treatment of a CCR2-related disorder.

[0421] Additionally, the present invention provides methods for treating, preventing, or managing a RAGE-related disorder in a patient in need of such treatment, comprising administering an effective amount of a combination of a modulator of RAGE ligand-independent RAGE activation by a certain co-localized activating GPCR of the invention and a modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway.

[0422] In a particularly preferred form of the invention, the method may comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation via a co-localized activated GPCR of the invention in combination with a modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway is administered at a dose lower than the dose typically administered for the treatment of RAGE-related disorders.

[0423] Additionally, the present invention provides methods for treating, preventing, or managing a RAGE-associated disorder in a patient in need of such treatment, comprising administering an effective amount of a modulator of RAGE ligand-independent RAGE activation by a certain colocalized activating GPCR of the invention in combination with a modulator of a certain colocalized GPCR and / or a modulator of a certain colocalized GPCR signaling pathway.

[0424] For example, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, comprising administering an effective amount of a combination of a modulator of RAGE ligand-independent RAGE activation via an activated angiotensin receptor of the present invention and a modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway and a modulator of AT1R and / or a modulator of the AT1R signaling pathway.

[0425] For example, the present invention provides methods for treating, preventing or managing a RAGE-related disorder in a patient in need of such treatment, comprising administering an effective amount of a combination of a modulator of RAGE ligand-independent RAGE activation via a certain activated chemokine receptor of the present invention and a modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway and a modulator of CCR2 and / or a modulator of the CCR2 signaling pathway.

[0426] In a particularly preferred form of the invention, the method may comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation by a colocalized activated GPCR of the invention in combination with a modulator of the colocalized GPCR and / or a modulator of a colocalized GPCR signaling pathway and a modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway is administered at a dose lower than the dose typically administered for the treatment of a RAGE-associated disorder, and / or the modulator of a colocalized GPCR and / or a modulator of a colocalized GPCR signaling pathway is administered at a dose lower than the dose typically administered for the treatment of a disorder associated with the GPCR.

[0427] In a particularly preferred form of the invention, the method may comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation via an activated angiotensin receptor of the invention in combination with a modulator of AT1R and / or a modulator of the AT1R signaling pathway and a modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway is administered at a dose lower than the dose typically administered for the treatment of RAGE-associated disorders, and / or the modulator of AT1R and / or a modulator of the AT1R signaling pathway is administered at a dose lower than the dose typically administered for the treatment of AT1R-associated disorders.

[0428] In a particularly preferred form of the invention, the method may comprise administering an effective amount of a modulator of RAGE ligand-independent RAGE activation by an activating chemokine receptor of the invention in combination with a modulator of CCR2 and / or a modulator of the CCR2 signaling pathway and a modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent RAGE activation and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway is administered at a dose lower than the dose typically administered for the treatment of RAGE-related disorders, and / or the modulator of CCR2 and / or a modulator of the CCR2 signaling pathway is administered at a dose lower than the dose typically administered for the treatment of CCR2-related disorders.

[0429] RAGE-related disorders are defined as disorders that are dependent on RAGE expression. This does not exclude disorders that are also dependent on RAGE expression and are associated with a co-localized GPCR, such as AT1R-related disorders or CCR2-related disorders. In fact, a disorder can be RAGE-related and associated with a co-localized GPCR, including AT1R-related or CCR2-related.

[0430] A disorder associated with a co-localized GPCR is defined as a disorder that is dependent on the expression of a co-localized GPCR. This does not exclude RAGE-related disorders that are also dependent on the expression of a co-localized GPCR. In fact, a disorder can be RAGE-related and associated with a co-localized GPCR, including AT1R-related or CCR2-related.

[0431] In one form of the invention, the RAGE-associated disorder is a disorder selected from the group consisting of cardiovascular disease, digestive system disease, cancer, nervous system disease, respiratory disease, connective tissue disease, kidney disease, reproductive disease, skin disease, eye disease, and endocrine disease.

[0432] In one form of the invention, the RAGE-associated disorder is a cardiovascular disease selected from the group consisting of atherosclerosis, ischemic heart disease, myocarditis, endocarditis, cardiomyopathy, acute rheumatic fever, chronic rheumatic heart disease, cerebrovascular disease / stroke, heart failure, vascular calcification, peripheral vascular disease, and lymphangitis.

[0433] In one form of the invention, the RAGE-associated disorder is a digestive disease selected from the group consisting of periodontitis, esophagitis, gastritis, gastro-duodenal ulcer, Crohn's disease, ulcerative colitis, ischemic colitis, enteritis and enterocolitis, peritonitis, alcoholic liver disease, hepatitis, toxic liver disease, biliary cirrhosis, liver fibrosis / cirrhosis, non-alcoholic fatty liver disease / non-alcoholic steatohepatitis (NAFLD / NASH), liver trauma and recovery from liver injury, trauma or surgery.

[0434] In one form of the invention, the RAGE-related disorder is a cancer selected from the group consisting of: malignancies of the lip, oral cavity, and pharynx, malignancies of the digestive organs, malignancies of the respiratory organs and thoracic organs, malignancies of the bone and articular cartilage, melanoma and other malignancies of the skin, malignancies of the mesothelial tissue and soft tissue, malignancies of the breast, malignancies of the female reproductive organs, malignancies of the male reproductive organs, malignancies of the urinary tract, malignancies of the eye, brain, and other parts of the central nervous system, malignancies of the thyroid and other endocrine glands, malignancies of lymphoid tissue, hematopoietic tissue, and related tissues, malignancies of disease-defined site, secondary, and / or unspecified site.

[0435] In one form of the invention, the RAGE-related disorder is a neurological disease and is selected from the group consisting of inflammatory diseases of the central nervous system, generalized atrophy primarily affecting the central nervous system, extrapyramidal and movement disorders, Parkinson's disease, demyelinating diseases of the central nervous system, Alzheimer's disease, focal brain atrophy, Lewy body disease, epilepsy, migraine, neuropathic pain, diabetic neuropathy, polyneuropathy, glioma formation and progression, spinal cord trauma, and ischemic brain injury / stroke, brain trauma and recovery from brain injury, trauma or surgery.

[0436] In one form of the invention, the RAGE-related disorder is a psychiatric disorder and is selected from the group consisting of dementia, Alzheimer's disease, vascular dementia, addiction, schizophrenia, major affective disorder, depression, mania, bipolar disorder, and anxiety.

[0437] In one form of the invention, the RAGE-related disorder is a respiratory (lung) disease and is selected from the group consisting of acute upper respiratory tract infection, rhinitis, nasopharyngitis, sinusitis, laryngitis, influenza and pneumonia, acute bronchitis, acute bronchiolitis, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, emphysema, chronic lung disease due to external agents, acute respiratory distress syndrome (ARDS), pulmonary eosinophilia and pleural, lung trauma and recovery from lung injury, trauma or surgery.

[0438] In one form of the invention, the RAGE-related disorder is a connective tissue disease and is selected from the group consisting of osteoarthritis, infectious arthritis, rheumatoid arthritis, psoriatic arthropathy and enteropathic arthropathy, juvenile arthritis, gout and other crystalline arthropathy, diabetic arthropathy, polyarteritis nodosa, Churg-Strauss syndrome, mucocutaneous lymph node syndrome [Kawasaki disease], hypersensitivity vasculitis, Goodpasture syndrome, thrombotic microangiopathy, Wegener's granulomatosis, aortic arch syndrome [Takayasu disease], giant cell arteritis, polymyalgia rheumatica, microscopic polyangiitis, hypocomplementemic vasculitis, systemic lupus erythematosus, cutaneous polymyositis, polymyositis, systemic sclerosis, CR(E)ST syndrome, Sjögren's syndrome [ disease], mixed connective tissue disease, illness, traumatic muscle injuries, sprains, strains and fractures.

[0439] In one form of the invention, the RAGE-related disorder is a kidney disease and is selected from the group consisting of glomerulonephritis, nephritis, diabetic kidney disease, interstitial nephritis, obstructive and reflux nephropathy, acute renal failure, and chronic kidney disease.

[0440] In one form of the invention, the RAGE-related disorder is a reproductive disease and is selected from the group consisting of prostatitis, prostatic hyperplasia, prostatic dysplasia, salpingitis, oophoritis, pelvic inflammatory disease (PID), polycystic ovary syndrome, cervicitis, cervical dysplasia, vaginitis, vulvitis.

[0441] In one form of the invention, the RAGE-associated disorder is a skin disorder selected from the group consisting of dermatitis, eczema, pemphigus / pemphigoid, psoriasis, pityriasis rosea, lichen planus, urticaria, erythema multiforme, erythema nodosum, sunburn, keratosis, photoaging skin ulcers, superficial skin lesions, and open wounds.

[0442] In one form of the invention, the RAGE-related disorder is an eye disease selected from the group consisting of keratitis, conjunctivitis, retinitis, glaucoma, scleritis, episcleritis, chorioretinal inflammation, diabetic retinopathy, macular edema, retinopathy of prematurity and optic neuritis, ocular trauma and recovery from ocular injury, trauma or surgery.

[0443] In one form of the invention, the RAGE-related disorder is an endocrinopathy selected from the group consisting of diabetes, insulin resistance, impaired glucose tolerance, and thyroiditis.

[0444] In one form of the present invention, the inhibitor that inhibits AT1R or inhibits the AT1R signaling pathway is selected from the group consisting of: eprosartan (marketed under the name Abbott Laboratories USA), Losartan (marketing name Merck & Co), valsartan (market name Novartis), telmisartan (marketing name Boehringer Ingelheim), irbesartan (marketing name SanofiAventis), Olmesartan (market name Daiichi Sankyo Inc), azilsartan (marketed as Edarbi, Takeda), candesartan (marketed as AstraZeneca), ZD-7115, vasopressin ((Sar1-Ala8)AngII), Sarthran ((Sar1-Thr8)AngII), and DuP753. This list also includes prodrugs of these inhibitors, including candesartan (candesartan cilexetil), azilsartan (Azilsartan medoxomi), and olmesartan (Olmesartan medoxomil), which can be the form in which they are administered, as well as active metabolites (e.g., EXP-3174, the active metabolite of losartan). Note: Partial agonists can act to inhibit endogenous Ang II because, even if they show agonism, partial agonists do not produce maximal efficacy and can act as inhibitors therapeutically.

[0445] In one form of the invention, the inhibitor of a chemokine receptor or a chemokine signaling pathway is selected from the group consisting of propagermanium (also known as 3-[(2-carboxyethyl-oxygermyl)oxy-germyl] propionic acid, proxigermanium, Ge-132, bis(2-carboxyethylgermanium) sesquioxide (CEGS), 2-carboxyethylgermanosine, SK-818, organic germanium, germanium sesquioxide, 3,3′-(1,3-dioxo-1,3-digermanoxanediyl)dipropionic acid, 3-oxygermylpropionic acid polymer, poly-trans-(2-carboxyethyl)germanosine, proxigermanium, repagermanium, and Serocion; CCR2), BMS CCR222 (CCR2), resveratrol (CCR2), RS504393 (CCR2), RS102895 (CCR2), MLN-1202 (Millennium Pharmaceuticals; CCR2), INCB8696 (Incyte Pharmaceuticals; CCR2), MK-0812 (Merck; CCR2), CCX140 (ChemoCentryx; CCR2), PF-4136309 (Pfizer; CCR2), BMS-741672 (Bristol-Myers Squibb; CCR2); Repertaxin (CXCR2), TAK-779 (CCR5), TAK-220 (CCR5), TAK-652 (CCR5), AK692 (CCR5), CMPD167 (CCR5), BX-471 (CCR1), AMD3100 (CXCR4), AMD11070 (CXCR4), FC131 (CXCR4), MLN3897 (CCR1), CP-481715 (CCR1), GW-873140 (CCR5), SB225002 (CXCR2), and SB265610 (CXCR2).

[0446] In one form of the invention, the inhibitor that inhibits CCR2 or inhibits the CCR2 signaling pathway is selected from the group consisting of propagermanium (also known as 3-[(2-carboxyethyl-oxygermyl)oxy-germyl] propionic acid, proxigermanium, Ge-132, bis(2-carboxyethylgermanium) sesquioxide (CEGS), 2-carboxyethylgermanesquioxane, SK-818, organic germanium, germanium sesquioxide, 3,3′-(1,3-dioxo-1,3-digermanoxanediyl)dipropionic acid, 3-oxygermylpropionic acid polymer, poly-trans-(2-carboxyethyl)germanesquioxane, proxigermanium, propagermanium, and Serocion), BMS CCR222 (CCR2), resveratrol (CCR2), RS504393, RS102895, MLN-1202 (Millennium Pharmaceuticals), INCB8696 (Incyte Pharmaceuticals), MK-0812 (Merck), CCX140 (ChemoCentryx), PF-4136309 (Pfizer), BMS-741672 (Bristol-Myers Squibb).

[0447] In one form of the invention, the inhibitor of RAGE ligand-dependent RAGE activation and / or inhibitor of constitutively active RAGE and / or inhibitor of the RAGE signaling pathway is selected from the following groups: Azeliragon (TTP488 / PF-04494700) (an oral small molecule inhibitor of RAGE ligand interaction targeting the V-domain); TTP4000 (a soluble fusion protein inhibitor of RAGE as described in US7981423 using the ligand-binding extracellular domain of RAGE linked to a human Ig Fc domain); Antibodies that specifically bind to RAGE and RAGE-binding fragments thereof as described in 2007109747; FPS-ZM127 (a tertiary amide that blocks Aβ / RAGE interaction with high affinity); peptides that antagonize RAGE ligand-induced signaling as described in US20100249038; lysophosphatidic acid (LPA) antagonists as described in WO2012109569; 2-aminopyrimidines as described in Han et al. (2012); and peptides as described in Han et al. (2014). ) as described in ; 4,6-biphenyl-2-(3-alkoxyanilino)pyrimidines as described in Han et al. (2015); small molecule inhibitors of ligand-stimulated RAGE-DIAPH1 signaling as described in Manigrasso et al. (2016); polypeptides consisting essentially of all or a portion of the RAGE cytoplasmic tail or consisting essentially of a portion of hyaline-1 bound to the RAGE cytoplasmic tail as described in US20090220484.

[0448] In specific embodiments, a modulator is administered to a subject based on its identification as a modulator of RAGE ligand-independent RAGE activation by a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), using screening methods or methods for identifying modulators as broadly described herein.

[0449] An AT1R-related disorder is defined as a disorder that is dependent on AT1R expression. It does not exclude RAGE-related disorders that are also dependent on AT1R expression. In fact, a disorder can be both RAGE-related and AT1R-related.

[0450] A chemokine receptor-associated disorder is defined as a disorder that is dependent on the expression of a chemokine receptor. It does not exclude RAGE-associated disorders that are also dependent on the expression of a chemokine receptor. In fact, a disorder can be both RAGE-associated and a chemokine receptor-associated.

[0451] A CCR2-related disorder is defined as a disorder that is dependent on CCR2 expression. It does not exclude RAGE-related disorders that are also dependent on CCR2 expression. In fact, a disorder can be both RAGE-related and CCR2-related.

[0452] For the combination agents, the following dosages are "normally" administered. Methods for screening candidate substances

[0453] In one form, the invention includes a method for screening a candidate substance for its ability to modulate RAGE activity, wherein such RAGE activity is induced by a co-localized, activated GPCR, the method comprising the steps of: contacting a RAGE polypeptide with a GPCR polypeptide in the presence of a candidate substance, wherein the GPCR polypeptide is constitutively active and / or activated by the addition of an agonist, partial agonist or allosteric modulator of such GPCR; and detecting whether the candidate substance is a modulator of RAGE ligand-independent RAGE activation via the co-localized, activated GPCR by detecting an effect indicative of RAGE activation modulated by the presence of the candidate substance and / or by detecting RAGE-dependent signaling modulated by the presence of the candidate substance.

[0454] In one form, the present invention comprises methods for screening candidate substances for their ability to modulate (i.e., activate, inhibit, or allosterically modulate) RAGE ligand-independent RAGE activation (also known as RAGE ligand-independent RAGE transactivation) via a co-localized activating GPCR, such as angiotensin receptors (e.g., AT1R) or chemokine receptors (e.g., CCR2). These methods generally comprise, consist of, or consist essentially of the following steps: a. contacting a RAGE polypeptide with a GPCR polypeptide in the presence of a candidate substance, wherein the GPCR polypeptide is constitutively active and / or activated by adding an agonist, partial agonist or allosteric modulator of such GPCR; and b. Testing whether the candidate substance is a regulator of RAGE ligand-independent RAGE activation via a colocalized activated GPCR by detecting an effect indicating that RAGE activation is modulated by the presence of the candidate substance and / or by detecting RAGE-dependent signaling modulated by the presence of the candidate substance.

[0455] In some embodiments, the screening method further comprises detecting whether the candidate substance is a modulator (e.g., an activator, inhibitor, or allosteric modulator) of a co-localized GPCR (e.g., angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)) or a signaling pathway of a co-localized GPCR, such as an angiotensin receptor signaling pathway (e.g., AT1R signaling pathway) or a chemokine receptor signaling pathway (e.g., CCR2 signaling pathway) in the presence or absence of RAGE. In some embodiments, a candidate substance that results in greater modulation of the signaling in the presence of the RAGE polypeptide relative to RAGE-independent signaling due to activation of the co-localized GPCR is selective for modulating RAGE ligand-independent RAGE activation via a co-localized activated GPCR.

[0456] In one form, the invention comprises a peptide identified as a modulator by the method. In one form, the invention comprises a compound identified as a modulator by the method.

[0457] In some embodiments, the screening method further comprises testing whether the candidate substance is a modulator (e.g., an activator, inhibitor, allosteric modulator, or functional surrogate) of RAGE or a RAGE signaling pathway in the presence or absence of a co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2). In some embodiments, a candidate substance that results in greater modulation of RAGE-dependent signaling in its presence compared to in the absence of the GPCR polypeptide is selective for modulating RAGE ligand-independent RAGE activation via a co-localized activated GPCR.

[0458] In some embodiments, the screening method further comprises detecting whether the candidate substance is a regulator (such as an activator, inhibitor, allosteric regulator or functional substitute) of a RAGE polypeptide or a RAGE signaling pathway and a co-localized GPCR (such as an angiotensin receptor (such as AT1R) or a chemokine receptor (such as CCR2)) or a signaling pathway of a co-localized GPCR such as an angiotensin receptor signaling pathway (such as AT1R signaling pathway) or a chemokine receptor signaling pathway (such as CCR2 signaling pathway).

[0459] In some embodiments, the screening method further comprises the step of using an inhibitor of a RAGE ligand that binds to the extracellular domain of RAGE that itself inhibits RAGE activation in a RAGE ligand-dependent manner.

[0460] In some embodiments, the screening methods further comprise the use of a RAGE polypeptide that is mutated and / or truncated such that it is unable to bind a RAGE ligand to its extracellular domain and is itself unable to activate in a RAGE ligand-dependent manner.

[0461] In some embodiments, binding of a RAGE ligand to the extracellular domain of RAGE is impaired by exposing the cell to a modulator that modulates binding of a RAGE ligand to RAGE.

[0462] In some embodiments, a RAGE polypeptide that is mutated and / or truncated so that it is unable to bind a RAGE ligand and is itself unable to activate in a RAGE ligand-dependent manner is used before, after, or in parallel with a screen for a RAGE polypeptide capable of binding a RAGE ligand.

[0463] Suitably, such candidate substances or candidate substance derivatives are particularly useful for treating, preventing or managing RAGE-related disorders, wherein the candidate substances modulate RAGE ligand-independent RAGE activation via a certain co-localized activated GPCR (such as angiotensin receptor (such as AT1R) or a certain chemokine receptor (such as CCR2)), and suitably modulate the signaling pathway of a certain co-localized GPCR (such as angiotensin receptor (such as AT1R) or a certain chemokine receptor (such as CCR2)) and / or a certain co-localized GPCR (such as angiotensin receptor signaling pathway (such as AT1R signaling pathway) or a certain chemokine receptor signaling pathway (such as CCR2 signaling pathway)) and / or inhibit RAGE ligand-dependent RAGE activation and / or inhibit constitutively active RAGE and / or RAGE signaling pathway.

[0464] In certain embodiments of the screening methods of the present invention, if the candidate substance modulates RAGE-dependent signaling detected when a RAGE polypeptide is contacted with a GPCR polypeptide, the method further comprises determining whether and / or to what extent the candidate substance modulates RAGE-dependent signaling in the absence of the GPCR polypeptide, such that the candidate substance resulting in greater modulation of RAGE-dependent signaling when the GPCR polypeptide is present is selective for modulating RAGE ligand-independent RAGE activation via a co-localized activated GPCR.

[0465] In certain embodiments of the screening methods of the present invention, if the candidate substance modulates a RAGE-dependent signal detected when a RAGE polypeptide is contacted with a GPCR polypeptide, the method further comprises determining whether and / or to what extent the signal is generated in the absence of the RAGE polypeptide, and if the signal is generated in the absence of the RAGE polypeptide, determining whether and / or to what extent the candidate substance modulates the signal in the absence of the RAGE polypeptide, such that a candidate substance that results in greater modulation of the signal when the RAGE polypeptide is present relative to RAGE-independent signaling due to activation of a colocalized GPCR is selective for modulating RAGE ligand-independent RAGE activation due to activation of a colocalized GPCR.

[0466] In certain embodiments, a proximity screening assay is used, wherein the screening method evaluates the proximity of a RAGE polypeptide to a co-localized GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2). In an illustrative example of this type, a RAGE polypeptide is coupled (e.g., conjugated or otherwise linked) to a first reporter component, and the co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)) is coupled (e.g., conjugated or otherwise linked) to a second reporter component. The proximity of the first reporter component and the second reporter component generates a signal that can be detected by a detector. The first reporter component and the second reporter component constitute a complementary pair, meaning that the first reporter component can be interchanged with the second reporter component without significantly affecting the function of the invention. The first reporter component and the second reporter component can be the same or different.

[0467] In one embodiment, the proximity screening assay is that described in patent WO2008055313 (Dimerix Bioscience Pty Ltd; see also US8283127, US8568997, EP2080012, CA2669088, CN101657715), also known as receptor heteromerization technology or receptor-HIT (Jaeger et al., 2014). In this method, RAGE is coupled to a first reporter component, a co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)) is unlabeled for proximity screening assays, and a GPCR-interacting group is attached to a complementary second reporter component. When a ligand that is selective for the unlabeled GPCR or heteromeric complex is bound, the interaction of the second reporter component with the complex is modulated. Preferred examples of GPCR-interacting groups are arrestins, G proteins, and ligands. Alternatively, a co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)) is coupled to a first reporter component, RAGE is unlabeled relative to the proximity screening assay, and a RAGE-interactive group is attached to a complementary second reporter component whose interaction with the complex is modulated upon binding of a ligand that is selective for unlabeled RAGE or the heteromeric complex. Preferred examples of RAGE-interactive groups are proteins that interact with the cytoplasmic tail of RAGE, such as IQGAP-1, Diaphanous 1, Dock7, MyD88, TIRAP, IRAK4, ERK1 / 2, and PKCζ (Jules et al., 2013; Ramasamy et al., 2016).

[0468] The reporter component can include an enzyme coupled to RAGE, a co-localized GPCR, or an interactive group via a linker incorporating an enzymatic cleavage site, a luminescent or bioluminescent molecule, a fluorescent molecule, and a transcription factor or other molecule. In short, any known organic or inorganic, proteinaceous or non-proteinaceous molecule or complex thereof that is capable of emitting a detectable signal due to its spatial proximity.

[0469] Preferably, the signal generated by the proximity of the first reporter component and the second reporter component in the presence of the reporter component initiator is selected from the group consisting of: luminescence, fluorescence, and a colorimetric change.

[0470] In some embodiments, luminescence is produced by a bioluminescent protein selected from luciferase, β-galactosidase, β-lactamase, peroxidase, or any protein capable of emitting light in the presence of a suitable substrate.

[0471] Preferred combinations of the first reporter component and the second reporter component include those detailed in US Pat. No. 8,283,127, however, useful combinations of the first reporter component and the second reporter component are in no way limited thereto.

[0472] In some embodiments, the screening method further comprises detecting the proximity of the first reporter component and the second reporter component to each other, thereby determining whether the candidate substance modulates the interaction between the RAGE polypeptide and a co-localized GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2). Typically, this is achieved when the proximity of the first reporter component and the second reporter component generates a proximity signal, wherein the proximity signal is altered by the candidate substance modulating the proximity between the RAGE polypeptide and a co-localized GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2).

[0473] One or both of RAGE and a co-localized GPCR, such as an angiotensin receptor (eg, AT1R) or a chemokine receptor (eg, CCR2), may be in soluble form or expressed on the cell surface.

[0474] In some embodiments, RAGE and a co-localized GPCR, such as an angiotensin receptor (eg, AT1R) or a chemokine receptor (eg, CCR2), are located in, partially located in, or located on a single membrane; for example, both are expressed on the surface of a host cell.

[0475] In another embodiment of the invention, a co-localized GPCR, such as an angiotensin receptor (eg, AT1R) or a chemokine receptor (eg, CCR2), is preassembled at the cell membrane with RAGE in a preformed complex.

[0476] In another embodiment of the invention, signaling involving the cytoplasmic tail of RAGE is triggered following activation of a co-localized GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), by engagement of a corresponding ligand, such as Ang II that binds to AT1R or MCP-1 that binds to CCR2.

[0477] In one embodiment of the invention, activation of the RAGE cytoplasmic tail is associated with changes in its structural conformation and / or affinity for binding partners.

[0478] In one embodiment of the invention, monitoring of the structural conformation and / or binding partner affinity of RAGE occurs when the RAGE cytoplasmic tail has been mutated and / or truncated such that it can no longer be activated by RAGE ligands or by RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs.

[0479] In one embodiment of the invention, monitoring of structural conformation and / or binding partner affinity occurs in the presence of a substance that inhibits RAGE binding and / or activation by RAGE ligands or RAGE ligand-independent RAGE activation by certain co-localized activating GPCRs.

[0480] In one embodiment of the invention, monitoring of binding partner recruitment occurs prior to RAGE activation by RAGE ligand or RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs.

[0481] In one embodiment of the invention, recruitment and activation of signaling mediators and / or binding partners to the RAGE cytoplasmic tail is monitored, following RAGE activation by RAGE ligands or RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs.

[0482] In one embodiment of the invention, monitoring of binding partner recruitment following RAGE activation by RAGE ligands or RAGE ligand-independent RAGE activation via certain colocalized activating GPCRs occurs in the presence of a substance that inhibits RAGE binding and / or activation by RAGE ligands.

[0483] Other embodiments of the present invention include methods for screening candidate substances for their ability to modulate (e.g., activate, inhibit, or otherwise regulate) RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), by detecting modulation of RAGE-mediated signaling. Such methods may include the step of measuring NFκB canonical activation by measuring one or more of the following: Detecting IkB kinase (IKK) activity by monitoring in vitro phosphorylation of substrates (e.g., GST-IκBα); Detection of IκB degradation kinetics, including phosphorylation / ubiquitination and / or degradation of IκB and / or IκB-α; Detection of p65(Re1-A) phosphorylation / ubiquitination, such as by use of antibodies, gel shift, EMSA, and / or mass spectrometry; Detection of cytoplasmic to nuclear shuttling / translocation of NFκB components / subunits (e.g., p65 / phospho-p65); Detection of NFκB subunit dimerization / complexation; Detection of active NFκB components / subunits by binding to immobilized DNA sequences / oligonucleotides containing NFκB response elements / NFκB consensus binding motifs, such as by using electrophoretic mobility shift assays or gel shift assays, SELEX, protein-binding microarrays, or sequencing-based methods; Chromatin immunoprecipitation (ChIP) analysis to detect in situ binding of NFκB to DNA at promoters and enhancers of specific genes; In vitro kinase assay for NFκB kinase activity; NFκB reporter assays to measure NFκB transcriptional activity using transgenic expression of reporter constructs (e.g., LacZ Fluc, eGFP SEAP, NF-gluc) using methods such as plasmid transfection, reporter cell lines, minicircles, retroviruses, or lentiviruses; Measurement of changes in expression of NFκB downstream targets (e.g., cytokines, growth factors, adhesion molecules, and mitochondrial anti-apoptotic genes by real-time PCR assays, protein assays, or functional assays) (Note: The pleiotropic nature of NFκB is reflected in its current list of >500 transcriptional targets (see http: / / www.bu.edu / nf-kb / gene-resources / target-genes / , accessed 2 August 2017) and; • Measurement of functional or structural changes induced by NFκB-dependent signaling, such as POLKADOTS in T cells, adhesion in endothelial cells, activation in leukocytes, or tumorigenicity.

[0484] Additionally or alternatively, such methods may comprise measuring a signal resulting from a non-canonical effect of NF-κB by measuring one or more of the following: Detection of NIK (NFκB-inducible kinase); Detect IKKα activation / phosphorylation; NIK kinase activity is measured by performing a kinase assay based on the ability to autophosphorylate or phosphorylate a substrate; Produces NFκB dimers containing p52, such as p52 / RelB; Detection of phospho-NFκB2 p100 (Ser866 / 870); Detects the partial degradation (called processing) of the precursor p100 into p52; Detecting the translocation of p52 / RelB to the nucleus; Detection of p52 / RelB binding to the κB site; NFκB reporter assays to measure NFκB transcriptional activity using transgenic expression of reporter constructs (e.g., LacZ Fluc, eGFP SEAP, NF-gluc) using methods such as plasmid transfection, reporter cell lines, minicircles, retroviruses, or lentiviruses; • Measure changes in expression of downstream targets of NFκB non-canonical signaling, such as CXCL12, by real-time PCR, protein expression, or by functional assays.

[0485] In another aspect, the present invention provides methods for identifying modulators (e.g., activators, inhibitors, allosteric modulators, or functional surrogates) that modulate (i.e., activate, inhibit, or otherwise modulate) RAGE ligand-independent RAGE activation following activation of a co-localized GPCR by a corresponding ligand (e.g., activation of AT1R by AngII or activation of CCR2 by MCP-1) or if a co-localized GPCR is constitutively active, and suitably modulates a co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)) and / or modulates a RAGE polypeptide or a RAGE signaling pathway. In preferred forms of the invention, such modulators are inhibitors of one or both of RAGE or a co-localized GPCR in the RAGE signaling pathway (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)). In a particularly preferred form of the invention, modulation of the RAGE signaling pathway is distinct from and / or occurs to a significantly different extent than modulation of certain classically co-localized GPCR signaling pathways (e.g., the AT1R signaling pathway, the Gq signaling pathway, or the CCR2 signaling pathway, the Gi signaling pathway). In a particularly preferred form of the invention, inhibition of the RAGE signaling pathway is distinct from or greater than inhibition of certain classically co-localized GPCR signaling pathways (e.g., the AT1R signaling pathway, the Gq signaling pathway, or the CCR2 signaling pathway, the Gi signaling pathway). Construct

[0486] In a related aspect, the invention provides a construct system for identifying adjacent modulators between RAGE and a co-localized GPCR, such as an angiotensin receptor (eg, AT1R) or a chemokine receptor (eg, CCR2).

[0487] In some embodiments, these construct systems comprise a first construct comprising a regulatory sequence operably linked to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a RAGE polypeptide and a nucleic acid sequence encoding a proximity signal or energy donor molecule; and a second construct comprising a regulatory sequence operably linked to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2) and a nucleic acid sequence encoding a proximity signal or energy acceptor molecule. In specific embodiments, the energy donor molecule is a bioluminescent molecule or a fluorescent molecule and the energy acceptor molecule is a fluorescent acceptor molecule.

[0488] In other embodiments, the construct system of the present invention comprises a first construct comprising a regulatory sequence operably linked to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2) and a nucleic acid sequence encoding a proximal signal or energy donor molecule; and a second construct comprising a regulatory sequence operably linked to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a RAGE polypeptide and a nucleic acid sequence encoding a proximal signal or energy acceptor molecule. In specific embodiments, the energy donor molecule is a bioluminescent molecule or a fluorescent molecule and the energy acceptor molecule is a fluorescent acceptor molecule.

[0489] In other embodiments, the construct system of the present invention comprises a first construct comprising a regulatory sequence operably linked to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a co-localized GPCR, such as an angiotensin receptor (such as AT1R) or a chemokine receptor (such as CCR2); and a second construct comprising a regulatory sequence operably linked to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a RAGE polypeptide, wherein the RAGE polypeptide has deleted one or more extracellular domains of the native sequence. Methods for modulating RAGE ligand-independent RAGE activation

[0490] In a related aspect, the present invention provides methods for modulating RAGE ligand-independent RAGE activation in an animal or animal-derived cell or tissue (which may or may not be human or human-derived) via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1 R) or a chemokine receptor (e.g., CCR2). Methods for specifically modulating RAGE ligand-independent RAGE activation

[0491] In another related aspect, the present invention provides methods for specifically modulating RAGE ligand-independent RAGE activation and subsequent downstream signaling pathways in cells via a co-localized activating GPCR, such as angiotensin receptors (e.g., AT1 R) or a chemokine receptor (e.g., CCR2). These methods include truncating or mutating RAGE so that it is unable to bind a RAGE ligand to its extracellular domain, or such that binding of a RAGE ligand to its extracellular domain is impaired by exposing the cell to a modulator that modulates the binding of a RAGE ligand to RAGE.

[0492] In a preferred form of the invention, modulation of a RAGE ligand-independent signaling pathway is distinct from and / or more pronounced than modulation of a RAGE ligand-dependent signaling pathway.

[0493] In a particularly preferred form of the invention, the inhibition of the RAGE ligand-independent signaling pathway is distinct from and / or more pronounced than the inhibition of the RAGE ligand-dependent signaling pathway. Methods for modulating both RAGE ligand-dependent and RAGE ligand-independent RAGE activation

[0494] In another related aspect, the present invention provides methods for inhibiting RAGE ligand-dependent RAGE activation and subsequent downstream signaling pathways by RAGE ligands (including AGE-modified proteins, lipids or DNA, S100 calgranulin protein family members, HMGB1, amyloid and Mac-1), in addition to modulating RAGE ligand-independent RAGE activation by certain co-localized activating GPCRs in cells, tissues or animals.

[0495] In one aspect of the invention, these methods comprise the use of a modulator as described herein, including a fragment, analog, or derivative of the RAGE cytoplasmic tail, to take over the RAGE cytoplasmic tail in a binding interaction and thereby prevent activation of RAGE ligand-dependent RAGE activation and RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs. In one aspect of the invention, RAGE-dependent signaling is impaired by exposing cells to an inhibitor that inhibits binding of a signaling element to the RAGE cytoplasmic tail, resulting in inhibition of RAGE ligand-mediated RAGE activation and RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs.

[0496] In one aspect of the invention, these methods include using a modulator as described herein, including a fragment, analog or derivative of the RAGE transmembrane domain, to replace the RAGE transmembrane domain and to prevent activation of RAGE ligand-dependent RAGE activation and RAGE ligand-independent RAGE activation by certain co-located activating GPCRs. In one aspect of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or a portion thereof and the RAGE extracellular domain. In one aspect of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or a portion thereof and the RAGE cytoplasmic tail. In one aspect of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or a portion thereof and the RAGE extracellular domain and a fragment of the RAGE cytoplasmic tail.

[0497] In one aspect of the invention, a modulator of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs comprises a fragment of the ligand-binding extracellular domain of RAGE that is no more than 40, no more than 20, no more than 10, or no more than 5 amino acids in length.

[0498] In one aspect, inhibition of RAGE ligand-dependent RAGE activation occurs simultaneously with inhibition of RAGE ligand-independent RAGE activation of a co-localized activated GPCR.

[0499] In one aspect, these methods comprise silencing, truncating, modifying or mutating RAGE such that RAGE or an analog, fragment or derivative thereof is a non-functional substitute for the cytoplasmic tail of wild-type RAGE or a portion thereof, which non-functional substitute is incapable of being activated by a RAGE ligand-dependent pathway or a RAGE ligand-independent pathway (such as the S391A-RAGE mutation) or is incapable of promoting downstream RAGE-dependent signaling, and thereby inhibiting signaling via the RAGE cytoplasmic tail and RAGE-dependent signaling.

[0500] In one aspect, these methods include silencing, truncating, modifying or mutating RAGE so that RAGE or its analogs, fragments or derivatives are non-functional substitutes for the transmembrane domain of wild-type RAGE or its portion thereof, and the non-functional substitutes cannot be activated by RAGE ligand-dependent pathways or RAGE ligand-independent pathways or cannot promote downstream RAGE-dependent signal transduction, and thereby inhibit the signal transduction and RAGE-dependent signal transduction that occur by means of the RAGE cytoplasmic tail. In one aspect of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or its portion and the RAGE extracellular domain. In one aspect of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or its portion and the RAGE cytoplasmic tail. In one aspect of the invention, the modulator comprises a fragment of the RAGE transmembrane domain or its portion and the RAGE extracellular domain and a fragment of the RAGE cytoplasmic tail.

[0501] In one aspect, a modulator of RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs comprises a fragment of the ligand-binding extracellular domain of RAGE that is no more than 40, no more than 20, no more than 10, or no more than 5 amino acids in length.

[0502] In one aspect, these methods comprise silencing, truncating, modifying or mutating RAGE such that RAGE or its analogs, fragments or derivatives modulate common elements involved in signal transduction mediated by the cytoplasmic tail of RAGE (e.g., PKCζ, Diaph1, MyD88, TIRAP, NFκB), associated with activation of RAGE via either a RAGE ligand-dependent activation pathway or a RAGE ligand-independent activation pathway.

[0503] In one aspect, these methods comprise modulators that modulate RAGE ligand-independent RAGE activation via a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), in addition to modulators that modulate RAGE ligand-dependent RAGE activation (e.g., via modulators that modulate binding of a RAGE ligand to the extracellular domain of RAGE). Methods for modulating RAGE ligand-independent RAGE activation via certain co-localized activating GPCRs while also modulating RAGE-independent signaling via certain co-localized GPCRs.

[0504] In one aspect, the present invention provides a method for modulating RAGE-independent signaling pathways induced by activation of a corresponding ligand and modulating RAGE ligand-independent RAGE activation via a colocalized activated GPCR.

[0505] In one form, the invention provides a method for modulating RAGE-independent signaling pathways induced by activation of a corresponding ligand by a colocalized GPCR, while simultaneously modulating RAGE ligand-independent RAGE activation by a colocalized activated GPCR.

[0506] In one form, the RAGE-independent co-localized GPCR signaling pathway induced by activation of the corresponding ligand is the Gq signaling pathway, such as for AT1R activated by Ang II. In another form, the RAGE-independent co-localized GPCR signaling pathway is the Gi signaling pathway, such as for CCR2 activated by MCP-1. In another form, the RAGE-independent co-localized GPCR signaling pathway is β-arrestin-mediated extracellular regulated kinase (ERK) signaling. In another form, the RAGE-independent co-localized GPCR signaling pathway is a change in intracellular signaling intermediates (such as phosphoinositides or calcium). BRIEF DESCRIPTION OF THE DRAWINGS Example 1.

[0507] Figure 1A Quantification of plaque area in male apoE KO mice and AGER / apoE double knockout (DKO) mice four weeks after infusion of Ang II (1 μg / kg / min) or vehicle control, expressed as the percentage of aortic arch surface area positive for Sudan IV staining.

[0508] Figure 1B Expression of pro-atherogenic mediators, including AGER itself, adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines (TNFα, MCP-1, and IL-6), and macrophage markers (Mac-1 / Cd11b), in aortic homogenates from apoE KO mice and AGER / apoE DKO mice four weeks after infusion of Ang II (1 μg / kg / min) or vehicle control, as measured by real-time RT-PCR.

[0509] Figure 1C Markers of oxidative stress in apoE KO mice and AGER / apoEDKO mice after 4 weeks of infusion of Ang II (1 μg / kg / min) or vehicle control, as assessed by: (i) plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, and (ii) induction of gene expression of the NADPH oxidase subunits NOX-1 and NOX-4 in the aorta of apoE KO mice and AGER / apoE DKO mice, as assessed by real-time RT-PCR in aortic homogenates.

[0510] Figure 1D Expression of RAGE ligands in apoE KO mice and AGER / apoE DKO mice 4 weeks after infusion of Ang II (1 μg / kg / min) or vehicle, including (i) circulating plasma levels of S100A8 / A9 as measured by commercial ELISA, (ii) plasma AGE levels as measured by our own ELISA, and (iii) circulating AGE-precursor (methylglyoxal) levels as measured by HPLC.

[0511] Figure 1E Systolic blood pressure as measured by tail-cuff plethysmography in apoE KO mice and AGER / apoEDKO mice 4 weeks after infusion of Ang II (1 μg / kg / min) or vehicle control.

[0512] Data are mean ± SEM; n = 8 per group, * vs control apoE KO mice, # vs apoE KO + Ang II, p < 0.05. Example 2.

[0513] Figure 2A Quantitative plaque area in apoE KO mice and AGER / apoE DKO mice after six weeks of a 0.05% (low) sodium diet or normal chow, expressed as the percentage of aortic arch surface area that stained positively for Sudan IV.

[0514] Figure 2B Expression of pro-atherogenic mediators, including AGER itself, adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines (TNFα, MCP-1, and IL-6), and macrophage markers (Mac-1 / Cd11b), in aortic homogenates of apoE KO mice and AGER / apoE DKO mice after six weeks of a 0.05% (low) sodium diet or normal chow, as measured by real-time RT-PCR.

[0515] Figure 2C Markers of oxidative stress in apoE KO mice and AGER / apoE DKO mice after six weeks of a 0.05% (low) sodium diet or normal chow, as assessed by: (i) plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, and (ii) induction of gene expression of the NADPH oxidase subunits NOX-1 and NOX-4 in the aorta of apoE KO mice and AGER / apoE DKO mice, as assessed by real-time RT-PCR in aortic homogenates.

[0516] Figure 2D Circulating levels of soluble MCP-1 and ICAM-1 as measured by ELISA in apoE KO mice and AGER / apoE DKO mice exposed to a 0.05% (low) sodium diet or normal chow for six weeks.

[0517] Figure 2E The number of labeled leukocytes adhering to the aortic surface of apoE KO mice and AGER / apoE DKO mice ex vivo, as measured by dynamic flowmetry, before and after 1 week of exposure to a low sodium diet or normal chow.

[0518] Figure 2F Expression of RAGE ligands in apoE KO mice and AGER / apoE DKO mice after six weeks of a 0.05% (low) sodium diet or normal chow, including (i) circulating plasma levels of S100A8 / A9 as measured by commercial ELISA, (ii) plasma AGE levels as measured by our own ELISA, and (iii) circulating AGE-precursor (methylglyoxal) levels as measured by HPLC.

[0519] Figure 2G Systolic blood pressure as measured by tail-cuff plethysmography in apoE KO mice and AGER / apoE DKO mice after six weeks of a 0.05% (low) sodium diet or normal chow.

[0520] Figure 2H Markers of RAAS activation, as measured by radioimmunoassay, included (i) decreased sodium excretion, (ii) increased plasma renin activity, and (iii) increased plasma aldosterone levels in apoE KO mice and AGER / apoE DKO mice after six weeks of a 0.05% (low) sodium diet or normal chow.

[0521] Data are mean ± SEM; n = 8 per group, *vs apoE KO mice fed a normal diet, p < 0.05. #vs apoE KO mice + low sodium. Example 3.

[0522] Figure 3A Circulating Ang II concentrations in apoE KO mice and AGER / apoE KO mice with or without genetic Ace2 deficiency as measured by radioimmunoassay.

[0523] Figure 3B Systolic blood pressure as measured by tail-cuff plethysmography in 18-week-old apoE KO mice and AGER / apoE KO mice with or without genetic Ace2 deficiency.

[0524] Figure 3C Quantified plaque area in 18-week-old apoE KO mice and AGER / apoE KO mice with or without genetic Ace2 deficiency, expressed as the percentage of aortic arch surface area positive for Sudan IV staining.

[0525] Figure 3D Aortic expression of pro-atherosclerotic mediators, including AGER itself, adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines (TNFα, MCP-1, and IL-6), and macrophage markers (Mac-1 / Cd11b), as measured by real-time RT-PCR, in aortic homogenates of apoE KO mice in the presence or absence of Ace2 and / or RAGE.

[0526] Figure 3E Oxidative stress in 18-week-old apoE KO mice, Ace2 / apoE DKO mice, AGER / apoE DKO, and Ace2 / AGER / apoE triple KO (TKO) mice, as estimated from plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage.

[0527] Figure 3F Expression of RAGE ligands in 18-week-old apoE KO mice, Ace2 / apoE DKO mice, AGER / apoE DKO and Ace2 / AGER / apoE TKO mice, including (i) circulating plasma levels of S100A8 / A9 as measured by commercial ELISA, (ii) plasma AGE levels as measured by our own ELISA.

[0528] Data are mean ± SEM; n = 8 per group, * vs apoE KO control; # vs Ace2 / apoE DKO mice. Example 4.

[0529] Figure 4A Aortic expression of pro-atherosclerotic mediators in apoE KO mice and AGER / apoE DKO, including AGER itself, adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines (TNFα, MCP-1, and IL-6), and macrophage markers (Mac-1 / Cd11b), as measured by real-time RT-PCR in aortic homogenates after ex vivo exposure to Ang II or vehicle. Data are mean ± SEM; n = 6 per group, * vs untreated apoE KO control; # vs apoE KO + Ang II; p < 0.05

[0530] Figure 4B The number of labeled leukocytes adhering to the aorta surface of apoE KO mice and AGER / apoE DKO mice as a marker of endothelial activation after four hours of ex vivo exposure to Ang II (1 μM) or vehicle control, as measured by dynamic flowmetry. Data are mean ± SEM; n = 6 per group, * vs untreated apoE KO control; # AGER / apoE DKO + Ang II vs apoE KO + Ang II; p < 0.05

[0531] Figure 4C Number of labeled THP-1 monocytes adherent to primary murine aortic endothelial cell (PMAEC) monolayers from C57b16 or AGER KO mice in the presence or absence of Ang II (1 μM for 2 h) pretreatment.

[0532] Figure 4D Expression of pro-atherogenic mediators, including AGER itself, key adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines (TNFα and MCP-1), in murine primary aortic endothelial cells (PMAEC) from C57b16 and PMAEC from AGER KO mice after exposure to Ang II (1 μM) or vehicle control, as measured by real-time RT-PCR.

[0533] Figure 4E Markers of oxidative stress in PMAECs from c57bl6 mice and AGER KO mice after exposure to Ang II or vehicle control, as estimated by: (i) induction of DCFH fluorescence in a flow chamber assay, and (ii) levels of the GTP-activated NADPH oxidase subunit (Rac-1) and (iii) oxidized glutathione.

[0534] Figure 4F Gene expression of VCAM-1 and markers of non-canonical and canonical signaling via NFκB (CXCL12 and CXCL2, respectively) in primary murine aortic endothelial cells (PMAEC) monolayers from C57b16 and AGER KO mice following exposure to Ang II. PMAEC as measured by real-time RT-PCR. TNFα is shown as a canonical specificity control. VCAM-1 is shown as a target specificity control. Repeat Figure 4D The data in .

[0535] Figure 4GExpression of pro-atherogenic mediators, including adhesion molecules (ICAM-1, VCAM-1) and inflammatory cytokines and chemokines (TNFα and MCP-1), in murine aortic primary endothelial cells (PMAECs) from C57b16 mice and AGER KO mice treated with the RAGE ligand S100A8 / A9 (5 ng / mL) as measured by real-time RT-PCR. Data are mean ± SEM; n = 6 per group, symbols indicate * vs untreated wild-type PMAEC; # vs S100A8 / A9-treated wild-type PMAEC, p < 0.05.

[0536] Figure 4H Gene expression of the key adhesion protein VCAM-1, as estimated by real-time RT-PCR, in PMAEC monolayers in which expression of RAGE or the NFκB subunit p65 had been selectively silenced using siRNA or was unchanged (scrambled RNA control) and subsequently exposed to Ang II (1 μM) or the RAGE ligands S100A8 / A9 (5 ng / mL).

[0537] Figure 4I Markers of Gq-mediated signaling induced upon AT1R activation by Ang II (1 μM) in PMAEC monolayers from c57bl6 and AGER KO mice, including (i) induction of phosphoinositide synthesis as assessed by IP-1 and (ii) downstream induction of early growth response genes (EGR1).

[0538] Data are mean ± SEM; n = 6 per group, *vs untreated wild-type control PMAEC and #vs Ang II-treated wild-type controls, p < 0.05 unless otherwise stated. Example 5.

[0539] Figure 5A Induction of phosphoinositide synthesis in response to Ang II (1 μM) in CHO cells as assessed by IP-1 levels, a marker of classical responsiveness to exogenous Ang II, in the presence or absence of human AT1R expression, with or without additional expression of full-length human RAGE.

[0540] Figure 5B Induction of EGR1 expression in CHO cells in response to Ang II (1 μM) as assessed by downstream induction of the EGR1 gene, a marker of responsiveness to exogenous Ang II, in the presence or absence of human AT1R expression, with or without additional expression of full-length human RAGE.

[0541] Figure 5CAs a control for the integrity of RAGE signaling in CHO cells, NFκB activation after exposure to Ang II (1 μM) was measured by: (i) chemiluminescent SEAP reporter gene assay and (ii) induction of gene expression of the NFκB subunit p65 in the presence or absence of human AT1R expression in CHO cells with or without additional expression of full-length human RAGE, and (iii) after exposure to the RAGE ligand S100A8 / A9 (5 ng / mL).

[0542] Figure 5D NFκB activation in AT1R-CHO cells following exposure to Ang II (1 μM) was measured by induction of gene expression of the NFκB subunit p65 and a chemiluminescent SEAP reporter assay of NFκB activity in the presence or absence of expression of full-length human RAGE and N-truncated mCherry-RAGE constructs. Data are mean ± SEM, n = 6 per group, * vs. vector (neo)-transfected AT1R-CHO, p < 0.05.

[0543] Figure 5E NFκB activation in AT1R-CHO cells after exposure to Ang II (1 μM) was assessed by induction of gene expression of the NFκB subunit p65 and a chemiluminescent SEAP reporter assay of NFκB activity in the presence or absence of expression of full-length human RAGE and C-truncated mCherry-RAGE constructs. Data are mean ± SEM, n = 6 per group, * vs. vector (neo)-transfected AT1R-CHO, p < 0.05.

[0544] Figure 5F NFκB activation in AT1R-CHO cells following exposure to S100A8 / A9 (5 ng / mL) or Ang II (1 μM) as estimated by induction of gene expression of the NFκB subunit p65 in the presence or absence of expression of full-length human RAGE and N-truncated mCherry-RAGE constructs. Data are mean ± SEM, n = 6 per group, * vs. vector (neo)-transfected AT1R-CHO, p < 0.05.

[0545] Figure 5G NFκB activation in AT1R-CHO cells following exposure to S100A8 / A9 (5 ng / mL) or Ang II (1 μM) as estimated by induction of gene expression of the NFκB subunit p65 in the presence or absence of expression of full-length human RAGE and C-truncated mCherry-RAGE constructs. Data are mean ± SEM, n = 6 per group, * vs. vector (neo)-transfected AT1R-CHO, p < 0.05.

[0546] Figure 5H NFκB activation in AT1R-CHO cells after exposure to Ang II (1 μM) was assessed by induction of gene expression of the NFκB subunit p65 in the presence of expression of an N-truncated RAGE construct not fused to mCherry.

[0547] Data are mean ± SEM, n = 6 per group, unless otherwise stated, * vs untreated AT1R-CHO, p < 0.05. Example 6.

[0548] Figure 6A RAGE neutralizing antibodies (RAGEab) targeting the extracellular domain of RAGE or decoy receptors with ligand binding affinity (soluble RAGE 22-331 ) inhibited the induction of proinflammatory signaling by the RAGE ligand S100A8 / A9, but not by Ang II (1 μM), in RAGE-AT1R-CHO cells, as estimated by expression of the NFκB subunit p65 measured by RT-PCR. Data are shown as mean ± SEM; n = 6 per group. *vs vehicle only, p < 0.05.

[0549] Figure 6B RAGE neutralizing antibodies (RAGEab) targeting the extracellular domain of RAGE or decoy receptors with ligand binding affinity (soluble RAGE 22-331 sRAGE) did not inhibit the induction of proinflammatory signaling by Ang II (1 μM) in PMAECs from wild-type mice, as estimated by induction of key adhesion genes (ICAM-1 and VCAM-1) and inflammatory chemokine genes (MCP-1). Data from AGER KO mice were shown as a negative control. Data are shown as mean ± SEM; n = 6 per group, * vs. vehicle-only control cells (white bars), # vs. Ang II-only control cells, p < 0.05.

[0550] Figure 6C RAGE neutralizing antibodies (RAGEab) targeting the extracellular domain of RAGE or decoy receptors with ligand binding affinity (soluble RAGE 22-331 ) inhibits the induction of proinflammatory signaling by the RAGE ligand S100A8 / A9 in PMAECs from wild-type mice, as estimated by induction of a key adhesion gene (ICAM-1). Data are shown as mean ± SEM; n = 6 per group, * vs. vehicle-treated control cells (white bars), # vs. S100A8 / A9-treated control cells, p < 0.05. Example 7.

[0551] Figure 7A .Also expresses full-length wild-type RAGE 22-404 or the selected S391-RAGE 22-404 RAGE ligands S100A8 / A9 (5 ng / ml; gray bars) or Ang II (1 μM; black bars) induce proinflammatory signaling in mutant AT1R-CHO cells, as measured by induction of gene expression of the NFκB subunit p65. Data are shown as mean ± SEM; n = 6-8 per group. *p < 0.05 vs. vehicle-treated AT1R-CHO cells expressing full-length RAGE.

[0552] Figure 7B RAGE ligands S100A8 / A9 (5 ng / ml) or Ang II (1 μM) induce proinflammatory signaling in AT1R-CHO cells that also express a chimeric RAGE lacking a phosphorylatable motif other than the S391 cytoplasmic tail (chimeric RAGE; cRAGE) and an S391 cRAGE mutant completely lacking any phosphorylatable motif in the cytoplasmic tail, as measured by induction of gene expression of the NFκB subunit p65 in AT1R-CHO cells. Data are shown as mean ± SEM; n = 6-8 per group. *vs vehicle-treated AT1R-CHO cells expressing full-length chimeric RAGE; p < 0.05.

[0553] Figure 7C Ang II (1 μM) induces proinflammatory signaling in AT1R-CHO cells expressing either full-length or the N-truncated S391A-RAGE mutant, as measured by induction of gene expression of the NFκB subunit p65. Data are shown as mean ± SEM; n = 6-8 per group. *vs vector-transfected AT1R-CHO cells, p < 0.05.

[0554] Figure 7D In wild-type mCherry-RAGE 362-404 In the presence of S391-RAGE 362-404 Ang II (1 μM) induces proinflammatory signaling in mutant AT1R-CHO cells, as measured by induction of gene expression of the NFκB subunit p65. Data are shown as mean ± SEM; n = 6-8 per group. *vs vehicle control; p < 0.05. Example 8.

[0555] Figure 8AEffect of selective inhibition of MyD88 expression using siRNA or a scrambled control in PMAEC monolayers from C57b16 mice on the induction of RAGE ligand-dependent proinflammatory signaling by the RAGE ligand S100A8 / A9 (5 ng / ml), as assessed by ICAM-1 expression measured by real-time RT-PCR. As a positive control, selective inhibition of p65 expression (another downstream mediator of RAGE signaling) using siRNA is shown.

[0556] Figure 8B Effect of selective inhibition of MyD88 expression in PMAEC monolayers from C57b16 mice using siRNA or a scrambled control on Ang II (1 μM)-induced RAGE ligand-independent induction of proinflammatory signaling, as assessed by ICAM-1 expression measured by real-time RT-PCR.

[0557] Figure 8C In RAGE 362-404 Effects of selective inhibition of MyD88 expression in HMEC monolayers using siRNA or scrambled control in the presence and absence of Ang II (1 μM) on RAGE ligand-independent induction of proinflammatory signaling as assessed by MCP-1 expression measured by real-time RT-PCR.

[0558] Data are mean ± SEM; n = 6-8 per group, *vs scrambled control, p < 0.05. Example 9.

[0559] Figure 9A Effects of selective inhibition of PKCζ using a pseudosubstrate for PKCζ (iPKCz) or siRNA targeting PKCζζ expression (sipKCz) or siRNA targeting RAGE expression (siRAGE) or a scrambled control in PMAEC monolayers from C57bl6 mice on RAGE ligand-dependent signaling induced by the RAGE ligand S100A8 / A9, as assessed by ICAM-1 expression measured by real-time RT-PCR.

[0560] Figure 9B Effect of selective inhibition of PKCζζ expression using a pseudosubstrate for PKCζ (iPKCζ) or siRNA targeting PKCζ (siPKCζ) in PMAEC monolayers from C57b16 mice on RAGE ligand-independent induction of proinflammatory signaling by Ang II (1 μM), as assessed by real-time RT-PCR measurement of ICAM-1 gene expression. Columns 1 and 2 contain scrambled siRNA controls.

[0561] Figure 9C.The effect of PKCζζ expression on RAGE ligand-independent p65 and PCNA induction by Ang II (1 μM) was selectively inhibited using a pseudosubstrate of PKCζ (PKCζi) in CHO cells expressing a chimeric RAGE lacking the phosphorylatable motif in the non-S391 cytoplasmic tail (cRAGE) and cRAGE also containing the S391Q-RAGE mutation (S319Q-cRAGE), thereby removing all phosphorylation sites in the cytoplasmic tail, as estimated by RelA / p65 and PCNA gene expression measured by real-time RT-PCR.

[0562] Figure 9D In RAGE 362-404 Effects of selective inhibition of PKCζ expression using siRNA or scrambled control in HMEC monolayers in the presence and absence of Ang II (1 μM) on RAGE ligand-independent induction of proinflammatory signaling as assessed by MCP-1 expression measured by real-time RT-PCR.

[0563] Data are mean ± SEM; n = 6-8 per group, *vs untreated control, p < 0.05. Example 10.

[0564] Figure 10A Effect of selective inhibition of Diaph1 expression using siRNA in PMAEC monolayers from C57b16 mice on RAGE ligand-dependent signaling induced by the RAGE ligand S100A8 / A9, as assessed by real-time RT-PCR measurement of ICAM-1 expression. Data using siRAGE were included as a control.

[0565] Figure 10B The R366A-Q367A-RAGE mutation in AT1R-CHO cells differentially affects RAGE ligand S100A8 / A9-induced signaling and Ang II-induced RAGE ligand-independent signaling. The mutation selectively disrupts the charged cluster through which Diaph1 and RAGE putatively interact.

[0566] Figure 10C Effects of selective inhibition of Diaph1 expression using siRNA in PMAEC monolayers from C57b16 mice on AngII (1 μM)-induced RAGE ligand-dependent signaling, as estimated by ICAM-1 and VCAM-1 expression as measured by real-time RT-PCR. Data are mean ± SEM; n = 6 per group, *vs scrambled control, p < 0.05.

[0567] Figure 10DEffects of selective knockdown of Diaph1 or AGER expression using siRNA on leukocyte adhesion to endothelial cell monolayers after exposure to Ang II in SVEC monolayers compared to scrambled controls. Data are mean ± SEM; n = 6-8 per group. *vs scrambled controls, p < 0.05.

[0568] Figure 10E Effects of transfection of AT1R-CHO cells with the R366A-Q367A-RAGE mutant, in which the charged clusters through which Diaph1 and RAGE putatively interact are disrupted or deleted, on Ang II-induced signaling, as measured by induction of gene expression of the NFκB subunit p65. Data are mean ± SEM; n = 6 per group, *vs mCherry control, p < 0.05.

[0569] Figure 10F In RAGE 362-404 Effects of selective inhibition of Diaph1 expression using siRNA or scrambled control on RAGE ligand-independent proinflammatory signaling induced by Ang II (1 μM) in HMEC monolayers in the presence and absence of Ang II, as estimated by MCP-1 expression measured by real-time RT-PCR. Data are mean ± SEM; n = 6-8 per group, * vs. scrambled siRNA-treated vehicle control, p < 0.05.

[0570] Figure 10G .Inhibitory peptide S391A-RAGE 362-404 Effects of transfection with full-length RAGE, truncated RAGE, or RAGE mutants in pretreated AT1R-CHO cells on Ang II-induced signaling, as measured by induction of gene expression of the NFκB subunit p65. Data are mean ± SEM; n = 6-8 per group, *vs mCherry control, p < 0.05

[0571] Unless otherwise stated, data are mean ± SEM; n = 6-8 per group, * vs vehicle control, p < 0.05. Example 11.

[0572] Figure 11A Effect of selective inhibition of IQGAP-1 expression using siRNA targeting IQGAP-1 on Ang II (1 μM)-induced RAGE ligand-independent induction of proinflammatory signaling in PMAEC monolayers from C57bl6 mice compared to scrambled controls, as estimated by ICAM-1 expression measured by real-time RT-PCR.

[0573] Figure 11BEffects of selective inhibition of IQGAP-1 expression using siRNA on RAGE ligand S100A8 / A9-induced RAGE ligand-dependent proinflammatory signaling in PMAEC monolayers from C57bl6 mice compared to scrambled controls, as assessed by real-time RT-PCR measurement of ICAM-1 and VCAM-1 expression.

[0574] Figure 11C Using mutant RAGE cytoplasmic tail (S391A-RAGE 362-404 )-coated columns, along with IQGAP-1-related proteins (ezrin / radixin / moesin) and the GPCR olfactory receptor 2T2, pulled down proteins identified as IQGAP-1 from other cytosolic fractions.

[0575] Figure 11D In RAGE 362-404 Effects of selective inhibition of IQGAP-1 expression using siRNA or scrambled control in HMEC monolayers in the presence and absence of Ang II (1 μM) on RAGE ligand-independent induction of proinflammatory signaling as assessed by real-time RT-PCR measurement of MCP-1 expression.

[0576] Data are mean ± SEM; n = 6-8 per group, * vs scrambled control, p < 0.05, # vs scrambled control + ligand (Ang II or s100A8 / A9 as needed), p < 0.05. Example 12.

[0577] Figure 12A Effect of transfection of murine SVEC with RAGE or RAGE mutants on Ang II induction of ICAM-1 compared to vector only (pc-Neo) control, as measured by RT-PCR. Data are mean ± SEM; n = 6-8 per group. * vs untreated control, # vs neo + Ang II, p < 0.05.

[0578] Figure 12B Effects of transfection with truncated RAGE mutants on Ang II-induced ICAM-1 in murine SVECs to identify the minimal fragment with inhibitory activity. Data are mean ± SEM; n = 6-8 per group, * vs untreated control, p < 0.05.

[0579] Figure 12CInhibition of NFκB activation in RAGE-AT1R-CHO cells after exposure to Ang II (1 μM), as estimated by induction of gene expression of the NFκB subunit p65 in the presence of mutant and N-truncated RAGE constructs not fused to mCherry. Data are mean ± SEM; n = 6-8 per group, *vs untreated control, p < 0.05.

[0580] Figure 12D .Use RAGE 370-390 Effects of transfection with single site-specific alanine or lysine mutants of α-glucose on Ang II-induced ICAM-1 in murine SVECs. Data are mean ± SEM; n = 6-8 per group, *vs untreated control, p < 0.05.

[0581] Figure 12E In RAGE 379-390 and sequence homology between Streptomyces anti-inflammatory proteins and proteins from other microorganisms. Example 13.

[0582] Figure 13A Compared with TAT-Cherry alone (8 μg), TAT-mCherry-RAGE without or with S391A-RAGE mutation in AT1R-CHO cells 362-404 Effect of Ang II (1 μM; black bar) (0.4 ng / ml) on NFκB subunit p65 gene expression induced by Ang II (1 μM). Data are mean ± SEM; n = 6-8 per group, *vs untreated control, p < 0.05.

[0583] Figure 13B Wild-type RAGE 362-404 Peptide pretreatment did not reverse the expression of S391A-RAGE in AT1R-CHO cells 362-404 Peptide-mediated signaling inhibition via Ang II-dependent induction of NFκB subunit p65 gene expression. Data are mean ± SEM; n = 6-8 per group, * vs. untreated control; p < 0.05.

[0584] Figure 13C . It was observed that S391A-RAGE 362-404 The peptide inhibited signaling by inducing Ang II-dependent gene expression of the NFκB subunit p65, regardless of whether it was followed by a thousand-fold excess of wild-type RAGE. 362-404 Data are mean ± SEM; n = 6-8 per group, *vs untreated control, p < 0.05.

[0585] Figure 13DIn AT1R-CHO cells transfected with full-length S391Q-cRAGE without available phosphorylation targets, S391A-RAGE 362-404 Inhibitory effects of peptides on the induction of p65 and PCNA gene expression in response to Ang II. Data are mean ± SEM; n = 6-8 per group, * vs Ang II, # vs full-length wild-type RAGE; p < 0.05.

[0586] Figure 13E Wild-type RAGE 362-404 Effects of peptide (0.4 ng / ml) on the induction of pro-inflammatory gene expression in response to Ang II in RAGE-deficient PMAECs. Data are mean ± SEM; n = 6-8 per group, *vs no treatment control, p < 0.05.

[0587] Figure 13F .S391A-RAGE 362-404 Effects of peptides on the induction of proinflammatory VCAM-1, CXCL2, and CXCL12 gene expression in PMAECs in response to Ang II, using the response to TNFα as a control. Data are mean ± SEM; n = 6-8 per group, * vs vehicle and control (TAT) treatment, # vs Ang II and control (TAT) treatment; p < 0.05.

[0588] Figure 13G .S391A-RAGE 362-404 Effects of peptides and the AT1R blocker irbesartan on proinflammatory gene expression in HAECs in response to Ang II.

[0589] Figure 13H (i) S391A-RAGE 362-404 The inhibitory effect of the peptide on the induction of proinflammatory gene expression (p65) in response to the RAGE ligand S100A8 / A9 in AT1R-CHO cells that also express full-length RAGE, and AT1R-CHO cells expressing the inactive full-length S391A-RAGE mutant as a control are also shown. (ii) S391A-RAGE 362-404 Inhibitory effects of peptides on the induction of pro-inflammatory gene expression (VCAM-1) in response to Ang II or the RAGE ligands S100A8 / A9 in endogenously RAGE-rich PMAECs.

[0590] Unless otherwise stated, data are mean ± SEM; n = 6-8 per group, * vs vehicle control, p < 0.05; # vs control + Ang II, p < 0.05. Example 14.

[0591] Figure 14AAfter ex vivo exposure of intact aorta from apoE KO mice to Ang II (1 μM), S391A-RAGE 362-404 Effects on Ang II-dependent induction of proinflammatory markers. Data are mean ± SEM; n = 6 per group, * vs apoE KO + vehicle + TAT-mCherry control, p < 0.05; # vs apoE KO + TAT-mCherry control + Ang II, p < 0.05.

[0592] Figure 14B After intact aorta from AGER / apoE KO mice was exposed to Ang II (1 μM) in vitro, wild-type RAGE 362-404 Effects on Ang II-dependent induction of proinflammatory markers. Data are mean ± SEM; n = 8 per group, *vsapoE KO + vehicle + TAT-mCherry control, p < 0.05. Example 15.

[0593] Figure 15A TAT-mCherry-RAGE, containing the C-terminal 42 amino acids of RAGE, was labeled with mCherry fluorescent protein and HIV-TAT motif to promote cell penetration in apoE KO mice and Ace2 / AGER / apoE triple KO mice. 362-404 Pro-atherogenic effects on AngII-dependent induced aortic atherosclerosis. 362-404 Comparison of the anti-atherosclerotic effects of Ang II-dependent aortic atherosclerosis in Ace2 / apoE DKO mice. Data are mean ± SEM; n = 8 per group; * vs apoE KO control; # vs Ace2 / apoE DKO control; p < 0.05.

[0594] Figure 15B TAT-mCherry-RAGE, containing the C-terminal 42 amino acids of RAGE, tagged with mCherry fluorescent protein and HIV-TAT motif to facilitate cell penetration, was used in diabetic apoE KO mice and diabetic AGER / apoE DKO mice. 362-404 The pro-atherogenic effect of the peptide on Ang II-dependent induced aortic atherosclerosis. 362-404Comparison of the antiatherosclerotic effects of Ang II-dependent aortic atherosclerosis in diabetic apoE KO mice. Data are mean ± SEM; n = 8 per group; * vs apoE KO controls, # vs diabetic apoE DKO controls; p < 0.05.

[0595] Figure 15C As indicated, TAT-mCherry-RAGE 362-404 and TAT-mCherry-S391A-RAGE 362-404 There was a lack of effect on systolic blood pressure in mice with or without AGER expression and in diabetic apoE KO mice. Example 16.

[0596] Figure 16A In the presence or absence of soluble RAGE 22-331 BRET saturation curves for AT1 / Rluc8 and RAGE / Venu generated 60 minutes after addition of Ang II or vehicle in the presence of sRAGE. Data were pooled from three independent experiments.

[0597] Figure 16B Ang II-induced recruitment of β-arrestin2 / Venus to AT1 / Rluc8 and CCL22-induced recruitment of β-arrestin2 / Venus to CCR4 / Rluc8 served as controls.

[0598] Figure 16C Ang II-induced recruitment of β-arrestin2 / Venu was close to that of RAGE / Rluc8 following exposure to Ang II in the presence of AT1 receptors, and was absent following exposure to CCL22 in the presence of CCR4.

[0599] Figure 16D When Gαi / Nluc and Gγ2 / Venu were co-expressed in the presence of untagged CCR4, a CCL22-induced BRET signal was observed. Example 17.

[0600] Figure 17A In the presence of the AT1 receptor, Ang II-induced recruitment of β-arrestin2 / Venus (β-arr2 / Venus) was close to RAGE / Rluc8, but in the absence of the AT1 receptor, this recruitment was absent. Ang II-induced recruitment of β-arrestin2 / Venus to AT1 / Rluc8 was included as a control.

[0601] Figure 17BIn the presence of TRH receptor 1 (TRHR1), thyrotropin-releasing hormone (TRH) induced weak recruitment of β-arrestin2 / Venus (β-arr2 / Vemus) to RAGE / Rluc8, but in the absence of TRH receptor 1 (TRHR1), this recruitment was absent. TRH-induced recruitment of β-arrestin2 / Venus to TRHR1 / Rluc8 was included as a control. The inset shows the same data with an expanded y-axis scale.

[0602] Figure 17C In the presence of orexin receptor 1 (OxR1), orexin A (OxA)-induced recruitment of β-arrestin2 / Venus (β-arr2 / Venus) to RAGE / Rluc8 was close, but in the absence of orexin receptor 1 (OxR1), no such recruitment was observed. OxA-induced recruitment of β-arrestin2 / Vemus to OxR1 / Rluc8 was included as a control.

[0603] Figure 17D In the presence of BDK receptor 2 (BDKR), bradykinin (BDK) induced weak recruitment of β-arrestin2 / Vemus (β-arr2 / Vemus) close to RAGE / Rluc8, but in the absence of BDK receptor 2 (BDKR), no such recruitment was observed. BDK-induced recruitment of β-arrestin2 / Venus to BDKR / Rluc8 was included as a control. The inset shows the same data with an expanded y-axis scale.

[0604] Figure 17E Arginine vasopressin (AVP)-induced recruitment of β-arrestin2 / Venus (β-arr2 / Venus) to RAGE / Rluc8 in the presence of vasopressin receptor 2 (V2R), but not in the absence of V2R. AVP-induced recruitment of β-arrestin2 / Venus to V2R / Rluc8 was included as a control.

[0605] Figure 17F In the presence of CCR2, CCL2 (MCP1)-induced recruitment of β-arrestin2 / Venus (β-arr2 / Venus) was close to RAGE / Rluc8, but in the absence of CCR2, this recruitment was absent. MCP1-induced recruitment of β-arrestin2 / Venus to CCR2 / Rluc8 was included as a control.

[0606] Figure 17GFollowing exposure to MIP1β, CCL4 (MIP1β) induced a particularly weak recruitment of β-arrestin2 / Venus (β-arr2 / Venus) to RAGE / Rluc8 in the presence of CCR5, particularly compared to controls in the absence of CCR5. MIP1β-induced recruitment of β-arrestin2 / Venus to CCR5 / Rluc8 was included as an additional control. The inset shows the same data with an expanded y-axis scale.

[0607] All data are means ± SEM of 3 independent experiments. Example 18.

[0608] Figure 18A In the presence of CCR1, CCL3-induced recruitment of β-arr2 / Venus is close to that of RAGE / Rluc8.

[0609] Figure 18B In the presence of CCR2, CCL2-induced recruitment of β-arr2 / Venus is close to RAGE / Rluc8.

[0610] Figure 18C In the presence of CCR4, CCL22-induced recruitment of β-arr2 / Venus to RAGE / Rluc8 is absent.

[0611] Figure 18D In the presence of CCR5, CCL4-induced recruitment of β-arr2 / Venus to RAGE / Rluc8 is absent.

[0612] Figure 18E In the presence of CCR6, CCL20-induced recruitment of β-arr2 / Venus is close to that of RAGE / Rluc8.

[0613] Figure 18F In the presence of CCR7, CCL19-induced recruitment of β-arr2 / Venus is close to that of RAGE / Rluc8.

[0614] Figure 18G In the presence of CCR10, CCL27-induced recruitment of β-arr2 / Venus to RAGE / Rluc8 is absent.

[0615] Figure 18H In the presence of CXCR1, CXCL8 induced a weak recruitment of β-arr2 / Venus close to RAGE / Rluc8.

[0616] Figure 18I In the presence of CXCR2, CXCL8-induced recruitment of β-arr2 / Venus is close to that of RAGE / Rluc8.

[0617] Figure 18J In the presence of CXCR3, CXCL11-induced recruitment of β-arr2 / Venus to RAGE / Rluc8 is absent.

[0618] Figure 18K In the presence of CXCR4, CXCL12-induced β-arr2 / Venus is similar to the reduction of RAGE / Rluc8.

[0619] Figure 18L In the presence of CXCR6, CXCL16-induced recruitment of β-arr2 / Venus is close to RAGE / Rluc8.

[0620] All data are means ± SEM of 3 independent experiments. Example 19.

[0621] Figure 19A -OO. Rluc8-tagged RAGE is in proximity to Venus-tagged markers of the indicated subcellular compartments in the presence of the indicated non-BRET-tagged GPCR activated by the indicated ligand at the indicated concentrations at time zero. Example 20.

[0622] Figure 20A Activation of NFκB by CCL2 (MCP-1) in CHO cells expressing CCR2 in the presence or absence of RAGE co-expression, as measured by induction of gene expression of the NFκB subunit p65.

[0623] Figure 20B Activation of NFκB by CXCL2 (IL-8) in CHO cells expressing CXCR2 in the presence or absence of RAGE co-expression, as measured by induction of gene expression of the NFκB subunit p65.

[0624] Figure 20C .S391A-RAGE, a peptide inhibitor of RAGE activation 362-404 Activation of NFκB in bone marrow-derived primary macrophages in the presence or absence of CCL2 (MCP-1), as measured by induction of gene expression of the NFκB subunit p65.

[0625] Figure 20D .S391A-RAGE, a peptide inhibitor of RAGE activation 362-404 Activation of NFκB in HMECs in the presence or absence of CCL2 (MCP-1), as measured by autoinduction of MCP-1 gene expression.

[0626] Figure 20E.S391A-RAGE, a peptide inhibitor of RAGE activation 362-404 Activation of NFκB by IL-8 in the presence or absence of CXCR2-expressing HMECs, as measured by induction of MCP-1 gene expression. Example 21

[0627] Figure 21A .mCherry / RAGE 338-361 BRET between AT1 and Nluc / AT1 increased with Ang II. Data are shown as mean ± SEM; n = 3-5.

[0628] Figure 21B BRET saturation curves for AT1 / Rluc8 and RAGE / Venu were generated 60 min after addition of vehicle or Ang II as indicated, where cells were also treated with 200 ng or 400 ng of mCherry / RAGE 338-361 cDNA transfection. Data are combined from three independent experiments.

[0629] Figure 21C Ang II induced modulation of the BRET signal between AT1 / Rluc8 and RAGE / Venu, where cells were also treated with 0, 50, 100, 200, 300, or 400 ng of mCherry / RAGE as indicated. 338-361 cDNA or pcDNA3 control plasmid transfection. Data are presented as mean ± SEM; n = 3-4. Filters: Venus 550nm / Rluc8450nm.

[0630] Figure 21D . Lack of AT1 / Rluc8 and mCherry / RAGE 338-361 Ang II-induced BRET signals were measured in cells in which cells were treated with 50 ng AT1 / Rluc8 cDNA, 300 ng RAGE / Venus cDNA, and 0, 50, 100, 200, 300, or 400 ng mCherry / RAGE cDNA as indicated. 338-361 Transfection with cDNA or pcDNA3 control plasmid. Data are presented as mean ± SEM; n = 3-4. Filters: mCherry 650nm / Rluc 8450nm.

[0631] Figure 21E . Figure 21C and Figure 21D Luminescence from AT1 / Rluc8, fluorescence from RAGE / Venu, and fluorescence from mCherry / RAGE in the experiments shown 338-361 Data are shown as mean ± SEM; n = 3-4.

[0632] Figure 21F Ligand-induced modulation of the BRET signal between GPCR / Rluc8 and RAGE / Venu, where cells were also stained with mCherry / RAGE as indicated. 338-361 cDNA or pcDNA3 control plasmid transfection. Data are presented as mean ± SEM; n = 2-4. Filter: Venus 550nm / Rluc8 450nm. Amount of cDNA transfected: 50ng GPCR / Rluc8 + 300ng RAGE / Venus + 400ng mCherry / RAGE 338-361 or pcDNA3. The indicated Rluc8-tagged GPCRs activated by the indicated ligands at the indicated concentrations at time zero.

[0633] Figure 21G .HMEC1 is expressed by mCherry / RAGE 338-361 Inhibition of AngII-mediated pro-inflammatory signaling (ICAM-1 expression) by mCherry / RAGE 362-404 Data are shown as mean ± SEM; n = 6-8.

[0634] Figure 21H Ang II induction of proinflammatory signaling in HMEC cells was monitored by fusion or nonfusion of the RAGE transmembrane domain expressed with or without N-terminal mCherry. 343-361 Inhibition of overexpression as represented by ICAM1 expression measured using real-time RT-PCR. Data are shown as mean ± SEM; n = 6-8.

[0635] Figure 21I Induction of proinflammatory signaling by RAGE ligand S100A8 / A9 in RAGE-CHO cells is regulated by the RAGE transmembrane domain 343-361 or RAGE 370-390 Overexpression inhibits p65 expression as measured by real-time RT-PCR. Data are presented as mean ± SEM; n = 6-8.

[0636] Figure 21J Ang II induction of proinflammatory signaling in HMEC cells was inhibited by siRNA targeting Diaph1 or PKCz, as represented by ICAM-1 expression measured by real-time RT-PCR. 362-404 Rescue this inhibition, but RAGE 343-404 No rescue. Data are shown as mean ± SEM; n = 6-8.

[0637] Sequence Description Sequence ID number sequence length SEQ ID NO: 1 <![CDATA[RAGE 362-404 Peptide sequence]]> 43aa SEQ ID NO: 2 <![CDATA[S391A-RAGE 362-404 Peptide sequence]]> 43aa SEQ ID NO: 3 <![CDATA[RAGE 338-361 Peptide sequence]]> 24aa SEQ ID NO: 4 HIV TAT cell penetration motif (YGRKKRRQRRR) 11aa SEQ ID NO: 5 <![CDATA[RAGE 370-390 Peptide sequence]]> 21aa SEQ ID NO: 6 <![CDATA[RAGE 379-390 Peptide sequence]]> 12aa SEQ ID NO: 7 <![CDATA[S391A-RAGE 362-391 Peptide sequence]]> 30aa SEQ ID NO: 8 <![CDATA[RAGE 362-390 Peptide sequence]]> 29aa SEQ ID NO: 9 <![CDATA[Q390R RAGE 379-390 Peptide sequence]]> 12aa SEQ ID NO: 10 <![CDATA[Q390K RAGE 379-390 Peptide sequence]]> 12aa SEQ ID NO: 11 <![CDATA[Q379K RAGE 379-390 Peptide sequence]]> 12aa SEQ ID NO: 12 <![CDATA[Q379K Q390K RAGE 379-390 Peptide sequence]]> 12aa SEQ ID NO: 13 <![CDATA[Q379K Q390R RAGE 379-390 Peptide sequence]]> 12aa SEQ ID NO: 14 Full-length polypeptide sequence of wild-type human RAGE. 404aa SEQ ID NO: 15 The full-length polynucleotide sequence of the human AGER gene. 1704nts SEQ ID NOs: 16 to 58 Full-length polypeptide sequence of wild-type human G protein-coupled receptor. Various SEQ ID NO: 59 Wild-type Renilla reniformi luciferase polypeptide sequence. 311aa SEQ ID NO: 60 Cys124Ala / Met185Val variant Renilla luciferase polypeptide sequence. 311aa SEQ ID NO: 61 Variant Renilla luciferase polypeptide sequence (RLuc8). 311aa Detailed Description of the Invention 1. Definition

[0638] Unless otherwise specified, all terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0639] A "receptor heteromer" is defined as "a macromolecular complex composed of at least two (functional) receptor units with biochemical properties that are distinct from those of its individual components" (Ferre et al., 2009).

[0640] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0641] By "about" is meant a value, amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length that varies by up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference value, amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length.

[0642] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when read in the alternative (or).

[0643] The terms "substance", "candidate substance", "modulator", "modulator", "substitute", "functional substitute", "non-functional substitute" or "inhibitor" include chemical compounds, mixtures of chemical compounds, biological macromolecules, extracts made from biological materials, biological organisms or parts thereof, or other materials that cause the desired pharmacological and / or physiological effect. These terms also cover pharmaceutically acceptable components and pharmacologically active components of those compounds specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, etc. When the above terms are used, it should be understood that this includes the active substance itself as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, prodrugs, metabolites and analogs. The terms "substance", "modulator", "substitute" or "inhibitor" are not to be interpreted narrowly, but extend to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins and compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof as well as cellular material. The terms "substance," "modulator," "surrogate," or "inhibitor" include a cell that produces and secretes a polypeptide as described herein, as well as a polynucleotide comprising a nucleotide sequence encoding such a polypeptide. Thus, the terms "substance," "modulator," "surrogate," or "inhibitor" extend to nucleic acid constructs for expression and secretion in a range of cells, including vectors such as viral or non-viral vectors, expression vectors, and plasmids.

[0644] The term "inhibitor" is used in its broadest sense and includes any compound that reduces at least one aspect of the activity, activation, or function of another molecule, including proteins, polypeptides, peptides, antibodies, antibody fragments, macromolecules, or small molecules (less than 10 kDa). For example, an inhibitor can reduce the activity, activation, or function of RAGE and / or a co-localized GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), and / or appropriately reduce RAGE ligand-independent RAGE activation by a co-localized activated GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2). Thus, an "inhibitor of RAGE ligand-independent RAGE activation by a co-localized activated GPCR" refers to a substance that is capable of significantly reducing, inhibiting, antagonizing, blocking, negatively regulating, and / or ameliorating RAGE ligand-independent RAGE activation by a co-localized activated GPCR. The inhibition of RAGE ligand-independent RAGE activation by a certain co-localized activating GPCR by an inhibitor appropriately reduces or inhibits its biological effects, including the production of proinflammatory mediators (including proinflammatory cytokines) by cells or the regulation of other cellular components associated with abnormal RAGE ligand-independent RAGE activation disease symptoms. Note: A partial agonist can act as an inhibitor because, even if it exhibits agonism, it does not produce maximal efficacy. Therefore, by competing for or modulating the agonistic activity of a more effective agonist (such as an endogenous agonist), it can effectively act as an inhibitor of the receptor polypeptide and / or its signaling pathway because it reduces signaling output compared to if it were absent. Therefore, a partial agonist can act as an inhibitor in a therapeutic context. An inhibitor does not necessarily inhibit all aspects of the activity, activation or function of another molecule, and in fact, can inhibit certain aspects while activating other aspects and / or not regulating other aspects. Therefore, an inhibitor can exhibit ligand bias.

[0645] The term "ligand bias" refers to a phenomenon in which different ligand-stabilized receptor states may exist for the same receptor that selectively promote or inhibit activation of different signaling pathways (Mustafa et al., 2010). This phenomenon has been given a number of names, including but not limited to: ligand-biased signaling, ligand-induced biased signaling, agonist exchange of receptor signaling, cell-based functional selectivity, selectivity based on receptor activity state, stimulus exchange, biased agonism, accessory efficacy, and ligand-induced selective signaling (Mustafa et al., 2010). By way of example, this includes the concept that not all agonists activate all signaling pathways normally activated by a reference agonist (which is often an endogenous agonist). Relative to a reference, an agonist may activate certain pathways but not others, thereby exhibiting a bias. Additionally, antagonists or inverse agonists or inhibitors may inhibit only certain pathways but not others, and may act at orthosteric ligand binding sites and / or allosteric binding sites. Orthosteric and allosteric binding sites are defined as known in the art, and allosteric binding sites can occur from one receptor to another across the complex, so that the binding of a ligand to one receptor can lead to allosteric regulation of another receptor in the same macromolecular complex. Allosteric modulators can also show ligand bias and regulate certain signal transduction pathways, but do not regulate other signal transduction pathways. It is also known in the art that a ligand can, for example, potentially act as an agonist of one signal transduction pathway while acting as an inhibitor of another signal transduction pathway and / or not affecting a third signal transduction pathway. In fact, multiple variations and combinations of signal transduction regulatory effects can occur. Ligand bias is not absolute either, in that a ligand can, for example, reduce signal transduction by means of one pathway, rather than completely inhibiting it, and / or incompletely activating another signal transduction pathway. Each pathway can be regulated to varying degrees and this situation can be measured according to multiple parameters, including but not limited to differences in potency and / or efficacy and / or the temporal aspect of signal transduction and / or the spatial aspect of signal transduction.

[0646] The term "functional surrogate" is used in its broadest sense and includes any compound, including proteins, polypeptides, peptides, antibodies, antibody fragments, macromolecules, or small molecules (less than 10 kDa), that, when substituted for another molecule, can mimic or increase at least one aspect of the activity, activation, or function of that molecule. For example, in a system that otherwise lacks RAGE expression, a functional surrogate for RAGE can replicate the activity, activation, or function of RAGE and / or a co-localized GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2), and / or modestly reduce RAGE ligand-independent RAGE activation by a co-localized activating GPCR, such as an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2). Thus, a "functional surrogate" for RAGE ligand-independent RAGE activation by a co-localized activating GPCR refers to a substance that can significantly increase, enhance, agonize, and / or positively regulate RAGE ligand-independent RAGE activation by a co-localized activating GPCR. Restoring the signaling ability of an inhibitor to RAGE ligand-independent RAGE activation via a co-localized activated GPCR by a functional surrogate appropriately restores or enhances its biological effects, including the production of proinflammatory mediators (including proinflammatory cytokines) by cells or the modulation of other cellular components associated with RAGE ligand-independent RAGE activation disease symptoms. A functional surrogate need not necessarily mimic all aspects of the activity, activation, or function of another molecule and, in fact, may inhibit certain aspects while activating other aspects and / or not modulate other aspects. Thus, a functional surrogate may also exhibit ligand bias.

[0647] The term "non-functional surrogate" is used in its broadest sense and includes any compound, including proteins, polypeptides, peptides, antibodies, antibody fragments, macromolecules, or small molecules (less than 10 kDa), that, when substituted for another molecule, can inhibit, antagonize, or reduce at least one aspect of the activity, activation, or function of that molecule. For example, a non-functional surrogate for RAGE can inhibit the activity, activation, or function of RAGE and / or a co-localized GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)), and / or modestly reduce RAGE ligand-independent RAGE activation by a co-localized activating GPCR (e.g., an angiotensin receptor (e.g., AT1R) or a chemokine receptor (e.g., CCR2)). Thus, a "non-functional surrogate" for RAGE ligand-independent RAGE activation by a co-localized activating GPCR refers to a substance that can significantly reduce, inhibit, antagonize, and / or negatively regulate RAGE ligand-independent RAGE activation by a co-localized activating GPCR. Reducing the signaling ability of an inhibitor to activate RAGE ligand-independent RAGE by a co-localized activated GPCR by a non-functional surrogate appropriately reduces or inhibits its biological effects, including the production of proinflammatory mediators (including proinflammatory cytokines) by cells or the regulation of other cellular components associated with RAGE ligand-independent RAGE activation disease symptoms. Note: A partial agonist can also act as an inhibitor because, even if it exhibits agonism, it does not produce maximal efficacy. Therefore, by competing for or modulating the agonistic activity of a more effective agonist (such as an endogenous agonist), it can effectively act as an inhibitor of the receptor polypeptide and / or its signaling pathway because it reduces the signaling output compared to if it were absent. Therefore, a partial agonist can act as an inhibitor in a therapeutic context. A non-functional surrogate does not necessarily reduce all aspects of the activity, activation, or function of another molecule, and in fact, can inhibit certain aspects while promoting other aspects and / or not modulating other aspects. Therefore, a non-functional surrogate can also exhibit ligand bias.

[0648] The term "binding" and its grammatical equivalents refer to the physical association between molecules under physiological conditions, such as due to covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions, and includes various interactions, such as salt bridges and water bridges, as well as any other conventional binding means. Binding can occur directly or through interactions with one or more other intermediary molecules.

[0649] Throughout this specification, unless the context requires otherwise, the words "comprising," "including," and "comprising" will be understood to indicate the inclusion of a recited step or element or recited group of steps or elements, but not the exclusion of any other step or element or any other group of steps or elements. Thus, use of the terms "comprising" and the like indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present. "Consisting of" is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and other elements may not be present. By "consisting essentially of" is meant to include any elements listed after the phrase and be limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0650] The term "construct" refers to a recombinant genetic molecule comprising one or more isolated nucleic acid sequences from different sources. Thus, a construct is a chimeric molecule in which two or more nucleic acid sequences from different sources are assembled into a single nucleic acid molecule and includes any construct containing (1) nucleic acid sequences that do not occur together in nature (i.e., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), including regulatory sequences and coding sequences, or (2) sequences encoding portions of functional RNA molecules or proteins that are not naturally contiguous, or (3) portions of promoters that are not naturally contiguous. Representative constructs include any recombinant nucleic acid molecule derived from any source that is capable of genomic integration or autonomous replication, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, including nucleic acid molecules in which one or more nucleic acid molecules have been operatively linked. The constructs of the present invention will generally contain the necessary elements to direct the expression of a nucleic acid sequence of interest, which is also contained in the construct, such as, for example, a target nucleic acid sequence or a regulator nucleic acid sequence. In some embodiments of the present invention, the construct can be contained in a vector. In some embodiments of the present invention, the construct can be contained in a vector. In addition to the component of the construct, the vector can also include one or more selective markers, one or more replication origins (such as prokaryotic replication origins and eukaryotic replication origins), at least one multiple cloning site and / or promote the stable integration of the construct into the host cell gene to promote the stable integration of the construct into the element group of the host cell gene. Two or more constructs can be contained in a single nucleic acid molecule (such as a single vector) inside, or can be contained in two or more independent nucleic acid molecules (such as two or more independent vectors) inside." expression construct" usually includes at least one control sequence that is effectively connected to the nucleotide sequence of interest. In this way, for example, a promoter effectively connected to the nucleotide sequence to be expressed is provided in the expression construct for expression in biology or its part (comprising host cell). Conventional compositions and methods for preparing and using constructs and host cells for practicing the present invention are well known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, Vols. 1, 2, and 3, JF Sambrook, DW Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0651] By "corresponding to" or "corresponding to" is meant exhibiting substantial sequence identity to a reference nucleic acid sequence (e.g., at least about 50%,...

Claims

1. An isolated or purified peptide that inhibits RAGE ligand-independent signaling, said peptide comprising or consisting of an amino acid sequence that is at least 90% identical to a fragment of residues 22 to 404 of wild-type RAGE as set forth in SEQ ID NO: 14, wherein said peptide comprises: (i) a contiguous sequence from C-terminal residue 390 to at least residue 379, wherein residue 379 is Q or a conservative amino acid substitution thereof; or (ii) a contiguous sequence from N-terminal residue 380 to at least residue 391, wherein residue 391 is S or a conservative amino acid substitution thereof.

2. The isolated or purified peptide of claim 1, wherein the peptide in (i) comprises a C-terminal residue 390 and a contiguous sequence to at least residue 379, wherein residue 379 is Q.

3. The isolated or purified peptide of claim 1, wherein the peptide in (i) comprises a contiguous sequence from C-terminal residue 390 to at least residue 379, wherein residue 379 is selected from the group consisting of K, R, D, N, and E.

4. The isolated or purified peptide of claim 3, wherein residue 379 is K.

5. The isolated or purified peptide of claim 1, wherein the peptide in (ii) comprises a contiguous amino acid sequence from N-terminal residue 380 to at least residue 391, wherein residue 391 is selected from the group consisting of S, W, T, M, Q and I.

6. An isolated or purified peptide that inhibits RAGE ligand-independent signaling, said peptide comprising or consisting of an amino acid sequence that is at least 90% identical to a fragment of residues 22 to 404 of wild-type RAGE as shown in SEQ ID NO: 14, wherein said peptide comprises at least residues 379 and 391, and wherein the residue at 379 is not Q or a conservative amino acid substitution thereof.

7. An isolated or purified peptide that inhibits RAGE ligand-independent signaling, said peptide: (i) consisting of an amino acid sequence greater than 90% identical to a fragment of residues 22 to 404 of wild-type RAGE as set forth in SEQ ID NO: 14, wherein the peptide comprises at least residues 379 and 391, wherein the residue at position 379 is not Q or a conservative amino acid substitution thereof, or Among them, 391 residues were not S or their conservative amino acid substitutions; (ii) comprising or consisting of an amino acid sequence that is at least 95% identical to a fragment of residues 22 to 404 of wild-type RAGE as set forth in SEQ ID NO: 14, wherein the peptide comprises at least residues 379 and 391, wherein the residue at position 379 is not Q or a conservative amino acid substitution thereof, or Among them, 391 residues were not S or their conservative amino acid substitutions; (iii) comprising or consisting of an amino acid sequence that is at least 90% identical to a fragment of residues 22 to 404 of wild-type RAGE as set forth in SEQ ID NO: 14, wherein the peptide comprises at least residues 379 and 391, wherein residue 379 is Q or a conservative amino acid substitution thereof, and residue 391 is not S or a conservative amino acid substitution thereof or E; or (iv) comprising or consisting of an amino acid sequence that is at least 90% identical to a fragment of residues 22 to 404 of wild-type RAGE as set forth in SEQ ID NO: 14, wherein the peptide comprises at least residues 379 and 391, and wherein residue 376, if present, is not W, wherein the residue at position 379 is not Q or a conservative amino acid substitution thereof, or Among them, residue 391 was not S or its conservative amino acid substitution.

8. The isolated or purified peptide of claim 6 or 7, wherein the residue at 379 is Q and the residue at 391 is selected from the group consisting of A, C, E, Y, V, R, N, K, H, G, F, and D.

9. The isolated or purified peptide of claim 8, wherein residue 391 is A or E.

10. The isolated or purified peptide of claim 9, wherein residue 379 is A and residue 391 is S.

11. The isolated or purified peptide according to any one of claims 1 to 10, wherein the peptide: - missing residues 366 and / or 367, or - comprising residues at 366 and / or 367, wherein one or both of said residues have a non-conservative amino acid substitution that prevents binding to Hyalin-1.

12. The isolated or purified peptide of claim 11, wherein the non-conservative amino acid substitution is A.

13. The isolated or purified peptide according to any one of claims 1 to 12, wherein position 388 is not alanine.

14. The isolated or purified peptide according to any one of claims 1 to 13, wherein position 388 is leucine.

15. The isolated or purified peptide of any one of claims 1 to 14, wherein the peptide comprises or consists of an amino acid sequence that is at least 95% identical to a fragment of residues 22 to 404 of wild-type RAGE as shown in SEQ ID NO:

14.

16. The isolated or purified peptide according to any one of claims 1 to 15, wherein the peptide comprises or consists of an amino acid sequence identical to a fragment of residues 22 to 404 of wild-type RAGE as shown in SEQ ID NO:

14.

17. A fusion polypeptide comprising the peptide according to any one of claims 1 to 16.

18. Use of an isolated or purified peptide according to any one of claims 1 to 16 or a fusion polypeptide according to claim 17 for the manufacture of a medicament for the treatment, prevention or management of a RAGE-related disease in a patient in need of such treatment.

19. A nucleic acid consisting of a nucleotide sequence encoding the peptide according to any one of claims 1 to 16.

20. A nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide of claim 17.

21. A vector comprising a nucleotide sequence encoding the peptide of any one of claims 1 to 16.

22. A pharmaceutical composition comprising a therapeutically effective amount of the isolated or purified peptide according to any one of claims 1 to 16, the fusion polypeptide according to claim 17, the nucleic acid according to claim 19 or 20, or the vector according to claim 21.

23. The pharmaceutical composition of claim 22, further comprising one or more excipients.

24. A modulator of RAGE activity, wherein such RAGE activity is induced by a co-localized active GPCR.

25. The modulator of claim 24, comprising two or more features selected from the group consisting of a first charged or hydrogen-bonding group (A), a second charged or hydrogen-bonding group (B), a third charged or hydrogen-bonding group (C), and a hydrophobic group (D), wherein at up to or Within the tolerance range of , the distances between the location points of the features are as follows, provided that the distances between the features are positive in value; 26. A method for screening a candidate substance based on its ability to modulate RAGE activity, wherein such RAGE activity is induced by a co-localized activated GPCR, the method comprising the steps of: contacting a RAGE polypeptide with a GPCR polypeptide in the presence of a candidate substance, wherein the GPCR polypeptide is constitutively active and / or activated by addition of an agonist, partial agonist or allosteric modulator of such GPCR; and detecting whether the candidate substance is a modulator of RAGE ligand-independent RAGE activation via the co-localized activated GPCR by detecting an effect indicative of RAGE activation modulated by the presence of the candidate substance and / or by detecting RAGE-dependent signaling modulated by the presence of the candidate substance.

27. Use of a modulator according to any one of claims 24 to 26 for the manufacture of a medicament for the treatment, prevention or management of a RAGE-related disease in a patient in need of such treatment.

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