Screening assays, modulators, and modulators for activation of the advanced glycation end product receptor (RAGE).

Abstract

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

Description

[Technical Field]

[0001] The present invention relates, in general, to screening assays for identifying modulators of receptor activation associated with specific diseases and / or conditions, such modulators, and treatment methods including administration of such modulators. More specifically, the present invention relates to modulators of RAGE receptor (RAGE) activation via a RAGE ligand-independent mechanism (also known as RAGE ligand-independent transactivation of RAGE) by specific co-existing activated G protein-coupled receptors (GPCRs), including activated angiotensin receptor 1 (AT1R) and activated CC chemokine receptor 2 (CCR2), with or without modulation of 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 for treating RAGE-related disorders using such modulators. [Background technology]

[0002] Advanced glycation end product receptors (RAGE) are multivalent type I transmembrane glycoproteins belonging to the immunoglobulin (Ig) superfamily (Neeper et al., 1992). Glycosylated RAGE proteins of 50-55 kDa are constitutively expressed in a limited range of cells (e.g., vascular endothelium, type II lung cells, leukocytes), but 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 disorders, 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] Genetic deletion of the AGER gene, which encodes RAGE, has been previously demonstrated to provide protection from numerous diseases and disease processes in mice, including several cancers (Malik et al., 2015) and inflammatory diseases including atherosclerosis and diabetic complications (Chuah et al., 2013). For example, in apolipoprotein E (apoE) knockout (KO) mice, RAGE deletion results in less plaque accumulation with age and reduced atherosclerosis accelerated by diabetes (Soro-Paavonen et al., 2008). Similarly, AGER deletion can reduce renal impairment in diabetic mice without affecting glucose regulation (Thomas et al., 2005).

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

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

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

[0007] The exact molecular mechanisms by which RAGE is activated and which are responsible for all of its biological effects are not well understood, and therefore, its ability to target these clinically important signaling pathways has not yet been demonstrated in clinical settings.

[0008] The renin-angiotensin-aldosterone system (RAAS) is a crucial homeostatic pathway involved in the development and progression of many common diseases and disease processes. Inhibition of the 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 have benefits in preventing diabetes (Tikellis et al., 2004), neuroprotection (Thoene-Reineke et al., 2011), mitigating the growth of certain cancers (Shen et al., 2016), and in aging (Benigni et al., 2009) by deleting the AT1R gene, which gives mice a longer lifespan.

[0009] These effects of RAAS blockers are additive to and independent of the blood pressure reduction mediated by RAAS blockers. This is because equivalent blood pressure reduction by other drugs does not provide the same benefit (Lee et al., 1993). In particular, activation of AT1R by angiotensin II (Ang II) induces oxidative stress, activation of nuclear factor κB (NFκB), and inflammation via a pathway different from the one that causes 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). Atheroma formation increases after angiotensin (Ang) II infusion and, in experimental models, is associated with physiological RAAS activation, including low-salt diet (Tikellis et al., 2012), diabetes mellitus (Goldin et al., 2006; and Soro-Paavonen et al., 2008), and genetic deletion of angiotensin-converting enzyme 2 (Ace2) (Thomas et al., 2010), independently of its effect on blood pressure homeostasis. Similarly, inhibition of RAAS has an anti-atherosclerotic effect that is in addition to and independent of the reduction in systemic blood pressure (Candido et al., 2002; Candido et al., 2004; and Knowles et al., 2000). Ang II has numerous direct atherosclerotic-promoting effects, including induction of oxidative stress (Rajagopalan et al., 1996), vascular adhesion (Grafe et al., 1997), and inflammation (Marvar et al., 2010) (Daugherty et al., 2000; Ferrario et al., 2006; and Ekholm et al., 2009).

[0011] These atherosclerotic-promoting effects are thought to be primarily mediated by the activation of type 1 angiotensin receptor (AT1R), followed by the induction of reactive oxygen species (ROS) and activation of NFκB signaling (Li et al., 2008). However, the underlying signaling mechanisms of these effects, including their relative independence from conventional vasoconstrictive signaling via AT1R, are not fully understood.

[0012] Furthermore, specific chemokine signaling pathways are involved in the pathogenesis of atherosclerosis, and macrophage infiltration into arterial lesions has been shown to directly contribute to this abnormal inflammatory disease (Boisvert et al., 2004). In fact, all known CC and CXC chemokine receptors, as well as CX3CR1 and XCR1, are associated with inflammation (Murphy et al. 2000; Zlotnik and Yoshie 2000). The primary physiological function of chemokine ligands (CCLs) is the regulation of "periodic immune surveillance, inflammation, and developmental cell migration" (Allen et al., 2007). CCLs are released in response to pro-inflammatory cytokines and selectively bind to a large family of G protein-coupled receptors that mediate physiological responses to chemokines. Chemokines were originally referred to as chemotactic cytokines.

[0013] Animal model studies of chronic inflammatory diseases have demonstrated that inhibition of the binding between MCP-1 (monocyte chemotactic protein 1, also known as monocyte chemoattractant protein 1, monocyte chemotactic and activating factor (MCAF) and chemokine (CC motif) ligand 2 (CCL2)) and CCR2 (chemokine (CC motif) receptor 2) by an antagonist suppresses the inflammatory response. The interaction between MCP-1 and its recognition receptor CCR2 is linked to uveitis, atherosclerosis, rheumatoid arthritis, multiple sclerosis, Crohn's disease, nephritis, organ graft rejection, pulmonary fibrosis, renal failure, diabetes and diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic retinitis, diabetic microangiopathy, tuberculosis, sarcoidosis, invasive staphylococcal infection, inflammation after cataract surgery, allergic rhinitis, allergic conjunctivitis, and chronic It has been linked to the conditions of urticaria, allergic asthma, periodontal disease, periodontitis, gingivitis, gingival disease, diastolic cardiomyopathy, myocardial infarction, myocarditis, chronic heart failure, vascular stenosis, restenosis, reperfusion injury, glomerulonephritis, solid tumors and cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, multiple myeloma, malignant myeloma, Hodgkin's disease, and inflammatory diseases such as cancers of the bladder, breast, cervix, colon, lung, prostate, or stomach (Rollins, 1996; Dawson et al., 2003).

[0014] Mice knocked out (KO) of both MCP-1 and CCR2 have demonstrated significantly reduced monocyte infiltration into inflammatory lesions in the absence of these signaling pathways. Furthermore, such KO mice are resistant to the development of experimental allergic encephalomyelitis (EAE, a model of human multiple sclerosis), cockroach allergen-induced asthma, atherosclerosis, and uveitis. Patients with rheumatoid arthritis and Crohn's disease improved during treatment with TNFα antagonists (e.g., monoclonal antibodies and soluble receptors) at dose levels correlated with reduced MCP-1 expression and infiltrating macrophage numbers.

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

[0016] Renal disease is associated with chronic inflammation characterized by the accumulation of renal macrophages. Production of monocyte chemoattractant protein 1 (MCP-1 / CCL2) by diabetic kidney has been identified as a major factor influencing macrophage accumulation in renal disease resulting from diabetic nephropathy (Tesch et al., 2008). In various animal models, inhibition of CCL2, and / or inhibition of specific CCL2 pathways, and / or inhibition of the CCL2 ligand MCP-1 have been shown to mitigate renal damage (Tesch et al., 2008; Rao V et a., 2006; Kang et al., 2010; Kitagawa et al., 2004; Park J et al., 2008).

[0017] Tesch (2008) noted that selective targeting of MCP-1 has been shown to be an effective treatment in suppressing kidney disease in animal models, including diabetic nephropathy. Treatment with a small molecule antagonist of CCR2 (INCB3344, propagermanium, RS-504393) has been shown to suppress inflammation in multiple sclerosis, renal ischemia-reperfusion injury, ureteral obstruction, and diabetic nephropathy in mouse models, as well as in arthritis in rat models. Artificial biological antagonists of CCR2 have also been shown to be effective. Subcutaneous injection of cells transduced with a vector expressing a deficient and inactive form of MCP-1 has been found to suppress the development of nephritis in a mouse model of lupus nephritis. Similarly, muscle transduction with 7ND (an MCP-1 mutant) reduces nephritis 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 led to drug approval. Anders HJ et al. investigated why single chemokine antagonist treatment was ineffective in treating the disease and discussed possible explanations, including redundancy of single chemokine mediators and variable expression patterns of chemokine receptors (Anders HJ et al. 2010). Therefore, there is a technical need for effective treatment of diseases caused by activation of the CCR2 pathway.

[0018] It is important to note that the concept, which is the subject of this invention, that RAGE can be activated in a RAGE ligand-independent manner via activated coexisting GPCRs, is meaningful for a large number of GPCRs, particularly those associated with inflammation and cell proliferation.

[0019] The explanation of novel, RAGE-ligand-independent functional interactions between certain activated coexisting GPCRs, including AT1R and CCR2, and RAGE contradicts this background technology. [Overview of the Initiative] [Problems that the invention aims to solve]

[0020] RAGE signaling, the renin-angiotensin-aldosterone system (RAAS), and certain chemokine signaling pathways are known to functionally interact in pathways involved in the development and progression of vascular complications. For example, binding of RAGE ligands to RAGE can induce pro-inflammatory signaling, which can be reduced by AT1R antagonists (inhibitors) (e.g., Fukami et al. 2004). Similarly, activation of the AT1R receptor by Ang II increases the formation and release of RAGE ligands, and interventions that inhibit RAGE ligand binding to RAGE or reduce RAGE ligands can attenuate Ang II-AT1R-induced injury (e.g., Thomas et al. 2005). Some of the downstream signaling pathways and mediators induced after RAGE activation by RAGE ligands, particularly those that cause inflammation, are similar to those induced after AT1R activation by Ang II (e.g., NFκB activation).

[0021] This prior art neither suggests nor discloses evidence regarding the formation of a complex between RAGE and GPCRs such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2. Furthermore, it does not foresee that activation of coexisting GPCRs by GPCR recognition ligands, such as Ang II for angiotensin receptors or MCP-1 for CCR2, would directly lead to activation of RAGE, particularly its cytoplasmic tail, nor would it foresee the induction of subsequent signaling mediated by RAGE in the absence of any RAGE ligand or without actually requiring the RAGE ligand-binding extradomain of RAGE. Therefore, it was not foreseeable that the regulation of ligand-independent activation of the cytoplasmic tail of RAGE would involve the regulation of signaling induced after activation of specific coexisting GPCRs, such as Ang II binding to AT1R or MCP-1 binding to CCR2.

[0022] One of the notable characteristics of RAGE is its activation by multiple ligands at multiple sites on its external domain, rather than by a single ligand and a single binding site that is susceptible to inhibition. RAGE can be activated by advanced glycation end products (AGEs) and other non-AGE ligands, including high-mobility box-1 (HMGB-1), S-100 / cargranulin, SAA, Aβ, C3a, heat shock protein 70 (HSP70), maternal injury-associated glycoprotein, acidic and cysteine-rich secretory protein (SPARC), β2-integrin Mac-1 (CD11b), phosphatidylserine (PS), double-stranded DNA (dsDNA), double-stranded RNA (dsRNA), lipopolysaccharide (LPS), and protein peroxides.

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

[0024] RAGE ligand-induced signaling also triggers a positive feedback loop in which the RAGE ligand-receptor interaction increases RAGE expression via NFκB activation, thereby enhancing subsequent RAGE-induced cell activation. In fact, the only means we know of that strongly downregulates RAGE expression is to reduce RAGE activation. This situation is in contrast to other receptors, such as the low-density lipoprotein (LDL) receptor, where receptor expression decreases as ligand levels increase.

[0025] Importantly, we have shown that, following activation of specific co-existing GPCRs, such as AT1R by Ang II or CCR2 by MCP-1, the cytoplasmic tail of RAGE is activated independently of any RAGE ligand or the extracellular domain of RAGE, initiating downstream signaling that leads to the activation of NFκB, a key transcription factor involved in inflammation, oxidative stress, fibrillation, cell proliferation, and cell survival. The absence of RAGE expression, specifically the absence of expression of key domains in the cytoplasmic tail of RAGE, prevents the induction of NFκB activation following activation of co-existing GPCRs, such as AT1R by Ang II or CCR2 by MCP-1, independently of the expression of the extracellular domain of RAGE. While we do not wish to be bound by theory, we believe that RAGE ligand-independent activation of the cytoplasmic tail of RAGE by specific co-existing activated GPCRs, including AT1R and CCR2, is the dominant pathway for RAGE activation. Furthermore, while we do not wish to be constrained by theory again, we believe that, for example, de novo expression of RAGE in cells exposed to injury, stress, or hypoxia provides a conduit for pro-inflammatory signaling that occurs via the activation of established GPCR signaling pathways.

[0026] The inventors have shown that RAGE ligand-independent activation of the cytoplasmic tail of RAGE after activation by specific coexisting GPCRs, such as AT1R by Ang II or CCR2 by MCP-1, also triggers signaling that increases RAGE expression.

[0027] RAGE is associated with many aspects of tumor biology, including tumor cell proliferation, migration, and invasion (Malik et al., 2015; Abe et al., 2008). Many cancers have higher levels of RAGE (e.g., breast cancer, colon cancer, kidney cancer, and gastric cancer; Taguchi et al., 2000). Lung cancer is an exception, as RAGE is a normal part of lung function and is lost when lung cells differentiate and become malignant (Marinakis et al., 2014). In C6 glioma cells, tumor volume is significantly reduced in tumors composed of cells with blocked RAGE. In contrast, tumors overexpressing RAGE grew rapidly and invaded surrounding tissue very efficiently (Taguchi et al., 2000). The demand for therapeutic agents to block RAGE signaling as a cancer treatment has been made for many common cancers, including, but not limited to, glioma / 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 (Logsdon et al., 2007; Volz et al., 2010); and colon cancer (Sparvero et al., 2009).

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

[0029] In a healthy state, RAGE expression in the lungs is the highest among all tissues. However, RAGE expression in the lungs is usually found only in type 1 lung cells. Upregulation of RAGE signaling in other cells and other sites of the lungs has been associated with a wide range of lung disorders, including but not limited to chronic obstructive pulmonary disease (COPD) / emphysema (Sukkar et al., 2012); asthma (Sukkar et al., 2012); smoking / contamination injury; acute lung injury / acute respiratory distress syndrome (ARDS) (Guo et al., 2012); and pulmonary fibrosis.

[0030] RAGE is critically involved in many inflammatory diseases and, as a result, is a potential therapeutic target for their treatment. Such conditions include: inflammatory arthritis (Sparvero et al., 2009; Chuah et al., 2013); osteoarthritis (Xie et al., 2013); retinal diseases (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 (Volz et al., 2010; Russo et al., 2016); ischemic heart disease / cardiac remodeling / fibrosis (Yan et al., 2010; Ramasamy et al., 2012); heart failure (Ramasamy et al., 2012); diabetic and non-diabetic kidney disease (Fukami et al. This includes, but is not limited to, the following conditions: al., 2015; Gugliucci et al., 2014; inflammatory bowel disease (Ott et al., 2014); pre-eclampsia (Daffu et al., 2013); polycystic ovary syndrome (Garg et al., 2015); hepatic steatosis, fibrosis, ischemic and non-ischemic 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). [Means for solving the problem]

[0031] This invention arises in part from the inventors' determination that RAGE forms receptor heteromer complexes within the cell membrane together with certain coexisting GPCRs, including AT1R and CCR2.

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

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

[0034] While there is published data suggesting that the cytoplasmic tail of RAGE is phosphorylated (Sakaguchi et al., 2011), we have shown that RAGE ligand-independent signaling induced after activation of certain coexisting GPCRs, such as the AT1R receptor by Ang II, does not require phosphorylation of the cytoplasmic tail of RAGE at serine 391 or other sites on the cytoplasmic tail of RAGE. Furthermore, we have shown that RAGE homologs from other mammals and RAGE mutants lacking any residues capable of maintaining phosphorylation can also be activated in response to ligand-dependent and ligand-independent activation of RAGE, inducing signaling, thus demonstrating that RAGE ligand-dependent signaling induced after binding of a RAGE ligand (e.g., S100A8 / A9) to the extracellular domain of RAGE does not necessarily require phosphorylation of the cytoplasmic tail at serine 391 or other sites on the cytoplasmic tail of RAGE. In addition, we have shown that the N-terminal truncated construct of RAGE (e.g., S391A-RAGE) can also be activated and induce signaling in response to ligand-dependent and ligand-independent activation of RAGE, thus demonstrating that RAGE ligand-dependent signaling induced after binding of a RAGE ligand (e.g., S100A8 / A9) to the extracellular domain of RAGE does not necessarily require phosphorylation of the cytoplasmic tail at serine 391 or other sites on the cytoplasmic tail of RAGE. 362-404The inhibitory function of ) is maintained even in the absence of the RAGE phosphorylation target, which confirms that the modulating effect of the RAGE construct described by the inventors is independent of RAGE phosphorylation.

[0035] Conventional techniques have demonstrated that PKCζ inhibitors inhibit RAGE-mediated RAGE ligand-dependent (e.g., s100-inducible) signaling and many other PKCζ-dependent pathways. In humans and animals, genetic deletions of PKCζ result in serious diseases. Researchers have shown that PKCζ inhibitors also inhibit RAGE ligand-independent (i.e., transactivation-inducible) signaling via full-length RAGE. However, the N-terminal truncated construct of RAGE (e.g., RAGE 362-404 The regulatory function of ) is not affected by the inhibition of PKCζ, and this confirms that the regulatory effect of the RAGE construct described by the inventors is independent of PKCζ.

[0036] Inhibitors of shared pathways induced following RAGE activation (e.g., myD88, TIRAP, interleukin-1 receptor-associated kinase 4 (IRAK4), or NFκB) nonspecifically block both RAGE ligand-dependent (e.g., s100-inducible) and RAGE ligand-independent (i.e., transactivation-inducible) RAGE-mediated signaling. Since other receptors (e.g., TLRs) also utilize these signaling molecules / pathways, inhibition of any of these mediators is not specific to RAGE signaling and affects many other functions of these signaling mediators, which can be harmful to human health (e.g., gene deletions of myD88, TIRAP, IRAK4, or NFκB are harmful to humans and animals, unlike RAGE deletions).

[0037] The inventors further demonstrated that selective modulation, such as inhibition of RAGE ligand-independent signaling, can be achieved by selectively targeting signaling via the cytoplasmic tail of RAGE, and that their assay and the modulator identified therefrom act on this transactivation (RAGE ligand-independent RAGE activation) process.

[0038] The inventors further demonstrated that dual inhibition of RAGE ligand-dependent activation and RAGE ligand-independent transactivation of RAGE signaling can also be achieved by selectively targeting signaling mediated via the cytoplasmic tail of RAGE, showing that the inventors' assay and the modulators identified therefrom can act simultaneously on both modes of RAGE activation by shared mediators. This is related to soluble RAGE 22-331 This is directly distinguishable from RAGE neutralizing antibodies and small molecules that selectively bind to the extracellular domain of RAGE and inhibit only RAGE ligand-dependent activation.

[0039] The inventors further demonstrated that the modulation of RAGE ligand-dependent signaling and / or RAGE ligand-independent transactivation by selectively targeting signaling mediated via the cytoplasmic tail of RAGE can be achieved without regulating the interaction between RAGE and Diaphanous-1 (Diaph1), which may be a modulator of ligand-dependent RAGE activation as suggested by prior art (Manigrasso, MB, et al 2016). Furthermore, the inventors demonstrated that the N-terminal truncated construct of RAGE (e.g., RAGE 362-404 The regulatory function of ) is maintained in the absence of Diaph1, thereby confirming that the regulatory effect of the RAGE construct described by the inventors is independent of Diaph1.

[0040] Sakaguchi and his collaborators found that when cells were treated with RAGE ligands, S100A11, S100A12, HMGB1, or AGE, the common pro-inflammatory adapter proteins TIRAP, MyD88, and IRAK co-precipitated with RAGE, which was overexpressed primarily in HEK293 cells, resulting in RAGE ligand-dependent activation of RAGE. Since TIRAP, MyD88, and IRAK also function as adapter proteins for all Toll-like receptors (TLRs) except TLR-3, which activates NFκB transcription, these interactions are not specific to RAGE.

[0041] Following this research, the group developed S391E-RAGE 387-395 A study proposed (RAGE(E)-I) as an inhibitor of specific aspects of RAGE ligand-dependent signaling (i.e., inhibition of apoptosis, cell migration, and invasion) by preventing endogenous RAGE signaling through the mimicking the phosphorylation state of RAGE and sequestering the adapter protein TIRAP (Putranto et al., 2013). However, the inventors showed that phosphorylation is not required for RAGE activation. Furthermore, sequestering these common adapter proteins also affects signaling via TLRs (e.g., TLR-2 and TLR-4), some of which may also be activated by RAGE ligands (e.g., the s100 protein), which seems to explain the findings of Puranto et al. In the same experiment, Puranto et al. found S391A-RAGE 387-395 However, it was argued that it was not a suitable inhibitor because it did not show sufficient binding to TIRAP to be recognizable and did not attenuate apoptosis induced by the RAGE ligand S100B (Putranto et al., 2013). Puranto et al. concluded that S391E-RAGE was not a suitable inhibitor because the growth of U-87MG cells was not significantly affected, as assessed by determining the intracellular adenosine triphosphate content. 387-395It was also noted that this did not inhibit all RAGE ligand-induced signaling pathways (Putranto et al., 2013). Therefore, the RAGE ligand-dependent pathways putatively inhibited by Putranto and his collaborators, and the fragment of the cytoplasmic tail of RAGE used by Putranto et al., are clearly different from the RAGE ligand-independent activation by coexisting activated GPCRs and modulators, which is the subject of this invention. In fact, S391A-RAGE 387-395 The negative findings of Putranto et al. regarding this matter teach us to move away from the present invention. Putranto et al. do not intend in any part of this publication to achieve RAGE ligand-independent activation of the cytoplasmic tail of RAGE by coexisting GPCRs.

[0042] European Patent Application Publication No. 1415997-A1 details the identification and use of polypeptides that directly or indirectly bind to the cytoplasmic tail of RAGE, thereby inhibiting or enhancing signaling 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 respects. Firstly, this teaching does not intend to achieve RAGE-mediated RAGE ligand-independent signaling or dual inhibition of RAGE-mediated RAGE ligand-dependent and RAGE ligand-independent signaling. Secondly, the claims of European Patent Application Publication No. 1415997-A1 relate to the use of polypeptides for binding to unidentified elements of the cytoplasmic tail of RAGE. In contrast, the inventors have demonstrated that polypeptides encoding the cytoplasmic tail of RAGE and their variants can be used to selectively bind RAGE-mediated RAGE ligand-independent signaling or RAGE-mediated RAGE ligand-dependent signaling and signaling molecules associated with RAGE ligand-independent signaling, leading to subsequent NFκB activation and modulation of downstream pathways resulting therefrom. Thirdly, the inventors have demonstrated the ability to modulate RAGE-mediated RAGE ligand-independent signaling using selectively modified polypeptides containing key elements of the cytoplasmic tail of RAGE. Fourthly, the modulation of RAGE-mediated RAGE ligand-dependent signaling is not described in European Patent Publication No. 1415997-A1. Furthermore, the only polypeptide specifically identified in European Patent Publication No. 1415997-A1 that can modulate 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 the action of their modulators.

[0043] After binding to CpG-DNA, RAGE is monoubiquitinated at cytosolic residue K374 by the F-box protein FBXO10, leading to endocytosis and lysosome-mediated degradation (Evankovich et al. 2017). Endocytosis and / or ubiquitination of RAGE have not been observed with other pro-inflammatory RAGE ligands.

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

[0045] These data suggest that K374R and S391A-RAGE mutants may be resistant to ubiquitination under certain conditions, thereby accumulating at higher levels than wild-type RAGE. However, this potentially increased stability / resistance to degradation cannot explain the rapid regulation of RAGE ligand-independent RAGE activation after activation of coexisting GPCRs and the inhibition of RAGE ligand-independent activation of RAGE signaling achieved by the S391A-RAGE mutant even in the presence of 1000-fold excess wild-type RAGE, as detailed below, and this regulation does not occur similarly in the presence or absence of K374.

[0046] Without limiting the universality of the following description of the present invention, the inventors have demonstrated that activation of certain coexisting GPCRs, such as AT1R by Ang II or CCR2 by MCP-1, triggers activation of the coexisting cytoplasmic tail of RAGE. This activation occurs in the absence of the extracellular domain of RAGE and is therefore completely independent of their interactions with the RAGE ligand or the extracellular domain of RAGE. While not wishing to be bound by theory, the inventors believe that this transactivation of RAGE by certain coexisting GPCRs represents the primary mechanism of RAGE activation. Consistent with this premise, the inventors have demonstrated that selective restoration of RAGE ligand-independent RAGE signaling in AGER / apoE double knockout (DKO) mice restores atherogenesis to a level not significantly different from that observed in RAGE-rich apoE-KO mice, even in the complete absence of RAGE-ligand-dependent signaling.

[0047] Many of the harmful signaling events induced by AT1R activation are attenuated when RAGE expression is absent (e.g., gene deletion or silencing, in healthy cells that do not express RAGE, or when ligand-independent activation of RAGE by activated AT1R is prevented or inhibited).

[0048] Simultaneously, RAGE-independent AT1R signaling pathways, such as the Gq signaling pathway induced by AT1R activation leading to the induction of inositol phosphate and calcium influx inhibited by AT1R antagonists, are unaffected by RAGE deletion, silencing of RAGE expression, or inhibition of RAGE function.

[0049] Therefore, the regulation, and especially the inhibition, of RAGE ligand-independent RAGE activation by specific activated coexisting GPCRs such as AT1R or CCR2 offers particular advantages to therapeutic interventions targeting pathogenic signaling induced via RAGE after activation of coexisting GPCRs. For example, such modulators, in certain embodiments, enable the aggressive targeting of the adverse effects of AT1R without impairing blood pressure regulation or inducing feedback "evasion" from AT1R inhibition, such as that occurring after AT1R inhibition which limits the use of RAAS inhibitors. Such modulators may affect only cells and tissues where this transactivation pathway is constitutively activated (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 do not express RAGE (e.g., healthy smooth muscle cells).

[0050] Activation of AT1R has both hemodynamic and non-hemodynamic effects. Hemodynamic effects lead to changes in blood flow and include changes in blood volume, blood pressure, flow rate or velocity, resistance, cardiac output, turbulence, and wall tension. AT1R blockers (inhibitors) can exhibit both hemodynamic (e.g., lowering blood pressure, altering resistance and cardiac output) and non-hemodynamic (e.g., causing oxidative stress and inflammation) effects. In conditions where the RAAS is activated (e.g., heart disease, kidney disease, hypertension), both hemodynamic and non-hemodynamic pathways are activated.

[0051] RAGE ligand-independent activation by activated AT1R is a mediator of only the non-hemodynamic (non-blood flow) effects of AT1R activation. We have observed that overall gene deletion of RAGE has no direct hemodynamic effects (e.g., does not affect blood pressure, vascular resistance, blood flow, or blood volume) and does not alter the hemodynamic effects of AT1R activation or inhibition. The main advantage of targeting RAGE ligand-independent activation by activated AT1R is that it is not limited by blood pressure regulation constraints that limit how much blood pressure can be lowered before the treatment becomes unsafe due to adverse hemodynamic effects.

[0052] Furthermore, changes in blood flow automatically trigger a feedback (homeostatic) response to maintain blood flow at a constant level. These feedback responses act to counteract or avoid the hemodynamic effects of RAAS inhibition by AT1R inhibition or angiotensin-converting enzyme (ACE) inhibition. In contrast, selective inhibition of non-hemodynamic pathways induced after RAAS activation, achieved through RAGE ligand-independent inhibition of RAGE activation by activated angiotensin receptors such as AT1R, is not associated with feedback / avoidance responses. The absence of such feedback responses underscores the durability and efficacy of such inhibition.

[0053] <Modulator for RAGE ligand-independent RAGE activation by activated coexisting GPCRs> In one embodiment, the present invention comprises a modulator of RAGE activity, such RAGE activity being induced by a specific active coexisting GPCR.

[0054] In one embodiment, the present invention includes a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR.

[0055] In one embodiment, the present invention includes a modulator that is a modulator of RAGE-dependent signaling induced by a specific activated coexisting GPCR.

[0056] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR acts in the absence of the RAGE ligand.

[0057] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR acts in the presence of a cleaved external domain of RAGE.

[0058] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR acts in the presence of a cleaved external domain of RAGE having a length of 40 amino acids or less, 20 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0059] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR contains the entire extracellular domain of RAGE conjugated to an analog, fragment, or derivative of the transmembrane domain of RAGE having a length greater than 5 amino acids, greater than 10 amino acids, or greater than 20 amino acids.

[0060] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR acts in the absence of the RAGE ligand-binding extradomain of RAGE.

[0061] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR does not contain the extracellular domain of RAGE.

[0062] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR does not contain analogs, fragments, or derivatives of the extracellular domain of RAGE.

[0063] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR contains a fragment of the extracellular domain of RAGE.

[0064] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR contains a fragment of the extracellular domain of RAGE having a length of 40 amino acids or less, 20 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0065] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR does not bind to the extracellular domain of RAGE.

[0066] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits or promotes signaling that occurs via the C-terminal cytoplasmic tail of RAGE, which is induced by the activated coexisting GPCR.

[0067] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits the binding that occurs to the C-terminal cytoplasmic tail of RAGE.

[0068] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits or promotes the interaction between the transmembrane domain of RAGE and the specific GPCR.

[0069] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits the interaction between the transmembrane domain of RAGE and the specific GPCR.

[0070] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits or promotes the ability of the activated GPCR to modulate RAGE-dependent signaling that depends on the proximity of the transmembrane domain of RAGE and the specific GPCR.

[0071] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits the ability of the activated GPCR to regulate RAGE-dependent signaling, which depends on the proximity of the transmembrane domain of RAGE and the specific GPCR.

[0072] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits or promotes the ability of the activated GPCR to modulate RAGE-dependent signaling that depends on the proximity of the transmembrane domain of RAGE and the specific GPCR, and inhibits or promotes signaling that occurs via the C-terminal cytoplasmic tail of RAGE induced by the activated coexisting GPCR.

[0073] In one embodiment of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR inhibits the activated GPCR's ability to regulate RAGE-dependent signaling that depends on the proximity of the RAGE transmembrane domain and the specific GPCR, and inhibits signaling that occurs via the C-terminal cytoplasmic tail of RAGE induced by the activated coexisting GPCR.

[0074] Throughout this specification, unless the context requires otherwise, co-expression of a G protein-coupled receptor superfamily (GPCR; also known as 7-transmembrane domain receptor, 7TM receptor, heptahelical receptor, serpentin receptor, and G protein-linked receptor; several other 7TM proteins are classified as members of the G protein-coupled receptor superfamily, including GPR107, GPR137, OR51E1, TPRA1, GPR143, and GPR157) in the same cells as RAGE, either endogenously or as a result of transduction. Not all members of this superfamily are G protein-coupled, and in this regard, it should be noted that the term GPCR includes members of the superfamily that are not G protein-coupled. Co-expression in the same cells can be demonstrated by many techniques known to those skilled in the art, including co-immunoprecipitation, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), and microscopy. The coexisting GPCR is preferably a GPCR that is sufficiently close to RAGE so that a functional interaction occurs between the GPCR and RAGE. More preferably, the coexisting GPCR is a GPCR that is sufficiently close to RAGE so that a suitable proximity assay can detect this proximity. Examples of suitable proximity assays are BRET, FRET, enzyme fragment complementation, split cyferase complementation, split fluorophore complementation, TANGO assay, NanoLuc Binary Technology (NanoBIT) assay, proximity ligation assay (PLA), or any other proximity assay that can detect the proximity of two proteins, whether one or more of these proteins are labeled or tagged to facilitate the use of the assay. Such proximity assays can be constructed in different ways, and the configuration and derivatives of receptor-heteromer investigation technology (Receptor-HIT) are preferred configurations of such proximity assays (International Publication No. 2008 / 055313; Jaeger et al., 2014).

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

[0076] Throughout this specification, unless the context requires otherwise, the specific activated coexisting GPCRs of the present invention are GPCRs that are expressed in the same cells as RAGE and whose effects on RAGE, indicating the modulation of RAGE activation and / or the modulation of RAGE-dependent signaling induction, are detected when the specific coexisting GPCR is activated by a recognition ligand or when the GPCR is constitutively active.

[0077] In one embodiment, the effect on RAGE that demonstrates the regulation of RAGE activation is detected by changes in intracellular transport of luciferase conjugate RAGE (e.g., RAGE / Rluc8) by changes in its proximity to intracellular compartment markers such as fluorophore-labeled Rabs (e.g., Venus-Rab1, Venus-Rab4, Venus-Rab5, Venus-Rab6, Venus-Rab7, Venus-Rab8, Venus-Rab9 and / or Venus-Rab11) and / or plasma membrane markers such as fluorophore conjugate fragments of K-ras (e.g., Venus-K-ras) using bioluminescence resonance energy transfer (BRET) upon addition of recognition ligands of coexisting GPCRs (Tiulpakov et al., 2016).

[0078] In another embodiment, the effect on RAGE is observed in luciferase conjugate RAGE (e.g., RAGE-Rluc8), IQGAP-1, protein kinase C zeta (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), carponin-3, drebrin, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipid protein 2, coronin, S100 These are changes in RAGE-dependent signaling, such as those detected by changes in proximity to RAGE interaction groups, including A11, succinyl-CoA ligase [GDP formation] subunit α, Hsc70 interacting protein, apoptosis inhibitor 5, neuropilin, cleavage stimulants, growth factor receptor binding protein 2, sec61β subunit or Nck1, and fluorophore-labeled proteins that interact with the cytoplasmic tail of RAGE.

[0079] In another embodiment, the effect on RAGE is a change in RAGE-dependent signaling, which is detected by changes in the standard activation of NFκB upon activation of a specific coexisting GPCR by a recognition ligand, measured by one or more of the following: • Monitoring the activity of IκB kinase (IKK) by observing the in vitro phosphorylation of substrates such as 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 by means of antibody, gel shift, EMSA and / or mass spectrometry; • Detection of cytosol-to-nuclear transport / transfer of NFκB components / subunits such as p65 / phosphop65; • Detection of dimerization / complex formation of NFκB subunits; Detection of active NFκB components / subunits by binding to immobilized DNA sequences / oligonucleotides containing NFκB response elements / consensus NFκB binding motifs, such as by electrophoretic mobility shift assays or gel shift assays, SELEX, protein-conjugated microarrays, or sequence-based approaches; • Chromatin immunoprecipitation (ChIP) assay to detect in-situ binding of NFκB to DNA, specifically to promoters and enhancers of certain genes; • In vitro kinase assay for NFκB kinase activity; Measurement of NFκB transcriptional activity using NFκB reporter assays via transgene expression of reporter constructs such as LacZ Fluc, eGFP SEAP, and NF-gluc, using approaches such as plasmid transduction, reporter cell lines, minicircles, retroviruses, or lentiviruses; • Measurement of changes in the expression of downstream targets of NFκB, such as cytokines, growth factors, adhesion molecules, and mitochondrial anti-apoptotic genes, proteins, or functional assays using real-time PCR (note that the multifaceted nature of NFκB is currently reflected in approximately 500 transcriptional targets (see http: / / www.bu.edu / nf-kb / gene-resources / target-genes / as of 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 tumorigenesis.

[0080] In another embodiment, the effect on RAGE is a change in RAGE signaling, which can be detected by a change in non-standard NFκB activation by measuring one or more of the following: • Detection of NIK (NFκB-induced kinase); • Detection of IKKα activation / phosphorylation; • Detection of NIK kinase activity by performing a kinase assay, based on its ability to autophosphorylate or phosphorylate substrates; • Generation of p52-containing NFκB dimers such as p52 / RelB; Detection of phospho-NFκB2p100 (Ser866 / 870); • Detection of the partial decomposition (called treatment) of precursor p100 to p52; • Detection of p52 / RelB transfer to the nucleus; • Detection of p52 / RelB binding to the NFκB site; Measurement of NFκB transcriptional activity using NFκB reporter assays via transgene expression of reporter constructs such as LacZ Fluc, eGFP SEAP, and NF-gluc, using approaches such as plasmid transduction, reporter cell lines, minicircles, retroviruses, or lentiviruses; and • Measurement of changes in the expression of downstream targets of non-standard NFκB signaling by functional assays such as real-time PCR, protein expression, or CXCL12.

[0081] <Coexisting GPCR> In one embodiment, the specific activated coexisting GPCR of the present invention is a GPCR that is expressed in the same cells as RAGE and is associated with RAGE-related disorders.

[0082] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs that are expressed in the same cells as RAGE, are associated with RAGE-related disorders, and whose removal and / or inhibition results in reduction or mitigation of RAGE-related disorders.

[0083] In one embodiment, the specific activated coexisting GPCR of the present invention is a GPCR associated with inflammation.

[0084] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs associated with inflammation, and their removal and / or inhibition results in reduction or mitigation of inflammation.

[0085] In one embodiment, the specific activated coexisting GPCR of the present invention is a GPCR associated with cell proliferation.

[0086] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs that are associated with cell proliferation and whose removal and / or inhibition results in a reduction or mitigation of cell proliferation.

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

[0088] Family A GPCRs (excluding olfactory, vomeronasal, and opsin) and the current level of evidence for their involvement in inflammation (see keys above):

[0089] [Table 1]

[0090] [Table 2]

[0091] [Table 3]

[0092] [Table 4]

[0093] [Table 5]

[0094] [Table 6]

[0095] [Table 7]

[0096] [Table 8]

[0097] Family A olfactory GPCRs and the current level of evidence regarding their involvement in inflammation (see keys above):

[0098] [Table 9]

[0099] [Table 10]

[0100] [Table 11]

[0101] [Table 12]

[0102] [Table 13]

[0103] [Table 14]

[0104] [Table 15]

[0105] [Table 16]

[0106] [Table 17]

[0107] Family A vomeronasal and opsin GPCRs and the current level of evidence regarding their involvement in inflammation (see keys above):

[0108] [Table 18]

[0109] Family B GPCRs and the current level of evidence regarding their involvement in inflammation (see keys above):

[0110] [Table 19]

[0111] [Table 20]

[0112] Family C GPCRs and the current level of evidence regarding their involvement in inflammation (see keys above):

[0113] [Table 21]

[0114] Frizzled family GPCRs and the current level of evidence regarding their involvement in inflammation (see keys above):

[0115] [Table 22]

[0116] Other 7TM proteins classified as members of the GPCR superfamily and the current level of evidence for their involvement in inflammation (see keys above):

[0117] [Table 23]

[0118] In one embodiment, the specific activated coexisting GPCRs of the present invention are group::ADGRA2,ADGRB2,ADGRB3,ADGRF3,ADGRG4,ADGRV1,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,GPR1 49, 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, OR4A15, OR4C11, OR 4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K15, OR4K17, OR4N5, OR5AC2, OR5AK2, OR5AP2, OR5AR1, OR5AS1, OR5B12, OR5B17, OR5B2, OR5B21, OR5 B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5J2, OR5K3, OR5L1, OR5L2, OR5M1, OR5M10, OR5M11, OR5M3, OR5M8, OR5M9, OR5R1, OR5T1, OR5T2, OR 5T3, 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, Mochi Phosphate 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, GnRH1 receptor, GPBA receptor, GPR1, GPR119, GPR12, GPR142, GPR143, GPR146, GPR1 48, 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, kispeptin 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, OR51A2, OR51A4, OR51A7, OR51B2, OR 51B4, 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, 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, oxoglutarate receptor, P2RY10, P2RY8, P2Y12 receptor, P2Y4 receptor, PrRP receptor, QRFP receptor, RXFP2 receptor, RXFP4 receptor, sst1 receptor, sst2 receptor, sst3 receptor, sst4 receptor, sst5 receptor, TA 1 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 receptor, C3a receptor, C5a1 receptor, C5a2 receptor, CaS receptor, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin 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 The GPCR is selected from the following receptors: 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, and μ receptor.

[0119] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of CCR4.

[0120] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of CCR5.

[0121] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0122] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of CCR4, CCR5, CCR10, and CXCR3.

[0123] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group set forth in paragraph

[0095] of the International Publication of this application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0124] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0125] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, M2 receptor, and OX1 receptor.

[0126] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, M2 receptor, and OX1 receptor.

[0127] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR5, M2 receptor, and OX1 receptor.

[0128] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, M2 receptor, and OX1 receptor.

[0129] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0130] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, M2 receptor, and OX1 receptor.

[0131] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph

[0095] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0132] In one embodiment, the specific activated coexisting GPCRs of the present invention are: 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, 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, OR5I1, OR5J2, OR5K3, OR5L 1, 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, LPA4 receptor, MC2 receptor, MC4 receptor, mGlu2 receptor, mGlu3 receptor, motilin receptor, MRGPRD, MRGPRX1, MRGPRX3, NK2 receptor, NPFF1 receptor, NPFF2 receptor, NPS receptor 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, ADG RG5, 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, kispeptin 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, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, OR51F 2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR52B6, OR 52D1, 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, oxoglutarate receptor, P2RY10, 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-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, chemerin 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, NMU This GPCR is selected from the following receptors: 1 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, and μ receptor.

[0133] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of CCR4.

[0134] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of CCR5.

[0135] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0136] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of CCR4, CCR5, CCR10, and CXCR3.

[0137] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0138] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0139] In one embodiment, the specific activated coexisting GPCR of the present invention is a GPCR selected from the group: OX1 receptor, OX2 receptor, PTH1 receptor, PTH2 receptor, AMY1 receptor, AMY2 receptor, AMY3 receptor, AM1 receptor, AM2 receptor.

[0140] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, M2 receptor, and OX1 receptor.

[0141] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, M2 receptor, and OX1 receptor.

[0142] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR5, M2 receptor, and OX1 receptor.

[0143] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, M2 receptor, and OX1 receptor.

[0144] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0145] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, M2 receptor, and OX1 receptor.

[0146] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000109] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0147] In one embodiment, the specific activated coexisting GPCRs of the present invention are: 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, OR4A15, OR4C11, OR4C12, OR4C13, OR4C15, OR4C16, OR4K13, OR4K14, OR4K1 5, OR4K17, OR4N5, OR5AC2, OR5AK2, OR5AP2, OR5AR1, OR5AS1, OR5B12, OR5B17, OR5B2, OR5B21, OR5B3, OR5D13, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR 5J2, 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, A DGRB1, 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 receptor, FSH receptor, FZD1, F ZD2, 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, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor Receptor, HCA3 receptor, kispeptin 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, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR5 1F1, OR51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1, OR51M1, OR51Q1, OR51S1, OR51T1, OR51V1, OR52A1, OR52A4, OR52A5, OR52B2, OR52B4, OR 52B6, 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, oxoglutarate 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, TAS2R41, TAS2 R42, 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, chemerin receptor, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXC R6, 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, This GPCR is selected from 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, and μ receptor.

[0148] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of CCR4.

[0149] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of CCR5.

[0150] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0151] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of CCR4, CCR5, CCR10, and CXCR3.

[0152] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0153] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0154] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, and M2 receptor.

[0155] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, and M2 receptor.

[0156] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR5, and M2 receptor.

[0157] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, and M2 receptor.

[0158] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0159] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, and M2 receptor.

[0160] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000124] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0161] In one embodiment, the specific activated coexisting GPCRs of the present invention are: 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, G PR161, 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 Body, 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, D GPA receptors, 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, GPR33, GPR35, GPR6, GPR61, GPR78,GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA3 receptor, kispeptin 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, 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, OR52D1, OR52E2, OR52E4, OR52E5, OR52E6, OR52E8, OR52H1, OR52I1, OR52I2, OR52J 3, OR52K1, OR52K2, OR52L1, OR52M1, OR52N1, OR52N2, OR52N4, OR52N5, OR52R1, OR52W1, OR56A1, OR56A3, OR56A4, OR56A5, OR56B1, OR56B4, OR6V1, OR7D2 OR9A2, oxoglutarate receptor, P2RY10, 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-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, Chemerin 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,It is a GPCR selected from β2-adrenergic receptors, κ receptors, and μ receptors.

[0162] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of CCR4.

[0163] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of CCR5.

[0164] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0165] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of CCR4, CCR5, CCR10, and CXCR3.

[0166] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0167] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0168] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, and M2 receptor.

[0169] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, and M2 receptor.

[0170] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR5, and M2 receptor.

[0171] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, and M2 receptor.

[0172] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0173] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, and M2 receptor.

[0174] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000138] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0175] In one embodiment, the specific activated coexisting GPCRs of the present invention are: 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, FZ D3, 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, GPR78, GPR82, GPR83, GPR84, GPR85, GPR87, GPRC5D, GPRC6 receptor, HCA2 receptor, HCA 3 receptors, kispeptin 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, OR2T1 1, OR2T34, OR2W3, OR3A3, OR4D10, OR4M1, OR4Q3, OR51A2, OR51A4, OR51A7, OR51B2, OR51B4, OR51B5, OR51B6, OR51D1, OR51E1, OR51E1, OR51E2, OR51F1, O R51F2, OR51G1, OR51G2, OR51I1, OR51I2, OR51J1, OR51L1, 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, oxoglutarate receptor, P2RY10, 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, T AS2R43, 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 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, Chemerin 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, M This GPCR is selected from the following receptors: T2 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, and μ receptor.

[0176] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of CCR4.

[0177] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of CCR5.

[0178] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0179] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of CCR4, CCR5, CCR10, and CXCR3.

[0180] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group in paragraph [000152] of the international publication of the present application, excluding adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline 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.

[0181] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group in paragraph [000152] of the international publication of the present application, excluding adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline 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.

[0182] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group in paragraph [000152] of the international publication of the present application, excluding adrenaline α1A receptor, CCR3 and M2 receptor.

[0183] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group in paragraph [000152] of the international publication of the present application, excluding adrenaline α1A receptor, CCR3, CCR4 and M2 receptor.

[0184] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group in paragraph [000152] of the international publication of the present application, excluding adrenaline α1A receptor, CCR3, CCR5 and M2 receptor.

[0185] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, and M2 receptor.

[0186] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0187] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of the adrenaline α1A receptor, CCR3, CCR4, CCR5, CCR10, CXCR3, and M2 receptor.

[0188] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000152] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α1A receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0189] In one embodiment, the specific activated coexisting GPCRs of the present invention are: 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, CC R1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, chemerin 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, FPR 3, 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, This GPCR is selected from 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, and μ receptor.

[0190] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR4.

[0191] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR5.

[0192] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0193] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR4, CCR5, CCR10, and CXCR3.

[0194] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline β2 receptor, apelin receptor, CCR8, CCR10, CXCR1, CXCR3, CXCR6, CXCR7, platelet-activating factor receptor, prostaglandin E2 receptor, prostaglandin E3 receptor, and prostaglandin E4 receptor.

[0195] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline 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.

[0196] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR3.

[0197] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR3 and CCR4.

[0198] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR3 and CCR5.

[0199] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR3, CCR4, and CCR5.

[0200] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0201] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000166] of the International Publication of this Application, with the exception of CCR3, CCR4, CCR5, CCR10, and CXCR3.

[0202] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group in paragraph [000166] of the international published patent application of the present application, excluding adenosine A2A receptor, adenosine A2B receptor, adenosine A3 receptor, adrenaline α2A receptor, adrenaline α2C receptor, adrenaline β1 receptor, adrenaline β2 receptor, adrenaline 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.

[0203] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group: angiotensin receptors including AT1R and specific chemokine receptors including CCR1, CCR2, CCR6, CCR7, CXCR2, CXCR4, CXCR6 and CXCR7.

[0204] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group: angiotensin receptors including AT1R and specific chemokine receptors including CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2, CXCR4, CXCR6 and CXCR7.

[0205] In one embodiment, the specific activated co-existing GPCR of the present invention is a GPCR selected from the group: angiotensin receptors including AT1R and specific chemokine receptors including CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2, CXCR4 and CXCR6.

[0206] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from angiotensin receptors including group AT1R and specific chemokine receptors including CCR1, CCR2, CCR6, CCR7, CXCR2, CXCR4, and CXCR6.

[0207] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from angiotensin receptors including group AT1R and specific chemokine receptors including CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2, and CXCR6.

[0208] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from angiotensin receptors including group AT1R and specific chemokine receptors including CCR1, CCR2, CCR6, CCR7, CXCR2, and CXCR6.

[0209] In one embodiment, the activated coexisting GPCR of the present invention is vasopressin receptor 2.

[0210] In one embodiment, the specific chemokine receptor is a chemokine receptor that is co-expressed in the same cell as RAGE.

[0211] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is a chemokine receptor selected from the group: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0212] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR3, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0213] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is a chemokine receptor selected from the group: CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0214] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is a chemokine receptor selected from the group: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0215] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0216] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0217] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR4, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0218] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0219] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0220] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0221] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0222] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0223] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR3, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0224] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR1, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0225] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0226] In one embodiment, a specific chemokine receptor is co-expressed in the same cells as RAGE, is associated with inflammation, and is selected from the group: CCR1, CCR2, CCR6, CCR7, CCR8, CCR9, CXCR2, CXCR5, CXCR6, CXCR7, CX3CR1, XCR1.

[0227] In one embodiment of the present invention, the specific activated coexisting GPCRs of the present invention are: adenosine 1A receptor, adrenaline α1A receptor, adrenaline α1B receptor, adrenaline 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, neuro It is a GPCR selected from peptide 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.

[0228] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of CCR4.

[0229] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of CCR5.

[0230] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0231] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of CCR4, CCR5, and CXCR4.

[0232] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, muscarinic M2 receptor, and orexin receptor 1.

[0233] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, CCR4, muscarinic M2 receptor, and orexin receptor 1.

[0234] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, CCR5, muscarinic M2 receptor, and orexin receptor 1.

[0235] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, CCR4, CCR5, muscarinic M2 receptor, and orexin receptor 1.

[0236] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000204] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, CXCR4, muscarinic M2 receptor, and orexin receptor 1.

[0237] In a preferred embodiment of the present invention, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group: adrenaline α1A receptor, adrenaline 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.

[0238] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000214] of the International Publication of this Application, with the exception of CCR4.

[0239] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000214] of the International Publication of this Application, with the exception of CCR4 and CXCR4.

[0240] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000214] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, muscarinic M2 receptor, and orexin receptor 1.

[0241] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000214] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, CCR4, muscarinic M2 receptor, and orexin receptor 1.

[0242] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000214] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, CCR4, CXCR4, muscarinic M2 receptor, and orexin receptor 1.

[0243] In a particularly preferred embodiment of the present invention, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group: adrenaline α1A receptor, adrenaline 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.

[0244] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000220] of the International Publication of this Application, with the exception of CXCR4.

[0245] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000220] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, muscarinic M2 receptor, and orexin receptor 1.

[0246] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000220] of the International Publication of this Application, with the exception of adrenaline α1A receptor, CCR3, CXCR4, muscarinic M2 receptor, and orexin receptor 1.

[0247] In one embodiment of the present invention, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group: adenosine A1 receptor (ADORA1), adrenaline 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), thyroid-stimulating hormone-releasing hormone receptor 1 (TRHR1), vasopressin receptor 1A (V1aR), vasopressin receptor 1B (V1bR), and vasopressin receptor 2 (V2R).

[0248] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000224] of the International Publication of this Application, with the exception of CCR4.

[0249] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000224] of the International Publication of this Application, with the exception of CCR5.

[0250] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000224] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0251] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000224] of the International Publication of this Application, with the exception of CXCR4.

[0252] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000224] of the International Publication of this Application, with the exception of CCR4, CCR5, and CXCR4.

[0253] In a preferred embodiment of the present invention, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group: adrenaline α2B receptor, angiotensin receptor AT1 (AT1R), bradykinin receptor 2 (B2R), CCR1, CCR2, CCR4, CCR5, CCR6, CCR9, CXCR2, CXCR4, neuropeptide Y1 receptor (NPY1R), orexin receptor 2, sphingosine-1-phosphate receptor 1 (S1PR1), thyroid-stimulating hormone-releasing hormone receptor 1 (TRHR1), vasopressin receptor 1A (V1aR), vasopressin receptor 1B (V1bR), and vasopressin receptor 2 (V2R).

[0254] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000230] of the International Publication of this Application, with the exception of CCR4.

[0255] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000230] of the International Publication of this Application, with the exception of CCR5.

[0256] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000230] of the International Publication of this Application, with the exception of CCR4 and CCR5.

[0257] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000230] of the International Publication of this Application, with the exception of CXCR4.

[0258] In one embodiment, the specific activated coexisting GPCRs of the present invention are GPCRs selected from the group in paragraph [000230] of the International Publication of this Application, with the exception of CCR4, CCR5, and CXCR4.

[0259] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is an angiotensin receptor.

[0260] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is AT1R.

[0261] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is a specific chemokine receptor selected from the group:CCR1, CCR2, CCR6, CCR7, CXCR2, CXCR4, CXCR6, and CXCR7.

[0262] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is a specific chemokine receptor selected from the group: CCR1, CCR2, CCR6, CCR7, CXCR1, CXCR2, and CXCR6.

[0263] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is a specific chemokine receptor selected from the group:CCR1, CCR2, CCR6, CCR7, CXCR2, and CXCR6.

[0264] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is a specific chemokine receptor selected from CCR2 and CCR6.

[0265] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is CCR2.

[0266] In a particular embodiment of the present invention, the activated coexisting GPCR of the present invention is CXCR4.

[0267] In one embodiment of the present invention, a RAGE ligand is a ligand that interacts with the extracellular domain of RAGE to regulate RAGE activation. Therefore, in this embodiment of the present invention, RAGE ligand-independent RAGE activation means RAGE activation that does not occur via a ligand that interacts with the extracellular domain of RAGE.

[0268] Preferably, the RAGE ligand is a ligand that interacts with the extracellular domain of RAGE to regulate RAGE activation, but does not interact with the transmembrane domain or cytoplasmic tail of RAGE or any motifs contained therein. Therefore, in this preferred embodiment of the present invention, RAGE ligand-independent RAGE activation means RAGE activation that does not occur via ligands that interact with the extracellular domain of RAGE, as long as the ligand does not interact with the transmembrane domain or cytoplasmic tail of RAGE or any motifs contained therein.

[0269] The extracellular domain of RAGE (also called the external domain) contains three immunoglobulin-like regions: an N-terminal V-type domain followed by two C-type domains (C and C', or alternatively C1 and C2). The primary ligand-binding site is the V-domain, but RAGE activation can also be mediated by ligand binding to the C-domain. Because ligands tend to be negatively charged, most ligands tend to bind to the V-domain and / or C1-domain, although there is at least one example of a ligand that binds to the C2-domain (S100A6; Leclerc et al., 2007). While the C1 and C2-domains may not generally bind directly to ligands, they may play an important role in stabilizing the V-domain to mediate interactions with ligands. RAGE has a single transmembrane domain and a cytoplasmic tail. In humans, the cytoplasmic tail of RAGE is 43 amino acids long (residues 362-404). This cytoplasmic tail contains motifs important for RAGE-dependent cell activation.

[0270] In one embodiment of the present invention, a RAGE ligand is a ligand that activates RAGE by interacting with the extracellular V, C1 and / or C2 domains of the extracellular domain of RAGE. In this embodiment of the present invention, RAGE ligand-independent RAGE activation means activation of RAGE that does not occur via a ligand that interacts with the extracellular V, C1 or C2 domains of the extracellular domain of RAGE.

[0271] Preferably, the RAGE ligand does not interact with the transmembrane domain or cytoplasmic tail of RAGE or any motifs contained therein. In this embodiment of the present invention, RAGE ligand-independent RAGE activation means RAGE activation that does not occur via ligands that interact with the extracellular V, C1, or C2 domains of the extracellular domain of RAGE, provided that the ligand also does not interact with the transmembrane domain or cytoplasmic tail of RAGE or any motifs contained therein.

[0272] In one embodiment of the present invention, a modulator that modulates RAGE ligand-independent RAGE activation by an activated angiotensin receptor such as AT1R or an activated coexisting GPCR such as CCR2 also modulates RAGE ligand-dependent activation of RAGE.

[0273] In preferred embodiments of the present invention, the modulator of the present invention modulates, or does not modulate, or modulates, or modulates to a different degree, a RAGE-independent signaling pathway associated with a specific activated coexisting GPCR.

[0274] In preferred embodiments, the modulator of the present invention inhibits, or less inhibits, one or more specific coexisting GPCR signaling pathways that are RAGE-independent.

[0275] In one embodiment of the present invention, a specific coexisting GPCR signaling pathway that is RAGE-independent is the Gq signaling pathway. In one embodiment of the present invention, a specific coexisting GPCR signaling pathway that is RAGE-independent is the Gi / o signaling pathway. In one embodiment of the present invention, a specific coexisting GPCR signaling pathway that is RAGE-independent is the Gs signaling pathway. In one embodiment of the present invention, a specific coexisting GPCR signaling pathway that is RAGE-independent is the calcium signaling pathway. In one embodiment of the present invention, a specific coexisting GPCR signaling pathway that is RAGE-independent is the phospholipase C signaling pathway. In another embodiment of the present invention, a specific coexisting GPCR signaling pathway that is RAGE-independent is the β-arrestin-mediated extracellular regulatory kinase (ERK) signaling pathway.

[0276] In a particularly preferred embodiment, where the activated coexisting GPCR is activated AT1R, the modulator of the present invention modulates one or more RAGE-independent AT1R signaling pathways either not or to a lesser extent.

[0277] In a particularly preferred embodiment, where the activated coexisting GPCR is activated AT1R, the modulator of the present invention inhibits, or less inhibits, one or more RAGE-independent AT1R signaling pathways.

[0278] In one embodiment of the present invention, the RAGE-independent AT1R signaling pathway is the Gq signaling pathway. In another embodiment of the present invention, the RAGE-independent AT1R signaling pathway is the β-arrestin-mediated extracellular regulatory kinase (ERK) signaling pathway.

[0279] In another particularly preferred embodiment, where the activated coexisting GPCR is activated CCR2, the modulator of the present invention modulates one or more RAGE-independent CCR2 signaling pathways either not or less.

[0280] In another particularly preferred embodiment, where the activated coexisting GPCR is activated CCR2, the modulator of the present invention inhibits, or less inhibits, one or more RAGE-independent CCR2 signaling pathways.

[0281] In one embodiment of the present invention, the RAGE-independent AT1R signaling pathway is a Gi / o signaling pathway. In another embodiment of the present invention, the RAGE-independent CCR2 signaling pathway is a β-arrestin-mediated extracellular regulatory kinase (ERK) signaling pathway. In yet another embodiment of the present invention, the RAGE-independent CCR2 signaling pathway is a phospholipase C signaling pathway.

[0282] <Modulator> In one embodiment of the present invention, the modulator is an activator, inhibitor, allosteric modulator, or functional or non-functional substitute for the cytoplasmic tail of RAGE. Functional substitutes are modulators that replace the cytoplasmic tail of RAGE in the presence of specific coexisting GPCRs, and can be activated by them to induce downstream RAGE-dependent signaling in the presence or absence of wild-type RAGE expression. Non-functional substitutes are modulators that replace the cytoplasmic tail of RAGE in the presence of specific coexisting GPCRs, and cannot be activated by them or induce downstream RAGE-dependent signaling, and inhibit signaling that normally occurs through activation of the cytoplasmic tail of RAGE and the resulting RAGE-dependent signaling.

[0283] In one embodiment of the present invention, the modulator of the present invention is an activator, an inhibitor, an allosteric modulator, or a non-functional substitute for the transmembrane domain or a part thereof of RAGE.

[0284] Non-functional substitutes are modulators that replace the transmembrane domain of RAGE in the presence of certain coexisting GPCRs, are unable to be activated by them or induce downstream RAGE-dependent signaling, and inhibit signaling that normally occurs through activation of the cytoplasmic tail of RAGE and the resulting RAGE-dependent signaling.

[0285] In one embodiment of the present invention, the modulator comprises the transmembrane domain or a portion thereof of RAGE and a fragment of the extracellular domain of RAGE.

[0286] In one embodiment of the present invention, the modulator comprises the transmembrane domain or a portion thereof of RAGE and a fragment of the cytoplasmic tail of RAGE.

[0287] In one embodiment of the present invention, the modulator includes the transmembrane domain or a portion thereof of RAGE, a fragment of the extracellular domain of RAGE, and a fragment of the cytoplasmic tail of RAGE.

[0288] In one embodiment of the present invention, the modulator of the present invention contains a fragment of the extracellular domain of RAGE that is 40 amino acids or less, 20 amino acids or less, 10 amino acids or less, or 5 amino acids or less in length.

[0289] As an example of a modulator, the inventors have shown that RAGE 362-404 is a functional substitute for RAGE, can be activated by certain co-existing GPCRs such as AT1R and CCR2, and induces downstream RAGE-dependent signaling resulting from RAGE ligand-independent RAGE activation in the presence or absence of wild-type RAGE expression. Further, when RAGE 362-404 is fused to the cell-penetrating peptide (TAT) and the marker protein (mCherry), treatment with the TAT-mCherry-RAGE 362-404 oligopeptide can restore Ang II-dependent inflammation and atherogenesis in AGER / apoE-DKO mice in the absence of wild-type RAGE expression.

[0290] The sequence of RAGE 362-404 is SEQ ID NO: 1.

Chemical formula

[0291] As a further example of a modulator, the inventors have shown that S391A-RAGE 362-404 is a non-functional substitute for RAGE that is not activated by them in the presence of certain co-existing GPCRs and inhibits RAGE-dependent signaling. Expression of S391A-RAGE 362-404 inhibits wild-type RAGE ligand-independent RAGE activation by activated AT1R and wild-type RAGE ligand-dependent activation of wild-type RAGE by the RAGE ligand S100A8 / A9. Further, S391A-RAGE 362-404When it fuses with the cell-permeable peptide (TAT) and the marker protein (mCherry), TAT-mCherry-S391A-RAGE is formed. 362-404 Treatment with oligopeptides inhibits RAGE ligand-independent RAGE activation by activated AT1R, thereby attenuating Ang II-dependent inflammation and atherogenesis in apolipoprotein E knockout mice. Further examples are provided below.

[0292] S391A-RAGE 362-404 The array is sequence number 2. [ka]

[0293] As another example of a modulator, the inventors of RAGE 338-361 However, we demonstrated that it inhibits wild-type RAGE ligand-independent RAGE activation by activated AT1R. This inhibition is mCherry-RAGE 362-404 This is overcome by co-expression.

[0294] RAGE 338-361 The sequence is sequence number 3. [L 338 GTLALALGILGGLGTAALLIGVI 361 ]

[0295] In one embodiment, the present invention includes a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, which modulates the transactivation of the cytoplasmic tail of RAGE induced by the activation of such a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a chemokine receptor like CCR2.

[0296] In one embodiment, the present invention relates to protein kinase C zeta (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), carponin-3, drebrin, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipid protein 2, coronin, S100 It includes a modulator of RAGE ligand-independent activation of the cytoplasmic tail of RAGE by specific activated coexisting GPCRs, which binds to Ras-GTPase activator-like protein (IQGAP1) or other RAGE-related proteins, or disrupts the binding of these elements to RAGE in order to modulate RAGE transactivation by specific activated coexisting GPCRs, such as angiotensin receptors like AT1R or chemokine receptors like CCR2, comprising A11, succinyl-CoA ligase [GDP formation] subunit α, Hsc70 interacting protein, apoptosis inhibitor 5, neuropilin, cleavage stimulant, growth factor receptor binding protein 2, sec61β subunit or Nck1.

[0297] In one embodiment of the present invention, the modulator of the present invention is 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), carponin-3, drebrin, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipid protein 2, coronin, S100 It binds to the cytosolic elements of specific activated coexisting GPCRs, RAGE and / or elements complexed with either A11, succinyl-CoA ligase [GDP formation] subunit α, Hsc70 interacting protein, apoptosis inhibitor 5, neuropilin, cleavage stimulant, growth factor receptor binding protein 2, sec61β subunit or Nck1, and modulates these signaling elements necessary for RAGE transactivation by specific activated coexisting GPCRs, such as angiotensin receptors like AT1R or chemokine receptors like CCR2, thereby regulating RAGE ligand-independent signaling via the cytoplasmic tail of RAGE.

[0298] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR is the cytosolic element of RAGE and / or the element that complexes with RAGE in the cytosol (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), carponin-3, drebrin, filamin B, Ras-related protein Rab-13, radixin / ezrin / moesin, proteolipidoprotein 2, coronin, S100) By binding to A11, succinyl-CoA ligase [GDP formation] subunit α, Hsc70 interacting protein, apoptosis inhibitor 5, neuropilin, cleavage stimulants, growth factor receptor binding protein 2, sec61β subunit, or Nck1, etc., they also regulate the RAGE ligand-dependent activation of the cytoplasmic tail of RAGE, thereby inhibiting RAGE ligand-mediated signaling through these elements.

[0299] In certain embodiments, the modulator comprises, consists of, or is essentially derived from the amino acid sequence described in Sequence ID No. 1 or its analogues, fragments, or derivatives.

[0300] In some embodiments, the modulator is introduced by gene delivery (such as by using viral or artificial nonviral gene delivery, including electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous injection, sonication, lipofection, liposomes, nanobubbles, and polymer gene carriers), and peptide fragments, biologically active analogs, or derivatives are generated by the cell as a result of the transcription and translation process.

[0301] In some embodiments of this model, the modulator has a modified ability to form complexes with specific coexisting GPCRs such as AT1R or CCR2, or elements that complex with them. For example, RAGE analogs or derivatives can be distinguished from wild-type RAGE polypeptides or fragment sequences by substitution, addition, or deletion of at least one amino acid residue, or by addition or substitution of abnormal or unconventional amino acids or non-amino acid residues.

[0302] In some embodiments, the modulator lacks or has the serine 391 modification normally present in the wild-type human RAGE polypeptide. In exemplary examples of this type, a fragment, analog, or derivative of the cytoplasmic tail of RAGE lacks the serine at position 391 of the wild-type RAGE sequence (e.g., RAGE 370-390 The construct is cleaved at Glu390). Preferably, the serine at position 391 is deleted or substituted with another amino acid residue, analog or derivative, so as to impair or disable the signaling mediated by the serine at this site after activation of the coexisting GPCR. In one embodiment, the serine at position 391 is deleted or substituted with another amino acid residue selected from the group: alanine, aspartic acid, phenylalanine, histidine, lysine, arginine, tyrosine, asparagine, valine, glycine, cysteine, or glutamic acid.

[0303] In some embodiments, the modulator either retains serine 391, which is normally present in the wild-type human RAGE polypeptide, or is replaced with another amino acid, its analogue, or a derivative that has the same function as serine at position 391. In this type of example, the cytoplasmic tail fragment of RAGE retains serine at position 391 of the wild-type RAGE sequence (e.g., RAGE 370-404(Construction). Preferably, the serine at position 391 is replaced with another amino acid residue or an analog or derivative thereof to replicate the signaling mediated after activation of a coexisting GPCR by a RAGE construct containing serine at this site. In one embodiment, the serine at position 391 is replaced with another amino acid residue selected from the group: proline, glutamine, threonine, leucine, isoleucine, methionine, or tryptophan.

[0304] In some embodiments, the modulator lacks or has reduced ability to bind to Diaphanous 1 (Diaph1) compared to human wild-type RAGE. In exemplary examples of this type, the peptide or its analogues, fragments, or derivatives have a RAGE-Diaph1 binding site (RAGE 370-390 , RAGE 374-390 or RAGE 379-390 They have a Diaph1 binding site (e.g., 366A / 367A) that lacks or has been modified to disable or impair this site. Ideally, residues 366 / 367 are deleted or substituted with other residues (e.g., alanine) to impair or disable this site, thereby improving the affinity for binding to other targets by reducing the constraints caused by wild-type binding to Diaph1.

[0305] In one aspect of the present invention, the modulator of the present invention is defined by formula I: Z1 M Z2 (I) (In the formula, Z1 is either absent or selected from at least one proteinaceous moiety containing approximately 1 to 50 amino acid residues; M is the amino acid sequence described in Sequence ID No. 1 or its analogues, fragments, or derivatives; and Z2 is either absent or a proteinaceous region containing approximately 1 to 50 amino acid residues. It includes isolated or purified peptides that contain, consist of, or are essentially derived from the amino acid sequence represented by .

[0306] In some embodiments of the present invention described above, the modulator (such as a fragment of the cytoplasmic tail of RAGE, its analogues, or derivatives, as broadly described above and elsewhere herein) can permeate the cell membrane. In this non-limiting example, the RAGE modulator is conjugated, fused, or otherwise linked to a cell membrane permeability molecule (e.g., the HIV-TAT motif described in Sequence ID No. 4 below).

[0307] Sequence ID 4: [YGRKKRRQRRR].

[0308] In some embodiments of the present invention, the modulator is a non-peptide molecule that shares with the peptide modulator the ability to bind to and / or interfere with elements related to RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR. These non-peptide modulators may or may not have structural similarities to functionally important domains contained in the peptide modulator.

[0309] In a preferred embodiment, the non-peptide modulator includes structural similarities to functionally important domains contained in the peptide modulator, as represented by the pharmacophore described in paragraph [000318] of the International Publication of this Application.

[0310] In a preferred embodiment of the present invention, the modulator is an inhibitor.

[0311] In certain embodiments of the present invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, the modulator is an inhibitor of a specific coexisting GPCR and / or an inhibitor of a specific coexisting GPCR signaling pathway.

[0312] In certain embodiments of the present invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, the modulator is 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.

[0313] In a particular embodiment of the present invention, where the specific coexisting GPCR is AT1R, the modulator is an AT1R inhibitor and / or an inhibitor of the AT1R signaling pathway, in addition to being a RAGE ligand-independent inhibitor of RAGE activation.

[0314] In certain embodiments of the present invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by an activated angiotensin receptor, preferably activated AT1R, the modulator is 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.

[0315] In certain embodiments of the present invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, the modulator is an inhibitor of a specific coexisting GPCR and / or an inhibitor of a specific coexisting GPCR signaling pathway, an inhibitor of RAGE ligand-dependent activation of RAGE, and / or an inhibitor of constitutively activated RAGE, and / or an inhibitor of the RAGE signaling pathway.

[0316] In certain embodiments of the present invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by an activated angiotensin receptor, preferably activated AT1R, the modulator is an AT1R inhibitor and / or an inhibitor of the AT1R signaling pathway, an inhibitor of RAGE ligand-dependent activation of RAGE, and / or an inhibitor of constitutively activated RAGE, and / or an inhibitor of the RAGE signaling pathway.

[0317] In a particular embodiment of the present invention, where the specific coexisting GPCR is a specific chemokine receptor, preferably CCR2, the modulator is, in addition to being a RAGE ligand-independent inhibitor of RAGE activation, a specific chemokine receptor inhibitor, preferably a CCR2 inhibitor, and / or an inhibitor of a specific chemokine signaling pathway, preferably a CCR2 signaling pathway.

[0318] In certain embodiments of the present invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by an activated specific chemokine receptor, preferably activated CCR2, the modulator is 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.

[0319] In certain embodiments of the present invention, in addition to being an inhibitor of RAGE ligand-independent RAGE activation by an activated chemokine receptor, preferably activated CCR2, the modulator is a specific chemokine receptor inhibitor, preferably a CCR2 inhibitor and / or an inhibitor of a specific chemokine signaling pathway, preferably a CCR2 signaling pathway, and is 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.

[0320] In certain embodiments of the present invention, the modulator is a functional substitute for the cytoplasmic tail or a portion thereof of RAGE, which can be activated by certain coexisting GPCRs such as activated AT1R and activated CCR2, and induces downstream RAGE-dependent signaling in the presence or absence of wild-type RAGE expression.

[0321] In certain embodiments of the present invention, the modulator is a non-functional substitute for the cytoplasmic tail of RAGE or a portion thereof, the non-functional substitute is unable to be activated by coexisting GPCRs or to promote downstream RAGE-dependent signaling, and inhibits signaling that occurs through the cytoplasmic tail of RAGE and RAGE-dependent signaling.

[0322] In certain embodiments of the present invention, the modulator is a non-functional substitute for the transmembrane domain or a portion thereof of RAGE, which is unable to be activated by coexisting GPCRs or to facilitate downstream RAGE-dependent signaling, and inhibits signaling that occurs through the cytoplasmic tail of RAGE and RAGE-dependent signaling.

[0323] In certain embodiments of the present invention, the modulator comprises the transmembrane domain or a portion thereof of RAGE and a fragment of the extracellular domain of RAGE. In certain embodiments of the present invention, the modulator comprises the transmembrane domain or a portion thereof of RAGE and a fragment of the cytoplasmic tail of RAGE.

[0324] In certain embodiments of the present invention, the modulator includes the transmembrane domain or a portion thereof of RAGE, a fragment of the extracellular domain of RAGE, and a fragment of the cytoplasmic tail of RAGE.

[0325] In certain embodiments of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR comprises a fragment of the ligand-binding extradomain of human wild-type RAGE having a length of 40 amino acids or less, 20 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0326] The inventors have further discovered that a peptide containing residues 370-390 of the cytoplasmic tail of RAGE (see SEQ ID NO: 5) is an inhibitory peptide that inhibits both wild-type RAGE ligand-independent and RAGE ligand-dependent activation.

[0327] Sequence ID 5: [G 370 EERKAPENQEEEEERAELNQ 390 ].

[0328] RAGE 363-404Regarding this, a solution NMR structure exists (Rai V et al., 2012), and it has been shown that the N-terminus of this peptide (residues 363-376) is ordered. RAGE- 362-404 A Rosetta-derived model (Model 4) exists, which matches the NMR structure (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=2LMB, accessed August 25, 2016), and also suggests that the rest of the peptide forms an α-helix.

[0329] RAGE 370-390 The initial model was the Model 4 (Model 4_ 370-390 Model 4 was constructed by cleaving the ) . Model 4 is a theoretical model of the cytoplasmic tail of RAGE 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 presented by the I-Tasser server predicted that region 370-390 forms a helix. The models and NMR structures were aligned by the Cα carbon of the peptide sequence backbone. Model 4 was selected as the preferred model because the predicted structure of the region corresponding to the Diaphanous 1 binding site in Model 4 is the closest to the NMR structure recorded for this region.

[0330] We performed a 20ns molecular dynamics simulation of Model 4 in water using GROMACS (Hess et al., 2008). The molecular dynamics simulation was performed using Model 4_ 370-390 This suggests that the α-helix region is stable. Strong interactions were observed between numerous charged side chains, suggesting that these interactions stabilize the folded structure, and that arbitrary conservation of these residues may contribute to the stabilization of the peptide structure.

[0331] Using Blast search, RAGE 370-390Homologous sequences were identified. The sequences were aligned as follows: CLUSTAL 2.0.10 Multiple Array Alignment [ka] [ka] [ka]

[0332] Based on this analysis, RAGE was marked as follows: 370-390 Numerous strongly conserved residues were identified in this matrix. An asterisk (*) indicates a position with a single, completely conserved residue. A colon (:) indicates conservation between groups with strongly similar properties (score > 0.5 in the Gonnet PAM 250 matrix). A period (.) indicates conservation between groups with weakly similar properties (score < 0.5 in the Gonnet PAM 250 matrix).

[0333] [Table 24]

[0334] Highly conserved residues may play a structural role. Underlined residues are located on one face of a helix and may represent a binding pharmacophore.

[0335] Model 4_RAGE 370-390 Examination of the structural and molecular dynamics simulation results reveals the presence of numerous salt bridges within the structure. Molecular dynamics simulations indicate that these interactions are important structural features. Structural function is a possible reason for the conserved properties of these amino acids.

[0336] Many strongly conserved amino acids are not involved in salt bridge formation. These are RAGE 370-390These are present on one side of the helix and may represent a binding interface. These are Glu380, Glu384, Glu387, and Leu388. Another highly conserved residue, Glu377, is also present on this side of the peptide and may be involved in binding in addition to forming an α-helix stabilizing salt bridge to Lys374.

[0337] Alanine at the major hydrophobic residue L388 (e.g., L388A-RAGE) 370-390 ) substitution, when it acts on wild-type RAGE, 370-390 This results in a loss of inhibition achieved by the N-terminal truncation of RAGE (e.g., RAGE) which eliminates both 380 and 384. 385-390 and RAGE 385-404 ) results in a loss of the regulatory effect of these RAGE constructs. In contrast, the N-terminal truncates of RAGE that eliminate both 374 and 377 are inhibitors (RAGE 379-390 ) or as a functional substitute for wild-type RAGE (e.g., RAGE 379-404 This does not result in a loss of function of the RAGE peptide as such, indicating that these conserved residues (374 and 377) are not essential for regulatory activity, even if they may play a role in stabilizing the α-helical tertiary structure of the cytoplasmic tail of RAGE.

[0338] The inventors have determined that, in accordance with the conserved properties of these four amino acids representing the binding surface, a peptide (i.e., RAGE) containing only residues 379-390 of the cytoplasmic tail of RAGE is formed. 379-390 ) is an inhibitory peptide that inhibits both ligand-independent and ligand-dependent activation of wild-type RAGE, and RAGE 379-404 We further discovered that this can be activated by specific coexisting GPCRs in CHO cells.

[0339] In a preferred embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is E peptide Q as described in SEQ ID NO: 6.379 EEEEERAELNQ 390 or a derivative thereof.

[0340] Sequence ID 6: [Q 379 EEEEERAELNQ 390 ]

[0341] Structural Model 4_RAGE 370-390 RAGE 379-390 The pharmacophores of the peptides are shown below. [ka]

[0342] H4 is a hydrophobic residue, and P1-P3 are polar residues; distances are expressed in angstroms. The matrix of distances between sites is as follows, where P represents a polar site (hydrogen bond or charge) and H represents a hydrophobic site. Distances are in angstroms. A tolerance should be applied to the position of each point.

[0343] [Table 25]

[0344] Molecular dynamics simulations are performed using RAGE. 379-390 This indicates that if the interacting groups are mobile and the distance between sites is positive, a tolerance of up to ±10 Å should be applied to the position of each group.

[0345] As those skilled in the art will understand, by taking the above subset, additional smaller pharmacophores can be generated, and the present invention encompasses such pharmacophores, methods of such use for identifying compounds, and compounds thus identified.

[0346] In one embodiment, the present invention further comprises a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, comprising two or more features selected from the group: 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 distance between site points of the features is as follows, within a tolerance of up to ±10 Å when the magnitude of the distance between site points is positive:

[0347] [Table 26]

[0348] In a preferred embodiment of the present invention, when the magnitude of the distance between points is positive, the tolerance is a maximum of ±5 Å. In a preferred embodiment of the present invention, when the magnitude of the distance between points is positive, the tolerance is a maximum of ±2 Å. In a preferred embodiment of the present invention, when the magnitude of the distance between points is positive, the tolerance is a maximum of ±1 Å.

[0349] In a preferred embodiment of the present invention, the modulator includes three or more features selected from the group identified above.

[0350] In a preferred embodiment of the present invention, the modulator includes four features from the group identified above.

[0351] In one embodiment of the present invention, a modulator is provided that includes at least two features selected from the following combinations: AB, AC, AD, BC, BD, and CD.

[0352] In one embodiment of the present invention, a modulator is provided that includes at least three features selected from the following combinations: ABC, ABD, ACD, and BCD.

[0353] In one embodiment of the present invention, a modulator is provided that includes at least four features selected from one of the following combinations: A, B, C, and D.

[0354] In one embodiment of the present invention, the modulator is RAGE 370-390 A modulator is provided which comprises an additional charged or hydrogen-bonding group (P1) consistent with the conserved stabilizing effect of E377, and thus comprises two or more features selected from the group: 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 distance between the site points of the features is within a tolerance of ±10 Å, as follows:

[0355] [Table 27]

[0356] [ka] RAGE ligand-independent modulators of RAGE activation may be peptides or non-peptidyl compounds.

[0357] In one embodiment of the present invention, the hydrophobic group is an amino acid residue selected from the group: Ala, Val, Leu, Ile, Phe, Trp, Tyr.

[0358] In one embodiment of the present invention, the hydrophobic group is group:C 1~8 Alkyl, C 1~8 Alkenil, C 3~6 This is a chemical moiety selected from cycloalkyl, aryl, substituted aryl, alkylaryl, heteroaryl, and alkylheteroaryl.

[0359] "Alkyl" refers to an aliphatic hydrocarbon group, which can be linear or branched, and contains about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups contain about 1 to about 12 carbon atoms in the chain. Preferred alkyl groups contain about 1 to about 6 carbon atoms in the chain. A branched chain means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are bonded to a linear alkyl chain.

[0360] "Lower alkyl" refers to a group having approximately 1 to 6 carbon atoms in the chain, which may be linear or branched. Alkyl groups may be substituted with one or more substituents, which may be identical or different by choice, each substituent independently selected from the group consisting of halo, alkyl, aryl, cycloalkyl, cyano, hydroxy, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), -N(alkyl)2, carboxy, and -C(O)O-alkyl. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, and t-butyl.

[0361] "Alkenyl" refers to an aliphatic hydrocarbon group that may be linear or branched, containing about 2 to about 15 carbon atoms in the chain, and containing at least one carbon-carbon double bond. Preferred alkenyl groups have about 2 to about 12 carbon atoms in the chain, more preferably about 2 to about 4 carbon atoms in the chain. A branched chain means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are bonded to the linear alkenyl chain.

[0362] "Lower alkenyl" refers to a chain consisting of approximately 2 to 6 carbon atoms, which may be linear or branched. Non-restrictive 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 identical or different, and each substituent is independently selected from the group consisting of alkyl, aryl, and cycloalkyl groups.

[0363] "Alkynyl" refers to an aliphatic hydrocarbon group that may be linear or branched, containing at least one carbon-carbon triple bond and having about 2 to about 15 carbon atoms in the chain. A preferred alkynyl group has about 2 to about 12 carbon atoms in the chain, more preferably about 2 to about 4 carbon atoms. A branched chain means that one or more lower alkyl groups, such as methyl, ethyl, or propyl, are bonded to the linear alkynyl chain.

[0364] "Lower alkynyl" refers to a chain consisting of approximately 2 to 6 carbon atoms, which may be linear or branched. Non-restrictive 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 identical or different, and each substituent is independently selected from the group consisting of alkyl, aryl, and cycloalkyl groups.

[0365] "Aliphatic" means, and includes, paraffins, olefins, or acetylene carbon atoms in a straight or branched chain. An aliphatic group can be optionally substituted with one or more substituents that may be identical or different, each substituent being H, halo, halogen, alkyl, aryl, cycloalkyl, cycloalkylamino, alkenyl, heterocyclic, alkynyl, cycloalkylaminocarbonyl, hydroxyl, 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, aralkoxy, acyl, aroyl, nitro, amino, amide, ester, aryloxycarbonyl carboxylate A molecule is independently selected from the group consisting of hydrogen, alkyl, aryl, and aralkyl, where Y1 and Y2 may be the same or different.

[0366] "Heteroaliphatic" refers to a material that contains at least one heteroatom (such as oxygen, nitrogen, or sulfur) and is otherwise an aliphatic group. The term heteroaliphatic includes substituted heteroaliphatic groups.

[0367] "Aryl" means an aromatic monocyclic or polycyclic ring system containing about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group may be identical or different and may be optionally substituted with one or more "ring system substituents" as defined herein. Non-limiting examples of suitable aryl groups include phenyl and naphthyl.

[0368] "Heteroalkyl" means an alkyl group as defined above, in which one or more hydrogen atoms are substituted with heteroatoms selected from N, S, or O.

[0369] "Heteroaryl" means an aromatic monocyclic or polycyclic ring system containing about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, where one or more ring atoms are elements other than carbon, such as nitrogen, oxygen, or sulfur, alone or in combination. Preferred heteroaryls contain about 5 to about 6 ring atoms. A "heteroaryl" may be optionally substituted with one or more "ring system substituents," which may be the same or different, as defined herein. The prefixes aza, oxa, or thia preceding the root name of a heteroaryl mean that at least one nitrogen, oxygen, or sulfur atom is present as a ring atom, respectively. The nitrogen atom of a heteroaryl may optionally be oxidized to the corresponding N-oxide. Non-limiting examples of appropriate heteroaryls include pyridyl, pyrazinyl, furanil, thienyl, pyrimidinyl, pyridone (including N-substituted pyridone), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, flazanil, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridadinyl, quinoxalinyl, phthalazinyl, oxyindolyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzoflazanil, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindryl, 1,2,4-triazinyl, and benzothiazolyl. The term "heteroaryl" also refers to partially saturated heteroaryl moieties, such as tetrahydroisoquinolyl and tetrahydroquinolyl.

[0370] "Aralkyl" or "arylalkyl" refers to an aryl-alkyl group where aryl and alkyl are as described above. Preferred aralkyls include lower alkyl groups. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyl, and naphthalenylmethyl. Bonding to the parent moiety is via alkyl.

[0371] "Alkylaryl" refers to an alkyl-aryl group where alkyl and aryl are as described above. Preferred alkylaryls include lower alkyl groups. A non-limiting example of a suitable alkylaryl group is a toryl. Bonding to the parent moiety is via the aryl group.

[0372] "Cycloalkyl" means a non-aromatic monocyclic or polycyclic ring system containing about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. A preferred cycloalkyl ring contains about 5 to about 7 ring atoms. A cycloalkyl can be optionally substituted with one or more "ring system substituents" which may be the same or different and as defined above. Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. Non-limiting examples of suitable polycyclic cycloalkyls include 1-decalinyl, norbornyl, adamantyl, etc., and partially saturated species such as indanyl, tetrahydronaphthyl, etc. "Halogen" means fluorine, chlorine, bromine, or iodine. Fluorine, chlorine, and bromine are preferred.

[0373] A "cyclic substituent" refers to a substituent bonded to an aromatic or non-aromatic ring system that replaces an available hydrogen on the ring system, for example. Cyclic substituents may be the same or different, and are each under the names alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, and heteroaryl. A molecule independently selected from the group consisting of sulfonyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, heterocyclyl, -C(=N-CN)-NH2, -C(=NH)-NH2, -C(=NH)-NH(alkyl), Y1Y2N-, Y1Y2N-alkyl-, Y1Y2NC(O)-, Y1Y2NSO2-, and -SO2NY1Y2, where Y1 and Y2 may be the same or different, and independently selected from the group consisting of hydrogen, alkyl, aryl, cycloalkyl, and aralkyl. A "cyclic substituent" may also mean a single molecule that simultaneously replaces two available hydrogens (one H on each carbon) on two adjacent carbon atoms in a cyclic system. Examples of such molecules are methylenedioxy, ethylenedioxy, -C(CH3)2-, etc., which form molecules such as: [ka]

[0374] It should be noted that in the heteroatom-containing ring system of the present invention, there are no hydroxyl groups on carbon atoms adjacent to N, O, or S1, and there are no N or S groups on carbon atoms adjacent to other heteroatoms. Therefore, for example, in the ring, [ka] There are no -OH groups directly bonded to the carbons labeled 2 and 5.

[0375] For example, the chemistry part: [ka] It should be noted that other tautomer forms are also considered equivalents in certain embodiments of the present invention.

[0376] "Alkynylalkyl" refers to an alkynyl-alkyl group where alkynyl and alkyl are as described above. Preferred alkynylalkyls include lower alkynyl and lower alkyl groups. Bonding to the parent moiety is via alkyl. A non-limiting example of a suitable alkynylalkyl group is propargylmethyl.

[0377] "Heteroaralkyl" refers to a heteroaryl-alkyl group where heteroaryl and alkyl are as described above. Preferred heteroaralkyls include lower alkyl groups. Non-limiting examples of suitable aralkyl groups include pyridylmethyl and quinoline-3-ylmethyl. Bonding to the parent moiety is via alkyl.

[0378] "Hydroxyalkyl" means an HO-alkyl group, as previously defined. Preferred hydroxyalkyls include lower alkyls. Non-limiting examples of suitable hydroxyalkyls include hydroxymethyl and 2-hydroxyethyl.

[0379] "Acyl" refers to an HC(O)-, alkyl-C(O)-, or cycloalkyl-C(O)- group, and the various groups are as described above. Bonding to the parent moiety is via a carbonyl group. Preferred acyls include lower alkyl groups. Non-limiting examples of suitable acyl groups include formyl, acetyl, and propanoyl.

[0380] "Aroyl" refers to the aryl-C(O)- group, as described above. The bond to the parent moiety is via a carbonyl group. Non-restrictive examples of suitable groups include benzoyl and 1-naphthoyl.

[0381] "Alkoxy" refers to an alkyl-O-group whose alkyl group is as described above. Non-exclusive examples of suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, and n-butoxy. Bonding to the parent moiety is via an ether oxygen.

[0382] "Aryloxy" refers to an aryl-O- group, where the aryl group is as described above. Non-restrictive examples of suitable aryloxy groups include phenoxy and naphthoxy. Bonding to the parent moiety is via an ether oxygen.

[0383] "Alkylthio" refers to an alkyl-S-group where the alkyl group is as described above. Non-restrictive examples of suitable alkylthio groups include methylthio and ethylthio. Bonding to the parent moiety is via sulfur.

[0384] "Arylthio" refers to an aryl-S group, where the aryl group is as described above. Non-restrictive examples of suitable arylthio groups include phenylthio and naphthylthio. Bonding to the parent moiety is via sulfur.

[0385] "Aralkylthio" refers to an aralkyl-S-group, where the aralkyl group is as described above. A suitable non-restrictive example of an aralkylthio group is benzylthio. The bond to the parent moiety is via sulfur.

[0386] "Alkoxycarbonyl" refers to an alkyl-O-CO- group. Non-exclusive examples of suitable alkoxycarbonyl groups include methoxycarbonyl and ethoxycarbonyl groups. Bonding to the parent moiety is via a carbonyl group.

[0387] "Aralkoxycarbonyl" refers to an aralkyl-OC(O)- group. A non-restrictive example of a suitable aralkoxycarbonyl group is benzyloxycarbonyl. Bonding to the parent moiety is via a carbonyl group.

[0388] "Alkylsulfonyl" refers to an alkyl-S(O2)- group. A preferred group is one in which the alkyl group is a lower alkyl group. Bonding to the parent moiety is via a sulfonyl group.

[0389] "Arylsulfonyl" refers to the aryl-S(O2)- group. The bond to the parent molecule is via a sulfonyl group.

[0390] The term "substitution" means that one or more hydrogen atoms on a given atom are replaced by a selection of a given group, provided that the substitution does not exceed the normal valence of the given atom in its current state and the substitution results in a stable compound. Substituents and / or combinations of variables are permitted only if such combinations result in a stable compound.

[0391] A "stable compound" or "stable structure" means a compound that is strong enough to withstand isolation from a reaction mixture to a useful purity and formulation into an effective therapeutic agent.

[0392] The term "optionally substituted" means optional substitution of a particular group, radical, or part.

[0393] When a functional group in a compound is referred to as "protected," it means that the group is in a modified form to prevent undesirable side reactions at the protected site when the compound is subjected to a reaction. Appropriate protecting groups will be recognized by those skilled in the art and will also be recognized by referring to standard texts such as Greene et al (1991).

[0394] If any component or any variable (e.g., aryl, complex algebra, R²) appears multiple times in the present invention, its definition in each appearance is independent of its definition in all other appearances.

[0395] In one embodiment of the present invention, each of the charged or hydrogen bonding groups is an amino acid residue independently selected from the groups Asp and Glu.

[0396] In one embodiment of the present invention, each of the charged or hydrogen bonding groups is an amino acid residue having a carboxylic acid moiety.

[0397] In one embodiment of the present invention, each of the charged or hydrogen bonding groups is a chemical moiety independently selected from other cyclic and acyclic structures, including the group: carboxylic acids, hydroxy acids, phosphonic acids and phosphinic acids, sulfonic acids and sulfinic acids, sulfonamides, acylsulfonamides and sulfonylureas, 2,2,2-trifluoroethane-1-ol and trifluoromethyl ketones, tetrazoles, 5-oxo-1,2,4-oxadiazole and 5-oxo-1,2,4-thiadiazole, thiazolidinediones, oxazolidinedione and oxadiazolidinediones, 3-hydroxyisoxazole and 3-hydroxyisothiazole, substituted phenols, squalic acid, 3- and 4-hydroxyquinoline-2-one, tetranic acid and tetramic acid, cyclopentan-1,3-dione and boronic acid, mercaptoazoles, and sulfonimidamides (Ballatore et al., 2013).

[0398] In one embodiment, the present invention provides a method for identifying modulators of RAGE ligand-independent RAGE activation by specific activated coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, the method comprising: (1) comparing the three-dimensional structure of a compound with a pharmacophore comprising two or more features selected from the group: 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 distance between the features is within a tolerance of ±10 Å, as follows:

[0399] [Table 28] (2) The step of selecting a compound having a hydrophobic and / or charged or hydrogen-bonding chemical moiety arranged in such a manner.

[0400] Modulators of RAGE ligand-independent RAGE activation identified by the methods described above, including comparison with pharmacophores, may be peptides or non-peptidyl compounds.

[0401] In a preferred embodiment of the present invention, when the magnitude of the distance between points is positive, the tolerance is a maximum of ±5 Å. In a preferred embodiment of the present invention, when the magnitude of the distance between points is positive, the tolerance is a maximum of ±2 Å. In a preferred embodiment of the present invention, when the magnitude of the distance between points is positive, the tolerance is a maximum of ±1 Å.

[0402] In a preferred embodiment of the present invention, the modulator includes three or more features selected from the group identified above.

[0403] In a preferred embodiment of the present invention, the modulator includes four features from the group identified above.

[0404] In one embodiment of the present invention, the comparison between the three-dimensional structure of a compound and a pharmacophore is accompanied by a comparison between the minimum energy structure of the compound and a pharmacophore.

[0405] An efficient means of selecting a compound from a potentially large number of compounds involves comparing the compound with the pharmacophore of the present invention using a computer program, such as Catalyst(MSl), to screen one or more computerized databases of the three-dimensional chemical structures of the compounds.

[0406] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is a peptide having the amino acid sequence described in SEQ ID NO: 1, or an analog, fragment, or derivative thereof containing at least residues 379-390.

[0407] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is a peptide of the formula of Sequence ID No. 1, or an analog or derivative thereof.

[0408] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is a peptide of the formula of Sequence ID No. 2, or an analog or derivative thereof.

[0409] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is a peptide of the formula of SEQ ID NO: 5, or an analog or derivative thereof.

[0410] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is a peptide of the formula of SEQ ID NO: 6, or an analog or derivative thereof.

[0411] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is the S391A-E392X-RAGE peptide described in Sequence ID No. 7, or an analog or derivative thereof.

[0412] Sequence ID 7: [L 362 WQRRQRRGEERKAPENQEEEEERAELNQA 391 ]

[0413] In one embodiment of the present invention, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is the S391X-RAGE peptide described in Sequence ID No. 8, or an analog or derivative thereof.

[0414] Sequence ID 8: [L 362 WQRRQRRGEERKAPENQEEEEERAELNQ 390 ]

[0415] A preferred specific derivative is Q as described in Sequence ID No. 9 below. 379 EEEEERAELNR 390 Q as described in Sequence ID No. 10 379 EEEEERAELNK 390 , K as described in Sequence ID No. 11 379 EEEEERAELNQ 390 , K as described in Sequence ID No. 12 379 EEEERAELNK 390 and K described in Sequence ID No. 13 379 EEEEERAELNR 390 Includes. Sequence ID 9: [Q 379 EEEEERAELNR 390 ] Sequence ID 10: [Q 379 EEEEERAELNK 390 ] Sequence ID 11:[K 379 EEEEERAELNQ 390 ] Sequence ID 12:[K 379 EEEEERAELNK 390 ] Sequence ID 13:[K 379 EEEEERAELNR 390 ]

[0416] As used herein in relation to modulators of the present invention such as SEQ ID NOs: 1, 2, 5-13, the term "derivative" means that its primary structure is obtained from or derived from the C-terminal cytoplasmic tail or fragment thereof of RAGE, but is subject to addition, substitution, cleavage, chemical and / or biochemical modification of amino acids (acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, side-chain methylation), labeling with radionucleotides or halogens, abnormal or artificial amino acids (D-amino acids, N-methylated amino acids, tetrasubstituted, β-peptides, pyroglutamic acid; 2-aminoadipic acid; 3-aminoadipic acid; β-alanine; β-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid; piperidine acid; 6-aminocaproic acid; 2-A This refers to a modulator characterized by comprising minoheptanoic 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, etc.), retroinverted sequences, cyclic peptides, peptoids or non-peptide drugs, non-peptide labeling, and linkage to non-peptide carriers or non-peptide resins.

[0417] The inventors have further discovered that a peptide containing 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 activation.

[0418] Substitutions encompass amino acid changes in which an amino acid is replaced by a different native or atypical amino acid residue. Such substitutions can be classified as “conservative,” in which case the amino acid residue in the polypeptide is replaced by another native amino acid with similar properties in terms of polarity, side-chain functionality, or size, for example, Ser⇔Thr⇔Pro⇔Hyp⇔Gly⇔Ala, Val⇔Ile⇔Leu, His⇔Lys⇔Arg, Asn⇔Gln⇔Asp⇔Glu, or Phe⇔Trp⇔Tyr. It should be understood that some atypical amino acids can also be suitable substitutes for naturally occurring amino acids. For example, ornithine, homoarginine, and dimethyllysine are related to His, Arg, and Lys, respectively.

[0419] Substitutions included in the present invention may be "non-conservative," in which an amino acid residue present in a polypeptide is replaced with an amino acid having different properties, such as a naturally occurring amino acid from a different group (for example, replacing a charged or hydrophobic amino acid with alanine), or a naturally occurring amino acid is replaced with an unconventional amino acid.

[0420] Amino acid substitutions are typically single-residue substitutions, but can also be clustered or dispersed substitutions of multiple residues. Preferably, amino acid substitutions are conserved.

[0421] Addition encompasses the addition of one or more native or unconventional amino acid residues. Deletion encompasses the deletion of one or more amino acid residues.

[0422] As described above, the present invention includes peptides in which one or more amino acids have undergone side-chain modification. Examples of side-chain modifications intended by the present invention include reductive alkylation by reaction with an aldehyde and subsequent reduction with NaBH4; amidine by methyl acetimidate; acylation by acetic anhydride; carbamoylation of the amino group by cyanate; trinitrobenzylation of the amino group by 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of the amino group by succinic anhydride and tetrahydrophthalic anhydride; and modification of the amino group by pyridoxylation of lysine with pyridoxal-5-phosphate and subsequent reduction with NaBH4.

[0423] The guanidine group of the arginine residue can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal.

[0424] The carboxyl group can be modified by carbodiimide activation via O-acyl isourea formation and subsequent derivatization, for example, by derivatization to the corresponding amide. The sulfhydryl group can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation of performic acid to cysteic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides; formation of mercury derivatives using 4-chloromercurybenzoic acid, 4-chloromercuryphenylsulfonic acid, phenylmercury chloride, 2-chloromercury-4-nitrophenol and other mercury agents; and carbamoylation with cyanate at an alkaline pH. In a preferred embodiment of the present invention, any modification of the cysteine ​​residue should not affect the peptide's ability to form the required disulfide bonds. It is also possible to replace the sulfhydryl group of cysteine ​​with a selenium equivalent so that the peptide forms a diselenium bond instead of one or more disulfide bonds.

[0425] The tryptophan residue can be modified, for example, by oxidation with N-bromosuccinimide or by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halide. On the other hand, the tyrosine residue can be altered by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0426] Modification of the imidazole ring of histidine residues can be achieved by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethyl pyrocarbonate. Proline residues can be modified, for example, by hydroxylation at position 4.

[0427] The following table lists some amino acids with modified side chains and other unnatural amino acids.

[0428] [Table 29]

[0429] These types of modifications may be important for stabilizing peptides when administered to individuals or used as diagnostic reagents.

[0430] Conservative amino acid substitutions used herein can include amino acid residues within a group having sufficiently similar physicochemical properties such that substitutions between members of the group preserve 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 acid is 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 chain, side chains capable of entering into a hydrogen bridge, e.g., amino acids with side chains having a hydroxyl function group). In the present case, conservative substitutions are, for example, substituting a basic amino acid residue (Lys, Arg, His) with another basic amino acid residue (Lys, Arg, His), substituting an aliphatic amino acid residue (Gly, Ala, Val, Leu, Ile) with another aliphatic amino acid residue, substituting an aromatic amino acid residue (Phe, Tyr, Trp) with another aromatic amino acid residue, substituting threonine with serine or leucine with isoleucine. Further conservative amino acid exchanges will be known to those skilled in the art. The isoform should preferably be maintained, for example, K preferably substitutes R or H, and k preferably substitutes r and h.

[0431] When considering amino acid substitutions, preferred substitutions of the present invention are those described by Grantham, R. (1974) as having a D of less than 100, the content of which is incorporated by reference. The most preferred alternatives are those described as having a D of less than 50.

[0432] The peptide modulators of the present invention include peptides formed by retro-inverso isomers or modified or substituted variants or additions or deletions of SEQ ID NO: 1, 2, 5, 6, 7, 8, 9, 10, 11, 12 or 13 (Li et al., 2010).

[0433] <Method for treating, preventing or managing RAGE-related disorders> In another related aspect, the present invention provides a method for treating, preventing or managing RAGE-related disorders in patients requiring such treatment, the method comprising administering an effective dose of a RAGE ligand-independent RAGE activation modulator by a specific activated coexisting GPCR of the present invention.

[0434] In another aspect, the present invention includes the use of a RAGE ligand-independent RAGE activation modulator by a specific activated coexisting GPCR for the manufacture of a drug for treating, preventing or managing RAGE-related disorders in patients requiring such treatment.

[0435] In another embodiment, the present invention includes the use of a RAGE ligand-independent RAGE activation modulator by a specific activated coexisting GPCR for treating, preventing, or managing RAGE-related disorders in patients requiring such treatment.

[0436] In a preferred embodiment of the present invention, the specific coexisting GPCR is an angiotensin receptor. In a preferred embodiment of the present invention, the specific coexisting GPCR is AT1R.

[0437] In a preferred embodiment of the present invention, the specific coexisting GPCR is a specific chemokine receptor. In a preferred embodiment of the present invention, the specific coexisting GPCR is CCR2.

[0438] Furthermore, the present invention provides a method for treating, preventing, or managing RAGE-related disorders in patients requiring such treatment, the method comprising administering an effective amount of a combination of the RAGE ligand-independent RAGE activation modulator by a specific activated coexisting GPCR of the present invention and a modulator of a specific coexisting GPCR and / or a modulator of a specific coexisting GPCR signaling pathway.

[0439] In a preferred embodiment of the present invention, the specific coexisting GPCR is an angiotensin receptor. In a preferred embodiment of the present invention, the specific coexisting GPCR is AT1R.

[0440] In a preferred embodiment of the present invention, the specific coexisting GPCR is a specific chemokine receptor. In a preferred embodiment of the present invention, the specific coexisting GPCR is CCR2.

[0441] The method may involve administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by a specific coexisting GPCR of the present invention and the modulator of the specific coexisting GPCR and / or the modulator of a specific coexisting GPCR signaling pathway, wherein the modulator of the specific coexisting GPCR and / or the modulator of the specific coexisting GPCR signaling pathway is administered at a lower dose than that typically administered for treating impairments related to the specific coexisting GPCR.

[0442] The method may involve administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by a specific coexisting GPCR of the present invention and the modulator of the specific coexisting GPCR and / or the modulator of a specific coexisting GPCR signaling pathway, wherein the modulator of the specific coexisting GPCR and / or the modulator of the specific coexisting GPCR signaling pathway is administered at a lower dose than that typically administered for the treatment of RAGE impairment.

[0443] In a particularly preferred embodiment of the present invention, the method comprises administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by the activated angiotensin receptor of the present invention and the modulator of AT1R and / or the modulator of the AT1R signaling pathway, wherein the modulator of AT1R and / or the modulator of the AT1R signaling pathway is administered at a lower dose than that typically administered for the treatment of AT1R-related disorders.

[0444] In another particularly preferred embodiment of the present invention, the method comprises administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by a specific activated chemokine receptor of the present invention and the modulator of CCR2 and / or the modulator of the CCR2 signaling pathway, wherein the modulator of CCR2 and / or the modulator of the CCR2 signaling pathway is administered at a lower dose than that typically administered for the treatment of CCR2-related disorders.

[0445] Furthermore, the present invention provides a method for treating, preventing or managing RAGE-related disorders in patients requiring such treatment, the method comprising administering an effective amount of a combination of the RAGE ligand-independent RAGE activation modulator by a specific coexisting GPCR of the present invention, a RAGE ligand-dependent activation modulator of RAGE, and / or a constitutively active RAGE modulator, and / or a RAGE signaling pathway modulator.

[0446] In a particularly preferred embodiment of the present invention, the method comprises administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by a specific coexisting GPCR of the present invention, the modulator of RAGE ligand-dependent activation of RAGE, and / or the modulator of constitutively active RAGE, and / or the modulator of the RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE, and / or the modulator of constitutively active RAGE, and / or the modulator of the RAGE signaling pathway is administered at a lower dose than that typically administered for the treatment of RAGE-related disorders.

[0447] Furthermore, the present invention provides a method for treating, preventing, or managing RAGE-related disorders in patients requiring such treatment, the method comprising administering an effective amount of a combination of the RAGE ligand-independent RAGE activation modulator of the present invention by an activated specific coexisting GPCR and a modulator of a specific coexisting GPCR and / or a modulator of a specific coexisting GPCR signaling pathway.

[0448] For example, the present invention provides a method for treating, preventing or managing RAGE-related disorders in patients requiring such treatment, the method comprising administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by the activated angiotensin receptor of the present invention, a modulator of RAGE ligand-dependent activation of RAGE, and / or a modulator of constitutively activated RAGE, and / or a modulator of the RAGE signaling pathway, and a modulator of AT1R and / or a modulator of the AT1R signaling pathway.

[0449] For example, the present invention provides a method for treating, preventing or managing RAGE-related disorders in patients requiring such treatment, the method comprising administering an effective amount of a combination of the RAGE ligand-independent RAGE activation modulator by a specific activated chemokine receptor of the present invention, a RAGE ligand-dependent activation modulator of RAGE, and / or a constitutively activated RAGE modulator, and / or a RAGE signaling pathway modulator, and a CCR2 modulator and / or a CCR2 signaling pathway modulator.

[0450] In a particularly preferred embodiment of the present invention, the method comprises administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by a specific coexisting GPCR of the present invention, the modulator of a specific coexisting GPCR and / or the modulator of a signaling pathway of a specific coexisting GPCR, and the modulator of RAGE ligand-dependent activation of RAGE and / or the modulator of constitutively active RAGE and / or the modulator of a RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE and / or the modulator of constitutively active RAGE and / or the modulator of a RAGE signaling pathway is administered at a lower dose than that normally administered for the treatment of RAGE-related disorders, and / or the modulator of a specific coexisting GPCR and / or the modulator of a signaling pathway of a specific coexisting GPCR is administered at a lower dose than that normally administered for the treatment of disorders related to GPCRs.

[0451] In a particularly preferred embodiment of the present invention, the method comprises administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by the activated angiotensin receptor of the present invention, the modulator of AT1R and / or the modulator of the AT1R signaling pathway, and the modulator of RAGE ligand-dependent activation of RAGE and / or the modulator of constitutively active RAGE and / or the modulator of the RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE and / or the modulator of constitutively active RAGE and / or the modulator of the RAGE signaling pathway is administered at a lower dose than that normally administered for the treatment of RAGE-related disorders, and / or the modulator of AT1R and / or the modulator of the AT1R signaling pathway is administered at a lower dose than that normally administered for the treatment of AT1R-related disorders.

[0452] In a particularly preferred embodiment of the present invention, the method comprises administering an effective amount of a combination of the modulator of RAGE ligand-independent RAGE activation by a specific activated chemokine receptor of the present invention, a modulator of CCR2 and / or a modulator of the CCR2 signaling pathway, and a modulator of RAGE ligand-dependent activation of RAGE and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway, wherein the modulator of RAGE ligand-dependent activation of RAGE and / or a modulator of constitutively active RAGE and / or a modulator of the RAGE signaling pathway is administered at a lower dose than that normally 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 lower dose than that normally administered for the treatment of CCR2-related disorders.

[0453] RAGE-related disorders are defined as disorders that depend on RAGE expression. This does not exclude disorders related to specific co-existing GPCRs, such as AT1R-related disorders or CCR2-related disorders, which also depend on RAGE expression. In fact, disorders can be RAGE-related or related to specific co-existing GPCRs, including AT1R-related or CCR2-related disorders.

[0454] Disorders associated with specific co-existing GPCRs are defined as disorders that depend on the expression of those specific co-existing GPCRs. This does not exclude RAGE-related disorders, which also depend on the expression of specific co-existing GPCRs. In fact, disorders can be RAGE-related or related to specific co-existing GPCRs, including AT1R-related or CCR2-related disorders.

[0455] In one embodiment of the present invention, RAGE-related disorders are disorders selected from the group: cardiovascular disorders; gastrointestinal disorders; cancer; neurological disorders, respiratory disorders, connective tissue disorders, renal disorders, reproductive disorders, skin disorders, ocular disorders, and endocrine disorders.

[0456] In one embodiment of the present invention, RAGE-related disorders are cardiovascular disorders selected from the group: 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.

[0457] In one embodiment of the present invention, RAGE-related disorders are gastrointestinal disorders selected from the following groups: periodontitis, esophagitis, gastritis, gastric and duodenal ulcers, Crohn's disease, ulcerative colitis, ischemic colitis, enteritis and enterocolitis, peritonitis, alcoholic liver disease, hepatitis, toxic liver disease, biliary cirrhosis, hepatic fibrosis / cirrhosis, non-alcoholic fatty liver / non-alcoholic steatohepatitis (NAFLD / NASH), hepatic trauma and liver injury, and recovery from trauma or surgery.

[0458] In one embodiment of the present invention, RAGE-related disorders are cancers selected from the group: malignant neoplasms of the lips, oral cavity and pharynx; malignant neoplasms of the digestive tract; malignant neoplasms of the respiratory tract and intrathoracic organs; malignant neoplasms of bone and articular cartilage; melanoma and other malignant neoplasms of the skin; malignant neoplasms of mesothelial and soft tissues; malignant neoplasms of the breast; malignant neoplasms of the female reproductive organs; malignant neoplasms of the male reproductive organs; malignant neoplasms of the urinary tract; malignant neoplasms of the eye, brain and other parts of the central nervous system; malignant neoplasms of the thyroid and other endocrine glands; malignant neoplasms of the lymphatic system, hematopoietic system and related tissues; and malignant neoplasms of unclear, secondary and / or unspecified sites.

[0459] In one embodiment of the present invention, RAGE-related disorders are neurological disorders and are selected from the following groups: inflammatory diseases of the central nervous system, generalized atrophy affecting the central nervous system, extrapyramidal and motor disorders, Parkinson's disease, demyelinating diseases of the central nervous system, Alzheimer's disease, focal cerebral atrophy, Lewy body disease, epilepsy, migraine, neuropathic pain, diabetic neuropathy, polyneuropathy, development and progression of glioma, spinal cord injury, ischemic brain injury / stroke, traumatic brain injury and brain injury, recovery from trauma or surgery.

[0460] In one embodiment of the present invention, RAGE-related disorder is a mental disorder selected from the group: dementia, Alzheimer's disease, vascular dementia, addiction, schizophrenia, major affective disorders, depression, mania, bipolar disorder, and anxiety disorders.

[0461] In one embodiment of the present invention, RAGE-related disorders are respiratory (lung) disorders and are selected from the following groups: acute upper respiratory tract infections, 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 causes, acute respiratory distress syndrome (ARDS), pulmonary eosinophilia and pleurisy, lung trauma and lung injury, recovery from trauma or surgery.

[0462] In one embodiment of the present invention, RAGE-related disorders are connective tissue disorders and are selected from the following groups: osteoarthritis, infectious arthritis, rheumatoid arthritis, psoriatic and enteric arthropathy, juvenile arthritis, gout and other crystalline arthropathy, diabetic arthropathy, polyarteritis nodosa, Churg-Strauss syndrome, mucocutaneous lymphadenopathy [Kawasaki], hypersensitivity vasculitis, Goodpasture syndrome, thrombotic microangiopathy, Wegener's granulomatosis, aortic arch syndrome [Takayasu], giant cell arteritis, polymyalgia rheumatica, microscopic polyangiitis, hypovasculitis, systemic lupus erythematosus, cutaneous polymyositis, polymyositis, systemic sclerosis, CR(E)ST syndrome, xerosis syndrome [Sjögren's], mixed connective tissue disease, Behçet's disease, traumatic muscle injury, sprain, muscle contusion and fracture.

[0463] In one embodiment of the present invention, RAGE-related disorder is a renal disorder selected from the following groups: glomerulonephritis, nephritis, diabetic nephropathy, interstitial nephritis, obstructive and reflux nephropathy, acute renal failure, and chronic renal disease.

[0464] In one embodiment of the present invention, RAGE-related disorders are reproductive disorders and are selected from the group: prostatitis, benign prostatic hyperplasia, prostatic dysplasia, salpingitis, oophoritis, pelvic inflammatory disease (PID), polycystic ovary syndrome, cervicitis, cervical dysplasia, vaginitis, and vulvitis.

[0465] In one embodiment of the present invention, RAGE-related disorders are skin disorders selected from the group: dermatitis, eczema, pemphigus / bullous pemphigoid, psoriasis, pityriasis rosea, lichen planus, urticaria, erythema multiforme, erythema nodosum, sunburn, keratosis, photoaging skin ulcers, superficial skin injuries, and open wounds.

[0466] In one embodiment of the present invention, RAGE-related disorders are ocular disorders selected from the group: keratitis, conjunctivitis, retinitis, glaucoma, scleritis, episcleritis, chorioretinal inflammation, diabetic retinopathy, macular edema, retinopathy of prematurity, and optic neuritis, as well as ocular trauma and recovery from ocular injury, trauma or surgery.

[0467] In one embodiment of the present invention, RAGE-related disorders are endocrine disorders selected from the group: diabetes mellitus, insulin resistance, impaired glucose tolerance, and thyroiditis.

[0468] In one embodiment of the present invention, inhibitors that inhibit AT1R or the AT1R signaling pathway are selected from the group: eprosartan (trade name Teveten®, Abbott Laboratories USA), losartan (trade name Cozaar®, Merck & Co), valsartan (trade name Diovan®, Novartis), telmisartan (trade name Micardis®, Boehringer Ingelheim), irbesartan (trade name Avapro®, SanofiAventis), olmesartan (trade name Benicar®, Daiichi Sankyo Inc), azilsartan (trade name Edarbi, Takeda), candesartan (trade name Atacand®, AstraZeneca), ZD-7115, salacin ((Sar1-Ala8)Ang II), sarthran ((Sar1-Thr8)Ang II), and DuP753. This list also includes prodrugs of these inhibitors, which include candesartan (candesartan cilexetil), azilsartan (azilsartan medoxomil), and olmesartan (olmesartan medoxomil), and may be in the form in which they are administered or as active metabolites (such as EXP-3174, the active metabolite of losartan). Note that partial agonists may not produce the maximum effect even if they exhibit agonism, so partial agonists can act to inhibit endogenous Ang II and therefore may act as therapeutic inhibitors.

[0469] In one embodiment of the present invention, inhibitors that inhibit a specific chemokine receptor or a specific chemokine signaling pathway are grouped as: propagermanium (also known as 3-[(2-carboxyethyl-oxogermyl)oxy-oxogermyl]propanoic acid, proxigermanium, Ge-132, bis(2-carboxyethylgermanium)sesquioxide (CEGS), 2-carboxyethylgermasesquioxane, SK-818, organic germanium, germanium sesquioxide, 3,3'-(1,3-dioxo-1,3-digermanoxanediyl)bispropionic acid, 3-oxygermylpropionic acid polymer, poly-trans-(2-carboxyethyl)germasesquioxane, proxigermanium, repagermanium and cerocion; CCR2), BMS CCR2 22 (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); selected from 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), SB 225002 (CXCR2), and SB 265610 (CXCR2).

[0470] In one embodiment of the present invention, inhibitors that inhibit CCR2 or the CCR2 signaling pathway include: propagermanium (also known as 3-[(2-carboxyethyl-oxogermyl)oxy-oxogermyl]propanoic acid, proxigermanium, Ge-132, bis(2-carboxyethylgermanium)sesquioxide (CEGS), 2-carboxyethylgermasesquioxane, SK-818, organic germanium, germanium trioxide, 3,3'-(1,3-dioxo-1,3-digermanoxanediyl)bispropionic acid, 3-oxygermylpropionic acid polymer, poly-trans-(2-carboxyethyl)germasesquioxane, proxigermanium, repagermanium, and celocion), BMS CCR2 The following are selected: 22 (CCR2), Resveratrol (CCR2), RS504393, RS102895, MLN-1202 (Millennium Pharmaceuticals), INCB8696 (Incyte Pharmaceuticals), MK-0812 (Merck), CCX140 (ChemoCentryx), PF-4136309 (Pfizer), and BMS-741672 (Bristol-Myers Squibb).

[0471] In one embodiment of the present invention, inhibitors of RAGE ligand-dependent activation of RAGE, and / or inhibitors of constitutively active RAGE, and / or inhibitors of the RAGE signaling pathway include: azerylagon (TTP488 / PF-04494700) (an oral small molecule inhibitor of RAGE-ligand interaction targeting the V domain); TTP4000 (human Ig as described in U.S. Patent No. 7981423). RAGE soluble fusion protein inhibitors using the ligand-binding extradomain of RAGE linked to the Fc domain; antibodies that specifically bind to RAGE and its RAGE-binding fragment as described in International Publication No. 2007109747; FPS-ZM127 (a tertiary amide with high affinity that blocks Aβ / RAGE interaction); peptides that antagonize RAGE ligand-induced signaling as described in U.S. Patent Application Publication No. 20100249038; lysophosphatidic acid (LPA) antagonists as described in International Publication No. 2012109569; 2-aminopyrimidine as described by Han et al (2012); pyrazole-5-carboxamide as described by Han et al (2014); 4,6-bisphenyl-2-(3-alkoxyanilino)pyrimidine as described by Han et al (2015); Manigrasso et al Small molecule inhibitors of ligand-stimulated RAGE-DIAPH1 signaling as described in al (2016); selected from polypeptides essentially comprising all or part of the cytoplasmic tail of RAGE, or essentially comprising part of Diaphanous-1 that binds to the cytoplasmic tail of RAGE, as described in U.S. Patent Application Publication No. 20090220484.

[0472] In certain embodiments, the modulator is administered to a subject based on its identification as a modulator of RAGE ligand-independent RAGE activation by a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, using a screening method or a method for identifying modulators as extensively described herein.

[0473] AT1R-related disorders are defined as disorders that depend on AT1R expression. This does not exclude RAGE-related disorders, which also depend on AT1R expression. In fact, disorders can be both RAGE-related and AT1R-related.

[0474] Specific chemokine receptor-related disorders are defined as disorders that depend on the expression of a specific chemokine receptor. This does not exclude RAGE-related disorders, which also depend on the expression of a specific chemokine receptor. In fact, disorders can be both RAGE-related and specific chemokine receptor-related.

[0475] CCR2-related disorders are defined as disorders that depend on CCR2 expression. This does not exclude RAGE-related disorders, which also depend on CCR2 expression. In fact, disorders can be both RAGE-related and CCR2-related.

[0476] For combination medications, the following dosages are "usually" administered.

[0477] [Table 30]

[0478] <Screening methods for candidate drugs> In one embodiment, the present invention includes a method for screening a candidate drug for its ability to modulate RAGE activity induced by an active coexisting GPCR, the method comprising the steps of contacting a RAGE polypeptide with a GPCR polypeptide in the presence of the candidate drug, wherein the GPCR polypeptide is constitutively active and / or activated by the addition of an agonist, partial agonist or allosteric modulator of the GPCR; and detecting whether the candidate drug is a modulator of RAGE ligand-independent RAGE activation by an activated coexisting GPCR by detecting an effect indicating the modulation of RAGE activation in the presence of the candidate drug and / or detecting RAGE-dependent signaling regulated in the presence of the candidate drug.

[0479] In one embodiment, the present invention includes a method for screening candidate drugs for their ability to modulate (i.e., activate, inhibit, or allosterically modulate) RAGE ligand-independent RAGE activation (also known as RAGE ligand-independent transactivation of RAGE) by a specific coexisting GPCR, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2. These methods generally include, a. Contacting a RAGE polypeptide with a GPCR polypeptide in the presence of a candidate drug, where the GPCR polypeptide is constitutively active and / or activated by the addition of a GPCR agonist, partial agonist or allosteric modulator; and b. To detect whether a candidate drug is a modulator of RAGE ligand-independent RAGE activation by activated coexisting GPCRs by detecting an effect indicating the modulation of RAGE activation by the presence of the candidate drug, and / or by detecting RAGE-dependent signaling regulated by the presence of the candidate drug. It includes, consists of, or is essentially derived from.

[0480] In some embodiments, the screening method further includes detecting whether a candidate drug is a modulator (activator, inhibitor, allosteric modulator, etc.) of a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, or a modulator (activator, inhibitor, allosteric modulator, etc.) of a specific coexisting GPCR signaling pathway, such as an angiotensin receptor signaling pathway like the AT1R signaling pathway, or a specific chemokine receptor signaling pathway like the CCR2 signaling pathway, in the presence or absence of RAGE. In some embodiments, candidate drugs that result in greater signal modulation in the presence or absence of RAGE polypeptides are selective for the modulation of RAGE ligand-independent RAGE activation by activated coexisting GPCRs rather than for RAGE-independent signaling resulting from the activation of coexisting GPCRs.

[0481] In one embodiment, the present invention comprises a peptide identified as a modulator by the method described above. In another embodiment, the present invention comprises a compound identified as a modulator by the method described above.

[0482] In some embodiments, the screening method further includes detecting whether a candidate drug is a modulator (such as an activator, inhibitor, allosteric modulator, or functional substitute) of RAGE or the RAGE signaling pathway, in the presence or absence of a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2. In some embodiments, candidate drugs that result in greater modulation of RAGE-dependent signaling in the presence of a GPCR polypeptide compared to its absence are selective for the modulation of RAGE ligand-independent RAGE activation by the activated coexisting GPCR.

[0483] In some embodiments, the screening method further includes detecting whether the candidate drug is a modulator (such as an activator, inhibitor, allosteric modulator, or functional substitute) of a specific coexisting GPCR signaling pathway, such as a RAGE polypeptide or the RAGE signaling pathway and a specific coexisting GPCR, such as an angiotensin receptor signaling pathway, such as an AT1R signaling pathway, or a specific chemokine receptor signaling pathway, such as a CCR2 signaling pathway.

[0484] In some embodiments, the screening method further includes using an inhibitor of a RAGE ligand that binds to the extracellular domain of RAGE and thus inhibits RAGE activation in a RAGE ligand-dependent manner.

[0485] In some embodiments, the screening method further includes the use of a RAGE polypeptide which has been mutated and / or cleaved such that it cannot bind a RAGE ligand to its external domain and therefore cannot be activated in a RAGE ligand-dependent manner.

[0486] In some embodiments, the binding of the RAGE ligand to the extracellular domain of RAGE is impaired by exposing cells to a modulator that modulates the binding of the RAGE ligand to RAGE.

[0487] In some embodiments, the use of mutated and / or cleaved RAGE polypeptides that are unable to bind to RAGE ligands and therefore cannot be activated in a RAGE ligand-dependent manner occurs before, after, or in parallel with screening involving RAGE polypeptides capable of binding to RAGE ligands.

[0488] Candidate drugs or derivatives of candidate drugs that appropriately modulate RAGE ligand-independent RAGE activation by specific coexisting GPCRs such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, appropriately modulate the signaling pathways of specific coexisting GPCRs such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, and / or angiotensin receptor signaling pathways such as the AT1R signaling pathway or specific chemokine receptor signaling pathways such as the CCR2 signaling pathway, and / or inhibit RAGE ligand-dependent activation of RAGE, and / or inhibit constitutively active RAGE and / or RAGE signaling pathways, are particularly useful for the treatment, prevention, or management of RAGE-related disorders.

[0489] In certain embodiments of the screening method of the present invention, if a candidate drug modulates a RAGE-dependent signal detected when a RAGE polypeptide is in contact with a GPCR polypeptide, the method further includes determining whether and / or to what extent a candidate drug modulates a RAGE-dependent signal in the absence of a GPCR polypeptide, such that a candidate drug that results in greater modulation of the RAGE-dependent signal in the presence of a GPCR polypeptide is selective for modulation of RAGE ligand-independent RAGE activation by an activated coexisting GPCR.

[0490] In certain embodiments of the screening method of the present invention, if a candidate drug modulates a signal detected when a RAGE polypeptide is in contact with a GPCR polypeptide, the method further includes 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 drug modulates the signal in the absence of the RAGE polypeptide, such that the candidate drug that results in greater modulation of the signal in the presence of the RAGE polypeptide is more selective for modulation of RAGE ligand-independent RAGE activation by activated coexisting GPCRs than for RAGE-independent signaling resulting from the activation of coexisting GPCRs.

[0491] In certain embodiments, the screening method uses a proximity screening assay to evaluate the proximity of the RAGE polypeptide to a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2. In this exemplary example, the RAGE polypeptide is coupled (e.g., conjugated or otherwise linked) to a first reporter component, and the specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is coupled (e.g., conjugated or otherwise linked) to a second reporter component. The proximity of the first and second reporter components generates a signal detectable by a detector. The first and second reporter components form a complementary pair in the sense that the first reporter component can be exchanged for the second reporter component without any apparent effect on the function of the present invention. The first and second reporter components may be the same or different.

[0492] In one embodiment, the proximity screening assay is described in International Publication No. 2008055313 (Dimerix Bioscience Pty Ltd; also known as Receptor Heteromer Investigation Technology or Receptor-HIT (Jaeger et al., 2014) (Dimerix Bioscience Pty Ltd; also U.S. Patent No. 8283127, U.S. Patent No. 8568997, European Patent No. 2080012, Canadian Patent No. 2669088, Chinese Patent No. 101657715). In this method, RAGE is coupled to a first reporter component, and a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, is unlabeled for the proximity screening assay, and a GPCR interacting group is linked to a complementary second reporter component, and its interaction with the complex is regulated when bound to a ligand selective to the unlabeled GPCR or, in particular, the heteromeric complex. Preferred examples of GPCR interacting groups are arrestins, G proteins, and ligands. Instead, specific coexisting GPCRs, such as angiotensin receptors like AT1R or certain chemokine receptors like CCR2, are coupled to the first reporter component, RAGE remains unlabeled for proximity screening assays, and RAGE interacting groups are linked to a complementary second reporter component, whose interaction with the complex is regulated when bound to a ligand selective to unlabeled RAGE or, in particular, the heteromeric complex. Preferred examples of RAGE interacting 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).

[0493] The reporter component may include RAGE, a specific coexisting GPCR, or a transcription factor or other molecule coupled to an interacting group by a linker incorporating an enzyme, a luminescent or bioluminescent molecule, a fluorescent molecule, and an enzyme cleavage site. In short, it is a known organic or inorganic protein or non-protein or complex thereof molecule that can emit a detectable signal as a result of their spatial proximity.

[0494] Preferably, the signal generated by the proximity of the first and second reporter components in the presence of a reporter component initiator is selected from the group consisting of emission, fluorescence, and colorimetric change.

[0495] In some embodiments, luminescence is produced by a bioluminescent protein selected from the group consisting of luciferase, galactosidase, lactamase, peroxidase, or any protein capable of luminescence in the presence of a suitable substrate.

[0496] Preferred combinations of the first and second reporter components include those detailed in U.S. Patent No. 8,283,127, but useful combinations of the first and second reporter components are by no means limited to these.

[0497] In some embodiments, the screening method further includes detecting the proximity of the first and second reporter components to determine whether the candidate drug modulates the interaction between the RAGE polypeptide and a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2. Generally, this is achieved when the proximity of the first and second reporter components generates a proximity signal that is altered by the modulation of the proximity between the RAGE polypeptide and a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, by the candidate drug.

[0498] RAGE and certain coexisting GPCRs, such as angiotensin receptors like AT1R or chemokine receptors like CCR2, may exist in a soluble form or be expressed on the cell surface.

[0499] In some embodiments, RAGE and certain co-existing GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, are located within, partially within, or on a single membrane, and are expressed, for example, on the surface of host cells.

[0500] In another embodiment of the present invention, certain coexisting GPCRs, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, are pre-assembled with RAGE in a complex pre-formed at the cell membrane.

[0501] In another embodiment of the present invention, activation of a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, by association with a recognition ligand such as Ang II for AT1R or MCP-1 for CCR2, triggers signaling involved in the cytoplasmic tail of RAGE.

[0502] In one embodiment of the present invention, activation of the cytoplasmic tail of RAGE is associated with changes in its structural conformation and / or affinity for its binding partner.

[0503] In one embodiment of the present invention, monitoring of the structural conformation and / or affinity of RAGE to its binding partner occurs when the cytoplasmic tail of RAGE is mutated and / or cleaved so that it can no longer be activated by RAGE ligand-independent RAGE activation by a RAGE ligand or a specific activated coexisting GPCR.

[0504] In one embodiment of the present invention, monitoring of structural conformation and / or affinity to binding partners occurs in the presence of a drug that inhibits RAGE binding and / or activation by RAGE ligand-independent RAGE activation by a RAGE ligand or a specific activated coexisting GPCR.

[0505] In one embodiment of the present invention, monitoring of the recruitment of binding partners occurs before RAGE activation by RAGE ligand-independent RAGE activation by a RAGE ligand or a specific activated coexisting GPCR.

[0506] In one embodiment of the present invention, the mobilization and activation of signaling mediators and / or binding partners to the cytoplasmic tail of RAGE are monitored after RAGE activation by RAGE ligand-independent RAGE activation mediated by a RAGE ligand or a specific activated coexisting GPCR.

[0507] In one embodiment of the present invention, monitoring of the recruitment of a binding partner after RAGE activation by RAGE ligand-independent RAGE activation by a RAGE ligand or a specific activated coexisting GPCR occurs in the presence of a drug that inhibits RAGE binding and / or activation by the RAGE ligand.

[0508] Further embodiments of the present invention include a method for screening candidate drugs by detecting the modulation of RAGE-mediated signaling to determine their ability to modulate (activate, inhibit, or otherwise modulate) RAGE ligand-independent RAGE activation by specific coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2. Such a method may include the step of measuring standard NFκB activation by measuring one or more of the following: • Monitoring the activity of IκB kinase (IKK) by observing the in vitro phosphorylation of substrates such as 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 by means of antibody, gel shift, EMSA, or mass spectrometry; • Detection of cytoplasmic-to-nuclear transport / transfer of NFκB components / subunits such as p65 / phosphop65; • Detection of dimerization / complex formation of NFκB subunits; Detection of active NFκB components / subunits by binding to immobilized DNA sequences / oligonucleotides containing NFκB response elements / consensus NFκB binding, such as by electrophoretic mobility shift assays or gel shift assays, SELEX, protein-conjugated microarrays, or sequence-based approaches; • Chromatin immunoprecipitation (ChIP) assay to detect in-situ binding of NFκB to DNA, specifically to promoters and enhancers of certain genes; • In vitro kinase assay for NFκB kinase activity; Measurement of NFκB transcriptional activity using NFκB reporter assays via transgene expression of reporter constructs such as LacZ Fluc, eGFP SEAP, and NF-gluc, using approaches such as plasmid transduction, reporter cell lines, minicircles, retroviruses, and lentiviruses; • Measurement of changes in the expression of downstream targets of NFκB (cytokines, growth factors, adhesion molecules, and mitochondrial anti-apoptotic genes, proteins, or functional assays by real-time PCR, etc.) (Note that the multifaceted nature of NFκB is currently reflected in more than 500 of its transcriptional targets (see http: / / www.bu.edu / nf-kb / gene-resources / target-genes / , accessed 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 tumorigenesis.

[0509] Additionally or alternatively, such methods may include measuring signals resulting from the non-standard effects of NF-κB by measuring one or more of the following: • Detection of NIK (NFκB-induced kinase); • Detection of IKKα activation / phosphorylation; • Detection of NIK kinase activity by performing a kinase assay, based on its ability to autophosphorylate or phosphorylate substrates; • Generation of p52-containing NFκB dimers such as p52 / RelB; Detection of phospho-NFκB2p100 (Ser866 / 870); • Detection of the partial decomposition (called treatment) of precursor p100 to p52; • Detection of p52 / RelB transfer to the nucleus; • Detection of p52 / RelB binding to the κB site; Measurement of NFκB transcriptional activity using NFκB reporter assays via transgene expression of reporter constructs such as LacZ Fluc, eGFP SEAP, and NF-gluc, using approaches such as plasmid transduction, reporter cell lines, minicircles, retroviruses, or lentiviruses; • Measurement of changes in the expression of NFκB non-standard signaling downstream targets (such as CXCL12) by real-time PCR, protein expression, or functional assays.

[0510] In another embodiment, the present invention provides a method for identifying modulators (such as activators, inhibitors, allosteric modulators, or functional substitutes) that modulate RAGE ligand-independent RAGE activation (i.e., activate, inhibit, or otherwise modulate) after activation of a specific coexisting GPCR by a recognition ligand such as Ang II, such as AT1R, or by MCP-1, such as CCR2, or when a specific coexisting GPCR is constitutively active, and that appropriately modulate a specific coexisting GPCR, such as an angiotensin receptor such as AT1R, or a specific chemokine receptor such as CCR2, and / or modulate the RAGE polypeptide or the RAGE signaling pathway. In a preferred embodiment of the present invention, such modulators are inhibitors of RAGE or a specific coexisting GPCR, or both, such as an angiotensin receptor such as AT1R, or a specific chemokine receptor such as CCR2, or inhibitors of the RAGE signaling pathway. In a particularly preferred embodiment of the present invention, the modulation of the RAGE signaling pathway occurs differently and / or to a significantly different degree from the modulation of specific classical coexisting GPCR signaling pathways such as the AT1R signaling pathway, such as the Gq signaling pathway, or the CCR2 signaling pathway, such as the Gi signaling pathway. In a particularly preferred embodiment of the present invention, the inhibition of the RAGE signaling pathway occurs differently and / or to a greater extent than the inhibition of specific classical coexisting GPCR signaling pathways such as the AT1R signaling pathway, such as the Gq signaling pathway, or the CCR2 signaling pathway, such as the Gi signaling pathway.

[0511] <Structure> In related embodiments, the present invention provides a construct system for identifying modulators of proximity between RAGE and specific coexisting GPCRs, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2.

[0512] In some embodiments, these construct systems include a first construct comprising a regulatory sequence operably connected 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 connected to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, and a nucleic acid sequence encoding a proximity signal or energy receptor molecule. In certain embodiments, the energy donor molecule is a bioluminescent or fluorescent molecule, and the energy receptor molecule is a fluorescent receptor molecule.

[0513] In other embodiments, the construct system of the present invention includes a first construct comprising a regulatory sequence operably connected to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a specific coexisting GPCR, such as an angiotensin receptor like AT1R or a specific chemokine receptor like CCR2, and a nucleic acid sequence encoding a proximity signal or energy donor molecule; and a second construct comprising a regulatory sequence operably connected 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 proximity signal or energy acceptor molecule. In certain embodiments, the energy donor molecule is a bioluminescent or fluorescent molecule, and the energy acceptor molecule is a fluorescent acceptor molecule.

[0514] In other embodiments, the construct system of the present invention is a first construct comprising a regulatory sequence operably connected to a first coding sequence, wherein the first coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to a specific co-existing GPCR such as an angiotensin receptor such as AT1R or a specific chemokine receptor such as CCR2; and a second construct comprising a regulatory sequence operably connected to a second coding sequence, wherein the second coding sequence comprises a nucleic acid sequence encoding a polypeptide corresponding to the RAGE polypeptide, thereby comprising a second construct in which one or more of the external domains of the RAGE polypeptide are deleted.

[0515] <Method for regulating RAGE ligand-independent RAGE activation> In related aspects, the present invention provides a method for regulating RAGE ligand-independent RAGE activation by a specific co-existing GPCR such as an angiotensin receptor such as AT1R or a specific chemokine receptor such as CCR2 in cells or tissues of an animal or of animal origin (which may or may not be human or of human origin).

[0516] <Specific method for regulating RAGE ligand-independent RAGE activation> In another related aspect, the present invention particularly provides a method for regulating RAGE ligand-independent RAGE activation by a specific co-existing GPCR such as an angiotensin receptor such as AT1R or a specific chemokine receptor such as CCR2 and the subsequent intracellular downstream signaling pathway. These methods include cleaving or mutating RAGE such that the RAGE ligand cannot bind to its external domain or such that binding of the RAGE ligand to its external domain is impaired by exposing the cell to a modulator that regulates the binding of the RAGE ligand to RAGE.

[0517] In a preferred form of the present invention, the regulation of the RAGE ligand-independent signaling pathway is different from and / or significantly greater than the regulation of the RAGE ligand-dependent signaling pathway.

[0518] In a particularly preferred embodiment of the present invention, the inhibition of the RAGE ligand-independent signaling pathway is different from and / or significantly greater than the inhibition of the RAGE ligand-dependent signaling pathway.

[0519] <Method for regulating both RAGE ligand-dependent and RAGE ligand-independent RAGE activation> In another related aspect, the present invention provides a method for inhibiting the RAGE ligand-dependent activation of RAGE (including AGE-modified proteins, lipids or DNA, members of the S100 calgranulin family of proteins, HMGB1, amyloid and Mac-1) by a RAGE ligand and the subsequent downstream signaling pathway thereof in cells, tissues or animals, in addition to regulating the RAGE ligand-independent activation of RAGE by a specific activated co-existing GPCR.

[0520] In one aspect of the present invention, these methods use a modulator as described herein, which in a binding interaction includes a fragment, analog or derivative of the cytoplasmic tail of RAGE instead of the cytoplasmic tail of RAGE, to prevent the activation of both the RAGE ligand-dependent activation of RAGE and the RAGE ligand-independent activation of RAGE by a specific activated co-existing GPCR. In one aspect of the present invention, RAGE-dependent signaling is impaired by exposing cells to an inhibitor that inhibits the binding of signaling elements to the cytoplasmic tail of RAGE, resulting in the inhibition of both the activation of RAGE mediated by a RAGE ligand and the RAGE ligand-independent activation of RAGE by a specific activated co-existing GPCR.

[0521] In one aspect of the present invention, these methods include preventing both RAGE ligand-dependent and RAGE ligand-independent activation of RAGE by a specific activated coexisting GPCR, by using a modulator described herein, which comprises a fragment, analog, or derivative of the transmembrane domain of RAGE instead of the transmembrane domain of RAGE. In one aspect of the present invention, the modulator comprises the transmembrane domain of RAGE or a portion thereof and a fragment of the extracellular domain of RAGE. In one aspect of the present invention, the modulator comprises the transmembrane domain of RAGE or a portion thereof and a fragment of the cytoplasmic tail of RAGE. In one aspect of the present invention, the modulator comprises the transmembrane domain of RAGE or a portion thereof, a fragment of the extracellular domain of RAGE, and a fragment of the cytoplasmic tail of RAGE.

[0522] In one aspect of the present invention, a modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR contains a fragment of the ligand-binding extradomain of RAGE having a length of 40 amino acids or less, 20 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0523] In one embodiment, inhibition of RAGE ligand-dependent activation occurs simultaneously with inhibition of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR.

[0524] In one embodiment, these methods include silencing, cleaving, modifying, or muting RAGE such that RAGE or its analogues, fragments, or derivatives become non-functional substitutes for the cytoplasmic tail or a portion thereof of wild-type RAGE, which are not activated by either RAGE ligand-dependent or RAGE ligand-independent pathways (such as the S391A-RAGE mutation) or cannot promote downstream RAGE-dependent signaling, and therefore inhibit signaling that occurs through the cytoplasmic tail of RAGE and RAGE-dependent signaling.

[0525] In one embodiment, these methods include silencing, cleaving, modifying, or muting RAGE such that RAGE or its analogues, fragments, or derivatives are non-functional substitutes for the transmembrane domain or portion thereof of wild-type RAGE, which is not activated by either RAGE ligand-dependent or RAGE ligand-independent pathways, or fails to promote downstream RAGE-dependent signaling, and therefore inhibits signaling that occurs through the cytoplasmic tail of RAGE and RAGE-dependent signaling. In one embodiment of the present invention, the modulator comprises the transmembrane domain or portion thereof of RAGE and a fragment of the extracellular domain of RAGE. In one embodiment of the present invention, the modulator comprises the transmembrane domain or portion thereof of RAGE and a fragment of the cytoplasmic tail of RAGE. In one embodiment of the present invention, the modulator comprises the transmembrane domain or portion thereof, a fragment of the extracellular domain of RAGE, and a fragment of the cytoplasmic tail of RAGE.

[0526] In one embodiment, the modulator of RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR contains a fragment of the ligand-binding extradomain of RAGE having a length of 40 amino acids or less, 20 amino acids or less, 10 amino acids or less, or 5 amino acids or less.

[0527] In one embodiment, these methods include silencing, cleaving, modifying, or muting RAGE such that RAGE or its analogues, fragments, or derivatives modulate common elements (such as PKCζ, Diaph1, MyD88, TIRAP, NFκB, etc.) involved in signal transduction mediated by the cytoplasmic tail of RAGE, in association with activation of RAGE via either a RAGE ligand-dependent or RAGE ligand-independent activation pathway.

[0528] In one embodiment, these methods include the use of a modulator that modulates RAGE ligand-dependent activation of RAGE (such as by a modulator that modulates the binding of RAGE ligand to the extracellular domain of RAGE), as well as a modulator that modulates RAGE ligand-independent activation by a specific coexisting GPCR that has been activated, such as an angiotensin receptor such as AT1R or a specific chemokine receptor such as CCR2.

[0529] <A method to regulate RAGE ligand-independent RAGE activation by specific activated coexisting GPCRs, while also regulating RAGE-independent signaling via specific coexisting GPCRs.> In one embodiment, the present invention provides a method for not only regulating RAGE ligand-independent RAGE activation by specific activated coexisting GPCRs, but also for regulating RAGE-independent specific coexisting GPCR signaling pathways that are induced after activation by recognition ligands.

[0530] In one embodiment, the present invention provides a method for regulating RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR, while simultaneously regulating a specific RAGE-independent coexisting GPCR signaling pathway induced after activation by a recognition ligand.

[0531] In one form, a specific RAGE-independent coexisting GPCR signaling pathway induced after activation by a recognition ligand is a Gq signaling pathway such as AT1R activated by Ang II. In another form, a specific RAGE-independent coexisting GPCR signaling pathway is a Gi signaling pathway such as CCR2 activated by MCP-1. In yet another form, a specific RAGE-independent coexisting GPCR signaling pathway is β-arrestin-mediated extracellular regulatory kinase (ERK) signaling. In yet another form, a specific RAGE-independent coexisting GPCR signaling pathway is a change in intracellular signaling mediators (such as inositol phosphate or calcium). [Brief explanation of the drawing]

[0532] [Figure 1A] Quantitative plaque area, expressed as the percentage of Sudan IV-stained aortic arch surface area after 4 weeks of Ang II (1 μg / kg / min) or vehicle-controlled infusion in male apoE-KO mice and AGER / apoE double knockout (DKO) mice. [Figure 1B] Expression of atherosclerosis-inducing 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 from apoE-KO mice and AGER / apoE-DKO mice after 4 weeks of Ang II (1 μg / kg / min) or vehicle-controlled infusion. [Figure 1C] (i) Plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, and (ii) Markers of oxidative stress after 4 weeks of Ang II (1 μg / kg / min) or vehicle-controlled injection in apoE-KO mice and AGER / apoE-DKO mice, as predicted by induction of gene expression of NADPH oxidase subunits NOX-1 and NOX-4 in the aorta of apoE-KO mice and AGER / apoE-DKO mice, as predicted by real-time RT-PCR in aortic homogenate. [Figure 1D] (i) circulating plasma levels of S100A8 / A9 as measured by a commercially available ELISA; (ii) plasma AGE levels as measured by our proprietary ELISA; (iii) RAGE ligand expression, including circulating levels of methylglyoxal, an AGE precursor, as measured by HPLC in apoE-KO mice and AGER / apoE-DKO mice after 4 weeks of infusion of Ang II (1 μg / kg / min) or vehicle. [Figure 1E]Systolic blood pressure, measured by tail cuff plethysmography, after 4 weeks of Ang II (1 μg / kg / min) or vehicle-controlled infusion in apoE-KO mice and AGER / apoE-DKO mice. Data are mean ± SEM; n=8 per group, * indicates comparison with control apoE-KO mice, # indicates comparison with apoE-KO + Ang II, p<0.05. [Figure 2A] Quantitative plaque area, expressed as the percentage of Sudan IV-stained aortic arch surface area in apoE-KO mice and AGER / apoE-DKO mice after a 6-week diet of 0.05% (low) sodium or normal feed. [Figure 2B] Expression of atherosclerosis-inducing 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 and AGER / apoE-DKO mice after 6 weeks of a 0.05% (low) sodium diet or normal diet. [Figure 2C] (i) Plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, and (ii) Markers of oxidative stress in apoE-KO mice and AGER / apoE-DKO mice after 6 weeks of a 0.05% (low) sodium diet or normal diet, as estimated by induction of gene expression of NADPH oxidase subunits NOX-1 and NOX-4 in the aorta of apoE-KO mice and AGER / apoE-DKO mice, as estimated by real-time RT-PCR in aortic homogenate. [Figure 2D] Circulating levels of soluble MCP-1 and ICAM-1, measured by ELISA in apoE-KO mice and AGER / apoE-DKO mice exposed to a 0.05% (low) sodium diet or normal feed for 6 weeks. [Figure 2E]The number of labeled leukocytes adhering ex vivo to the aortic surface of apoE-KO mice and AGER / apoE-DKO mice one week prior to exposure to a low-sodium diet or normal feed, as measured by a dynamic flow assay. [Figure 2F] (i) circulating plasma levels of S100A8 / A9 as measured by a commercially available ELISA, (ii) plasma AGE levels as measured by our proprietary ELISA, and (iii) RAGE ligand expression, including circulating levels of methylglyoxal, an AGE precursor, as measured by HPLC in apoE-KO mice and AGER / apoE-DKO mice after a 6-week 0.05% (low) sodium diet or normal diet. [Figure 2G] Systolic blood pressure, measured by tail cuff plethysmography in apoE-KO mice and AGER / apoE-DKO mice after a 6-week diet of 0.05% (low) sodium or normal feed. [Figure 2H] Markers of RAAS activation, including (i) decreased sodium excretion, (ii) increased plasma renin activity, and (iii) increased plasma aldosterone levels, are measured by radioimmunoassay in apoE-KO mice and AGER / apoE-DKO mice after a 6-week 0.05% (low) sodium diet or normal diet. Data are mean ± SEM; n=8 per group. * indicates p<0.05 compared to apoE-KO mice on a normal diet. # indicates compared to apoE-KO mice with low sodium. [Figure 3A] Serum Ang II concentrations in apoE-KO mice and AGER / apoE-KO mice with or without genetic Ace2 deficiency, as measured by radioimmunoassay. [Figure 3B] Systolic blood pressure, measured by tail cuff plethysmography, in 18-week-old apoE-KO mice and AGER / apoE-KO mice with or without genetic Ace2 deficiency. [Figure 3C]Quantitative plaque area, expressed as the percentage of Sudan IV-stained aortic arch surface area, in 18-week-old apoE-KO mice and AGER / apoE-KO mice with or without genetic Ace2 deficiency. [Figure 3D] Aortic expression of atherosclerosis-inducing 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 from apoE-KO mice, in the presence or absence of Ace2 and / or RAGE. [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 by plasma 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage. [Figure 3F] (i) Circulating plasma levels of S100A8 / A9 as measured by a commercially available ELISA; (ii) RAGE ligand expression, including plasma AGE levels as measured by our proprietary ELISA in 18-week-old apoE-KO mice, Ace2 / apoE-DKO mice, AGER / apoE-DKO and Ace2 / AGER / apoE TKO mice. Data are mean ± SEM; n=8 per group; * indicates relative to apoE-KO control; # indicates relative to Ace2 / apoE-DKO mice. [Figure 4A]Aortic expression of atherosclerosis-inducing 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 apoE-KO mice and AGER / ApoE-DKO mice, 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; * compared to untreated apoE-KO control; # compared to apoE-KO + Ang II; p<0.05. [Figure 4B] The number of labeled leukocytes adhering to the aortic surface of apoE-KO mice and AGER / apoE-DKO mice as a marker of endothelial activation after 4 hours of ex vivo exposure to Ang II (1 μM) or vehicle control, as measured by dynamic flow assay. Data are mean ± SEM; n=6 per group; * is against untreated apoE-KO control; # is AGER / apoE-DKO + Ang II compared to apoE-KO + Ang II; p<0.05. [Figure 4C] The number of labeled THP-1 monocytes attached to a monolayer of primary mouse aortic endothelial cells (PMAEC) from C57bl6 or AGER-KO mice, in or without pretreatment with Ang II (1 μM for 2 hours). [Figure 4D] Expression of atherosclerosis-inducing mediators, including AGER itself, key adhesion molecules (ICAM-1, VCAM-1), inflammatory cytokines and chemokines (TNFα and MCP-1), as measured by real-time RT-PCR in primary mouse aortic endothelial cells (PMAEC) from C57bl6 and PMAEC from AGER-KO mice after exposure to Ang II (1 μM) or vehicle controls. [Figure 4E]Markers of oxidative stress after exposure to Ang II or vehicle control in PMAEC from c57bl6 mice and AGER-KO mice, 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) levels of oxidized glutathione. [Figure 4F] Gene expression of markers of non-standard and standard signaling mediated by VCAM-1 and NFκB (CXCL12 and CXCL2, respectively) in monolayers of primary mouse aortic endothelial cells (PMAEC) from C57bl6 and AGER-KO mice after exposure to Ang II. PMAEC measured by real-time RT-PCR. TNFα is shown as a standard specific control. VCAM-1 is shown as a target-specific control replicating the data in Figure 4D. [Figure 4G] Expression of atherosclerosis-inducing mediators, including adhesion molecules (ICAM-1, VCAM-1) and inflammatory cytokines and chemokines (TNFα and MCP-1), in primary mouse aortic endothelial cells (PMAECs) from C57bl6 and AGER-KO mice treated with RAGE ligand, S100A8 / A9 (5 ng / mL), as measured by real-time RT-PCR. Data are mean ± SEM; n=6 per group; symbols * for untreated wild-type PMAECs; # for S100A8 / A9-treated wild-type PMAECs; p<0.05. [Figure 4H] Genetic expression of VCAM-1, a key adhesion protein in the PMAEC monolayer, was determined by real-time RT-PCR, after selective silencing or no alteration of RAGE or NFκB subunit p65 expression using siRNA (scrambled RNA control), followed by exposure to Ang II (1 μM) or RAGE ligand, S100A8 / A9 (5 ng / mL). [Figure 4I]Markers of Gq-mediated signaling induced after AT1R activation by Ang II (1 μM) in the monolayer of PMAEC from c57bl6 mice and AGER-KO mice, including (i) induction of inositol phosphate synthesis as predicted by IP-1, and (ii) downstream induction of the early growth response gene (EGR1). Data are mean ± SEM; n=6 per group; unless otherwise specified, * is relative to untreated wild-type control PMAEC, # is relative to Ang II-treated wild-type control, p<0.05. [Figure 5A] Induction of inositol phosphate synthesis in response to Ang II (1 μM), a marker of classical responsiveness to exogenous Ang II as estimated by IP-1 levels in CHO cells, in or without additional expression of full-length human RAGE, in the presence or absence of human AT1R expression. [Figure 5B] Induction of EGR1 expression in response to Ang II (1 μM), a marker of responsiveness to exogenous Ang II, which is presumed to be caused by downstream induction of the EGR1 gene in CHO cells, with or without additional expression of full-length human RAGE, in the presence or absence of human AT1R expression. [Figure 5C] (i) Chemiluminescent SEAP reporter gene assay; (ii) Induction of NFκB subunit p65 gene expression in the presence or absence of human AT1R expression in CHO cells with or without additional expression of full-length human RAGE; and (iii) NFκB activation after exposure to Ang II (1 μM), as measured after exposure to RAGE ligand S100A8 / A9 (5 ng / mL) as a control for the completeness of RAGE signaling in CHO cells. [Figure 5D]NFκB activation in AT1R-CHO cells after exposure to Ang II (1 μM) was measured by a chemiluminescent SEAP reporter gene assay for induction of NFκB subunit p65 gene expression and NFκB activity in the presence or absence of full-length human RAGE and N-terminal truncated mCherry-RAGE constructs. Data are mean ± SEM, n=6 per group, * indicates p<0.05 compared to vector (neo)-transduced AT1R-CHO. [Figure 5E] NFκB activation in AT1R-CHO after exposure to Ang II (1 μM), as estimated by chemiluminescent SEAP reporter gene assay, in the presence or absence of full-length human RAGE and C-deficient mCherry-RAGE constructs, and induction of NFκB subunit p65 gene expression and NFκB activity. Data are mean ± SEM, n=6 per group, * indicates p<0.05 compared to vector (neo)-transduced AT1R-CHO. [Figure 5F] NFκB activation in AT1R-CHO after exposure to S100A8 / A9 (5 ng / mL) or Ang II (1 μM), as estimated by induction of NFκB subunit p65 gene expression in the presence or absence of full-length human RAGE and N-terminal truncated mCherry-RAGE constructs. Data are mean ± SEM, n=6 per group, * indicates p<0.05 compared to vector (neo)-transduced AT1R-CHO. [Figure 5G] NFκB activation in AT1R-CHO after exposure to S100A8 / A9 (5 ng / mL) or Ang II (1 μM), as estimated by induction of NFκB subunit p65 gene expression in the presence or absence of full-length human RAGE and C-deficient mCherry-RAGE constructs. Data are mean ± SEM, n=6 per group, * indicates p<0.05 compared to vector (neo)-transduced AT1R-CHO. [Figure 5H]NFκB activation after exposure to Ang II (1 μM) in AT1R-CHO, as predicted by induction of NFκB subunit p65 gene expression in the presence of an N-terminal truncated RAGE construct not fused to mCherry. Data are mean ± SEM; n=6 per group; * indicates p<0.05 compared to untreated AT1R-CHO unless otherwise specified. [Figure 6A] RAGE neutralizing antibodies targeting the extracellular domain of RAGE (RAGEab) or decoy receptors with ligand-binding affinity (soluble RAGE22-331) inhibit the induction of pro-inflammatory signaling in RAGE-AT1R-CHO cells, mediated by RAGE ligand S100A8 / A9 but not by Ang II (1 μM), as can be inferred from the expression of NFκB subunit p65 measured by RT-PCR. Data are shown mean ± SEM; n=6 per group, * indicates p<0.05 compared to vehicle alone. [Figure 6B] RAGE neutralizing antibodies targeting the extracellular domain of RAGE (RAGEab) or decoy receptors with ligand-binding affinity (soluble RAGE22-331; sRAGE) do not inhibit the induction of pro-inflammatory signaling by Ang II (1 μM) in PMAEC from wild-type mice, as presumably by the induction of the major adhesion genes ICAM-1 and VCAM-1 and the inflammatory chemokine gene (MCP-1). Data from AGER-KO mice are shown as a negative control. Data are shown mean ± SEM; n=6 per group, * indicates control cells treated with vehicle alone (white bar), # indicates control cells treated with Ang II alone, p<0.05. [Figure 6C]RAGE neutralizing antibodies targeting the extracellular domain of RAGE (RAGEab) or decoy receptors with ligand-binding affinity (soluble RAGE22-331) inhibit the induction of pro-inflammatory signaling by RAGE ligand S100A8 / A9 in PMAEC from wild-type mice, as predicted by the induction of the major adhesion gene ICAM-1. Data are shown mean ± SEM; n=6 per group, * indicates control cells treated with vehicle alone (white bar), # indicates control cells treated with S100A8 / A9 alone, p<0.05. [Figure 7A] Induction of inflammatory signaling by RAGE ligand S100A8 / A9 (5 ng / ml; gray bar) or Ang II (1 μM; black bar) in AT1R-CHO cells expressing full-length wild-type RAGE22-404 or selected S391-RAGE22-404 mutants, as measured by induction of NFκB subunit p65 gene expression. Data show mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to vehicle-treated AT1R-CHO cells expressing full-length RAGE. [Figure 7B] In AT1R-CHO cells, induction of pro-inflammatory signaling by RAGE ligand S100A8 / A9 (5 ng / ml) or Ang II (1 μM) was measured by induction of NFκB subunit p65 gene expression, including chimeric RAGE lacking phosphorylated motifs on the cytoplasmic tail other than S391 (chimeric RAGE; cRAGE), and S391-cRAGE mutants completely lacking any phosphorylated motifs on the cytoplasmic tail. Data are shown mean ± SEM; n=6-8 per group, * indicates comparison with vehicle-treated AT1R-CHO cells expressing full-length chimeric RAGE; p<0.05. [Figure 7C]Induction of pro-inflammatory signaling by Ang II (1 μM) in AT1R-CHO cells expressing full-length or N-terminal truncated S391A-RAGE mutants, as measured by induction of NFκB subunit p65 gene expression. Data are shown mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to vector-transduced AT1R-CHO cells. [Figure 7D] Induction of pro-inflammatory signaling by Ang II (1 μM) in the presence of wild-type mCherry-RAGE362-404 in AT1R-CHO cells also expressing the S391-RAGE362-404 mutant, as measured by induction of NFκB subunit p65 gene expression. Data are shown mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to vehicle control. [Figure 8A] The effect of selective suppression of MyD88 expression using siRNA or a scrambled control on the induction of RAGE ligand-dependent induction of inflammatory signaling by RAGE ligand S100A8 / A9 (5 ng / ml) in a monolayer of PMAEC from C57bl6 mice, as estimated by ICAM-1 expression measured by real-time RT-PCR. Selective suppression of p65 expression, another downstream mediator of RAGE signaling, using siRNA is shown as a positive control. [Figure 8B] The effect of selective suppression of MyD88 expression using siRNA or scrambled control in a monolayer of PMAEC from C57bl6 mice on the induction of RAGE ligand-independent induction of inflammatory signaling by Ang II (1 μM), as estimated by ICAM-1 expression measured by real-time RT-PCR. [Figure 8C]The effect of selective suppression of MyD88 expression in a monolayer of HMEC using siRNA or a scrambled control on the induction of RAGE ligand-independent induction of inflammatory signaling by Ang II (1 μM) in the presence and absence of RAGE362-404, as estimated by MCP-1 expression measured by real-time RT-PCR. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to scrambled control. [Figure 9A] The effect of selective suppression of PKCζ using siRNA targeting the expression of a pseudosubstrate of PKCζ (iPKCz) or PKCζ (siPKCz), RAGE (siRAGE), or a scrambled control in a monolayer of PMAEC from C57bl6 mice on induction of RAGE ligand-dependent signaling by RAGE ligand S100A8 / A9, as predicted by ICAM-1 expression measured by real-time RT-PCR. [Figure 9B] The effect of selective suppression of PKCζ expression using siRNA targeting a pseudosubstrate of PKCζ (iPKCζ) or PKCζ (siPKCζ) on RAGE ligand-independent induction of inflammatory signaling by Ang II (1 μM) in a monolayer of PMAEC from C57bl6 mice. Columns 1 and 2 include scrambled siRNA controls. [Figure 9C] The effect of selective repression of PKCζ expression using a pseudosubstrate of PKCζ (PKCζi) on RAGE ligand-independent induction of p65 and PCNA by Ang II (1 μM) in CHO cells expressing cRAGE, which includes chimeric RAGE and S391Q-RAGE mutations (S319Q-cRAGE) lacking phosphorylated motifs on the cytoplasmic tail other than S391(cRAGE), as predicted by the gene expression of RelA / p65 and PCNA measured by real-time RT-PCR. [Figure 9D]The effect of selective suppression of PKCζ expression using siRNA or scrambled control in a monolayer of HMEC on the induction of RAGE ligand-independent induction of inflammatory signaling by Ang II (1 μM) in the presence and absence of RAGE362-404, as estimated by MCP-1 expression measured by real-time RT-PCR. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to untreated control. [Figure 10A] The effect of selective suppression of Diaph1 expression using siRNA in a monolayer of PMAEC from C57bl6 mice on induction of RAGE ligand-dependent signaling by RAGE ligand S100A8 / A9, as predicted by ICAM-1 expression measured by real-time RT-PCR. siRAGE data are included as a control. [Figure 10B] The specific effect of the R366A-Q367A-RAGE mutation on RAGE ligand S100A8 / A9-induced signaling and Ang II-induced RAGE ligand-independent signaling in AT1R-CHO cells, specifically disrupting charged patches through which Diaph1 and RAGE interact putatively. [Figure 10C] The effect of selective suppression of Diaph1 expression using siRNA in a monolayer of PMAEC from C57bl6 mice on the induction of Ang II (1 μM)-induced RAGE ligand-independent signaling, as estimated by the expression of ICAM-1 and VCAM-1 as measured by real-time RT-PCR. Data are mean ± SEM; n=6 per group, * indicates p<0.05 compared to scrambled control. [Figure 10D] Selective suppression of Diaph1 or AGER expression using siRNA in a monolayer of SVEC, in contrast to scrambled control, compared to induction of leukocyte adhesion to the endothelial monolayer after exposure to Ang II. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to scrambled control. [Figure 10E]The effect of transduction by R366A-Q367A-RAGE mutants in which a charged patch, through which Diaph1 and RAGE putatively interact, is disrupted or deleted, on Ang II-induced signaling, as measured by the induction of NFκB subunit p65 gene expression, in AT1R-CHO cells. Data are mean ± SEM; n=6 per group, * indicates p<0.05 compared to mCherry control. [Figure 10F] The effect of selective suppression of Diaph1 expression using siRNA or scrambled control in a monolayer of HMEC on the induction of RAGE ligand-independent induction of inflammatory signaling by Ang II (1 μM) in the presence and absence of RAGE362-404, as estimated by MCP-1 expression measured by real-time RT-PCR. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to vehicle control treated with scrambled siRNA. [Figure 10G] The effects of transduction with full-length RAGE, truncated RAGE, or RAGE mutants on Ang II-induced signaling, as measured by induction of NFκB subunit p65 gene expression, in AT1R-CHO cells pretreated with the inhibitory peptide S391A-RAGE362-404. Data are mean ± SEM; n=6-8 per group; * indicates p<0.05 compared to mCherry control. Unless otherwise specified, data are mean ± SEM; n=6-8 per group; * indicates p<0.05 compared to vehicle control. [Figure 11A] The effect of selective suppression of IQGAP-1 expression using IQGAP-1-targeted siRNA in a monolayer of PMAEC from C57bl6 mice, compared to a scrambled control, on the induction of RAGE ligand-independent induction of inflammatory signaling by Ang II (1 μM), as estimated by ICAM-1 expression measured by real-time RT-PCR. [Figure 11B]The effect of selective suppression of IQGAP-1 expression using siRNA in a monolayer of PMAEC from C57bl6 mice, compared to a scrambled control, on the induction of RAGE ligand-dependent induction of inflammatory signaling by RAGE ligand S100A8 / A9, as predicted by the expression of ICAM-1 and VCAM-1 as measured by real-time RT-PCR. [Figure 11C] Pulldown of proteins identified as IQGAP-1, as well as IQGAP-1-related proteins, ezrin / radixin / moesin, and GPCR olfactory receptor 2T2, from other cytosolic components using a column coated with the cytoplasmic tail of mutant RAGE (S391A-RAGE362-404). [Figure 11D] The effect of selective suppression of IQGAP-1 expression in a monolayer of HMEC using siRNA or a scrambled control on the induction of RAGE ligand-independent induction of inflammatory signaling by Ang II (1 μM) in the presence and absence of RAGE362-404, as estimated by MCP-1 expression measured by real-time RT-PCR. Data are mean ± SEM; n=6-8 per group. * indicates p<0.05 compared to scrambled control, and # indicates p<0.05 compared to scrambled control + ligand (Ang II or s100A8 / A9 where appropriate). [Figure 12A] The effect of transduction of mouse SVEC with RAGE or RAGE mutants on ICAM-1 induction by Ang II, compared to vector alone (pc-Neo) as a control, as measured by RT-PCR. Data are mean ± SEM; n=6-8 per group; * indicates compared to untreated control; # indicates compared to neo + Ang II, p<0.05. [Figure 12B] Effect of transduction with truncated RAGE mutants on Ang II-mediated induction of ICAM-1 in mouse SVEC to identify the smallest fragment with inhibitory activity. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to untreated control. [Figure 12C]Inhibition of NFκB activation after exposure to Ang II (1 μM) in RAGE-AT1R-CHO, presumably by induction of NFκB subunit p65 gene expression, in the presence of non-mCherry fused mutants and N-terminal truncated RAGE constructs. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to untreated control. [Figure 12D] Effect of transduction with single-site-directed alanine or lysine mutants of RAGE370-390 on Ang II-mediated induction of ICAM-1 in mouse SVEC. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to untreated control. [Figure 12E] Sequence homology between RAGE379-390 and anti-inflammatory proteins from Streptomyces and other microorganisms. [Figure 13A] Effect of TAT-mCherry-RAGE362-404 (0.4 ng / ml) with or without the S391A-RAGE mutation on the induction of NFκB subunit p65 gene expression by Ang II (1 μM; black bar) in AT1R-CHO cells compared to TAT-Cherry alone (8 μg). Data are mean ± SEM; n=6-8 per group; * indicates p<0.05 compared to untreated control. [Figure 13B] Inhibition of signaling achieved by the S391A-RAGE362-404 peptide on Ang II-dependent induction of NFκB subunit p65 gene expression in AT1R-CHO cells is not reversed by pretreatment with wild-type RAGE362-404 peptide. Data are mean ± SEM; n=6-8 per group, * compared to untreated control; p<0.05. [Figure 13C] Inhibition of signaling achieved by the S391A-RAGE362-404 peptide on Ang II-dependent induction of NFκB subunit p65 gene expression in AT1R-CHO cells is observed regardless of post-treatment with 1000-fold excess wild-type RAGE362-404 peptide. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to untreated control. [Figure 13D] Inhibitory effect of S391A-RAGE362-404 peptide on the induction of p65 and PCNA gene expression in response to Ang II in AT1R-CHO cells transduced with full-length S391Q-cRAGE, which lacks targets available for phosphorylation. Data are mean ± SEM; n=6-8 per group; * indicates relative to Ang II, # indicates relative to full-length wild-type RAGE; p<0.05. [Figure 13E] Effect of wild-type RAGE362-404 peptide (0.4 ng / ml) on induction of pro-inflammatory gene expression in RAGE-deficient PMAEC in response to Ang II. Data are mean ± SEM; n=6-8 per group, * indicates p<0.05 compared to untreated control. [Figure 13F] Effect of S391A-RAGE362-404 peptide on the response to TNFα as a control and on the induction of pro-inflammatory VCAM-1, CXCL2, and CXCL12 gene expression in PMAEC in response to Ang II. Data are mean ± SEM; n=6-8 per group; * indicates vehicle and treatment control (TAT), # indicates Ang II and treatment control (TAT); p<0.05. [Figure 13G] Effects of S391A-RAGE362-404 peptide, AT1R blocker, and irbesartan on the induction of pro-inflammatory gene expression in response to Ang II in HAEC. [Figure 13H](i) Inhibitory effect of the S391A-RAGE362-404 peptide on the induction of pro-inflammatory gene expression (p65) in response to RAGE ligand S100A8 / A9 in AT1R-CHO cells expressing an inactive full-length S391A-RAGE mutant (shown as a control) and in AT1R-CHO cells also expressing full-length RAGE. (ii) Inhibitory effect of the S391A-RAGE362-404 peptide on the induction of pro-inflammatory gene expression (VCAM-1) in response to Ang II or RAGE ligand S100A8 / A9 in PMAEC cells where RAGE is endogenously sufficient. Unless otherwise specified, data are mean ± SEM; n=6-8 per group; * indicates p<0.05 compared to vehicle control; # indicates p<0.05 compared to control + Ang II. [Figure 14A] Effect of S391A-RAGE362-404 on Ang II-dependent induction of pro-inflammatory markers after ex vivo exposure of the entire aorta to Ang II (1 μM) from apoE-KO mice. Data are mean ± SEM; n=6 per group. * indicates p<0.05 compared to apoE-KO + vehicle + TAT-mCherry control, and # indicates p<0.05 compared to apoE-KO + TAT-mCherry control + Ang II. [Figure 14B] Effect of wild-type RAGE362-404 on Ang II-dependent induction of pro-inflammatory markers after ex vivo exposure of the entire aorta to Ang II (1 μM) from AGER / apoE-KO mice. Data are mean ± SEM; n=8 per group, * indicates p<0.05 compared to apoE-KO + vehicle + TAT-mCherry control. [Figure 15A]The atherosclerotic effect of TAT-mCherry-RAGE362-404, which contains 42 C-terminal amino acids of mCherry fluorescent protein-labeled RAGE and an HIV-TAT motif that promotes cell permeability, on Ang II-dependent induction of aortic atherosclerosis in apoE-KO and Ace2 / AGER / apoE triple KO mice. This is compared to the anti-atherosclerotic effect of TAT-mCherry-S391A-RAGE362-404 on Ang II-dependent induction of aortic atherosclerosis in Ace2 / apoE-DKO mice. Data are mean ± SEM; n=8 per group; * is against apoE-KO control; # is against Ace2 / apoE-DKO control; p<0.05. [Figure 15B] The atherosclerotic effect of the TAT-mCherry-RAGE362-404 peptide, containing 42 C-terminal amino acids of mCherry fluorescent protein-labeled RAGE and a cell permeability-promoting HIV-TAT motif, on Ang II-dependent induction of aortic atherosclerosis in diabetic apoE-KO and diabetic AGER / apoE-DKO mice. This is compared to the anti-atherosclerotic effect of TAT-mCherry-S391A-RAGE362-404 on Ang II-dependent induction of aortic atherosclerosis in diabetic apoE-KO mice. Data are mean ± SEM; n=8 per group; * is against apoE-KO control, # is against diabetic apoE DKO control; p<0.05. [Figure 15C] As shown, there is no effect of TAT-mCherry-RAGE362-404 and TAT-mCherry-S391A-RAGE362-404 on systolic blood pressure in mice with or without AGER expression and in diabetic apoE-KO mice. [Figure 16A] BRET saturation curves using AT1 / Rluc8 and RAGE / Venus were generated 60 minutes after the addition of Ang II or vehicle with or without soluble RAGE22-331 (sRAGE). Data are combined from three independent experiments. [Figure 16B]Ang II-induced mobilization of βarrestin2 / Venus to AT1 / Rluc8 and CCL22-induced mobilization of βarrestin2 / Venus to CCR4 / Rluc8 were used as controls. [Figure 16C] Ang II-induced recruitment of β-arrestin 2 / Venus adjacent to RAGE / Rluc8, in the presence of AT1 receptors after exposure to Ang II, and not in the presence of CCR4 after exposure to CCL22. [Figure 16D] When Gαi / Nluc and Gγ2 / Venus are co-expressed in the presence of unlabeled CCR4, a CCL22-induced BRET signal is observed. [Figure 17A] Ang II-induced recruitment of β-arrestin2 / Venus (β-arr2 / Venus) to RAGE / Rluc8 in proximity, not in the absence of the AT1 receptor. Ang II-induced recruitment of β-arrestin2 / Venus to AT1 / Rluc8 is included as a control. [Figure 17B] Weakly thyrotropin-releasing hormone (TRH)-induced recruitment of β-arrestin 2 / Venus (β-arr2 / Venus) adjacent to RAGE / Rluc8 in the presence, rather than the absence, of TRH receptor 1 (TRHR1). TRH-induced recruitment of β-arrestin 2 / Venus to TRHR1 / Rluc8 is included as a control. The inset shows the same data with an enlarged y-axis scale. [Figure 17C] Orexin A (OxA)-induced recruitment of β-arrestin 2 / Venus (β-arr2 / Venus) adjacent to RAGE / Rluc8 in the presence, not in the absence, of orexin receptor 1 (OxR1). OxA-induced recruitment of β-arrestin 2 / Venus to OxR1 / Rluc8 is included as a control. [Figure 17D]Weak bradykinin (BDK)-induced recruitment of β-arrestin 2 / Venus (β-arr2 / Venus) to RAGE / Rluc8 in the presence, rather than the absence, of BDK receptor 2 (BDKR). BDK-induced recruitment of β-arrestin 2 / Venus to BDKR / Rluc8 is included as a control. The inset shows the same data with an enlarged y-axis scale. [Figure 17E] Arginine vasopressin (AVP)-induced recruitment of β-arrestin 2 / Venus (β-arr2 / Venus) adjacent to RAGE / Rluc8 in the presence, not in the absence, of vasopressin receptor 2 (V2R). AVP-induced recruitment of β-arrestin 2 / Venus to V2R / Rluc8 is included as a control. [Figure 17F] CCL2 (MCP1)-induced recruitment of β-arrestin2 / Venus (β-arr2 / Venus) adjacent to RAGE / Rluc8 in the presence, not in the absence, of CCR2. MCP-1-induced recruitment of β-arrestin2 / Venus to CCR2 / Rluc8 is included as a control. [Figure 17G] In particular, the CCL4(MIP1β)-induced, especially weak, recruitment of β-arrestin2 / Venus (β-arr2 / Venus) adjacent to RAGE / Rluc8 in the presence of CCR5 after exposure to MIP1β, compared to a control in the absence of CCR5. MIP1β-induced recruitment of βarrestin2 / Venus to CCR5 / Rluc8 is included as a further control. The inset shows identical data with an enlarged y-axis scale. All data are mean ± SEM from three independent experiments. [Figure 18A] CCL3-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CCR1. [Figure 18B] CCL2-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CCL2. [Figure 18C] Lack of CCL22-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CCR4. [Figure 18D]Absence of CCL4-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CCR5. [Figure 18E] CCL20-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CCR6. [Figure 18F] CCL19-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CCR7. [Figure 18G] Absence of CCL27-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CCR10. [Figure 18H] CXCL8-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CXCR1. [Figure 18I] CXCL8-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CXCR2. [Figure 18J] Absence of CXCL11-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CXCR3. [Figure 18K] CXCL12-induced decrease in the proximity of β-arr2 / Venus to RAGE / Rluc8 in the presence of CXCR4. [Figure 18L] CXCL16-induced recruitment of β-arr2 / Venus adjacent to RAGE / Rluc8 in the presence of CXCR6. All data are mean ± SEM from three independent experiments. [Figure 19A-OO] Proximity of Rluc8-labeled RAGE to a Venus-labeled intracellular compartment marker in the presence of a indicated non-BRET-labeled GPCR activated by the indicated ligand at time zero. [Figure 20A] Activation of NFκB, as measured by induction of NFκB subunit p65 gene expression by CCL2(MCP-1) in CHO cells expressing CCR2, either in the presence or absence of RAGE co-expression. [Figure 20B]Activation of NFκB, as measured by induction of NFκB subunit p65 gene expression by CXCL2(IL-8) in CHO cells expressing CXCR2, in or without the presence of RAGE co-expression. [Figure 20C] NFκB activation is measured by induction of NFκB subunit p65 gene expression by CCL2 (MCP-1) in myeloid-derived primary macrophages, in the presence or absence of the RAGE activation peptide inhibitor S391A-RAGE362-404. [Figure 20D] Activation of NFκB, measured by self-induction of MCP-1 gene expression by CCL2(MCP-1) in HMEC, in the presence or absence of the RAGE activation peptide inhibitor, S391A-RAGE362-404. [Figure 20E] Activation of NFκB, as measured by induction of MCP-1 gene expression by IL-8 in HMECs expressing CXCR2, in or without the RAGE activation peptide inhibitor S391A-RAGE362-404. [Figure 21A] The BRET between mCherry / RAGE338-361 and Nluc / AT1 increases with Ang II. Data are expressed as mean ± SEM, with n=3-5. [Figure 21B] As shown, BRET saturation curves for AT1 / Rluc8 and RAGE / Venus were generated 60 minutes after the addition of vehicle or Ang II when cells were also transduced with 200 ng or 400 ng of mCherry / RAGE338-361 cDNA. Data are combined from three independent experiments. [Figure 21C] As shown, Ang II-inducible regulation of the BRET signal between AT1 / Rluc8 and RAGE / Venus when cells are also transduced by 0, 50, 100, 200, 300 or 400 ng of mCherry / RAGE338-361 cDNA or pcDNA3 regulatory plasmid. Data are expressed as mean ± SEM, n=3-4. Filter: Venus 550nm / Rluc8 450nm. [Figure 21D]As shown, the absence of Ang II-inducible BRET signal between AT1 / Rluc8 and mCherry / RAGE338-361 when cells are transduced with 50 ng of AT1 / Rluc8 cDNA, 300 ng of RAGE / Venus cDNA, and 0, 50, 100, 200, 300 or 400 ng of mCherry / RAGE338-361 cDNA or pcDNA3 control plasmid. Data are expressed as mean ± SEM, n=3-4. Filter: mCherry 650 nm / Rluc8 450 nm. [Figure 21E] Figures 21C and 21D show the emission from AT1 / Rluc8, fluorescence from RAGE / Venus, and fluorescence from mCherry / RAGE338-361 in the experiment. Data are expressed as mean ± SEM, with n=3-4. [Figure 21F] Ligand-induced regulation of the BRET signal between GPCR / Rluc8 and RAGE / Venus when cells are also transduced by mCherry / RAGE338-361 cDNA or pcDNA3 regulatory plasmids, as shown. Data are expressed as mean ± SEM, n=2-4. Filter: Venus 550nm / Rluc8 450nm. Amount of transduced cDNA: 50ng GPCR / Rluc8 + 300ng RAGE / Venus + 400ng mCherry / RAGE338-361 or pcDNA3. Shown Rluc8-labeled GPCR activated by the shown ligand at the shown concentration, time zero. [Figure 21G] Ang II-mediated pro-inflammatory signaling (ICAM-1 expression) in HMEC1 inhibited by mCherry / RAGE338-361 is rescued by mCherry / RAGE362-404. Data are expressed as mean ± SEM, n=6-8. [Figure 21H]Induction of pro-inflammatory signaling by Ang II in HMEC cells is inhibited by overexpression of RAGE343-361, a RAGE transmembrane domain with or without an N-terminal mCherry fusion, as represented by ICAM-1 expression measured using real-time RT-PCR. Data are expressed as mean ± SEM, n=6-8. [Figure 21I] As indicated by p65 expression measured using real-time RT-PCR, induction of pro-inflammatory signaling by RAGE ligand S100A8 / A9 in RAGE-CHO cells is inhibited by overexpression of RAGE transmembrane domains RAGE343-361 alone or RAGE370-390. Data are expressed as mean ± SEM, n=6-8. [Figure 21J] As indicated by ICAM-1 expression measured by real-time RT-PCR, induction of pro-inflammatory signaling by Ang II in HMEC cells is inhibited by siRNA targeting Diaph1 or PKCz. This inhibition is rescued by RAGE362-404 but not by RAGE343-404. Data are expressed as mean ± SEM, n=6-8. [Modes for carrying out the invention]

[0533] [A brief explanation of arrays]

[0534] [Table 31]

[0535] [Detailed description of the invention] <1.Definition> Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below:

[0536] A "receptor heteromer" is defined as "a macromolecular complex composed of at least two (functional) receptor units that possess biochemical properties distinctly different from those of their individual components" (Ferre et al., 2009).

[0537] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. For example, “element” means one or more elements.

[0538] "Approximately" means a measurement, quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% different from the baseline measurement, quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0539] As used herein, "and / or" means any combination of one or more of the related enumerated items and the absence of any combination as interpreted by alternative (or).

[0540] The terms “drug,” “candidate drug,” “modulator,” “alternative,” “functional alternative,” “non-functional alternative,” or “inhibitor” include compounds, mixtures of compounds, biomolecules, extracts from biological materials, biological organisms or parts thereof, or other materials that induce a desired pharmacological and / or physiological effect. These terms also include, but are not limited to, salts, esters, amides, prodrugs, active metabolites, analogs, and other pharmaceutically acceptable pharmacologically active components of the compounds specifically referred to herein. When the above terms are used, it should be understood that this includes the active drug itself as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, prodrugs, metabolites, and analogs. The terms “drug,” “modulator,” “alternative,” or “inhibitor” should not be interpreted narrowly and extend to protein molecules such as small molecules, peptides, polypeptides, and proteins, and compositions containing them, as well as genetic molecules such as RNA, DNA and their mimes and chemical analogs, and cell agents. The terms “drug,” “modulator,” “alternative,” or “inhibitor” include cells capable of producing and secreting polypeptides and polynucleotides comprising nucleotide sequences encoding such polypeptides, as referred to herein. Therefore, the terms “drug,” “modulator,” “alternative,” or “inhibitor” extend to nucleic acid constructs, including vectors such as viral or nonviral vectors, expression vectors, and plasmids, for expression and secretion within the cellular range.

[0541] The term “inhibitor” is used in its broadest sense to include any compound, including proteins, polypeptides, peptides, antibodies, antibody fragments, macromolecules, or small molecules (less than 10 kDa), that reduces at least one aspect of the activity, activation, or function of another molecule. For example, an inhibitor may reduce the activity, activation, or function of RAGE and / or specific coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, and / or appropriately reduce RAGE ligand-independent activation by specific activated coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2. Thus, “inhibitor of RAGE ligand-independent activation by specific activated coexisting GPCRs” refers to a drug that can substantially reduce, inhibit, antagonize, block, negatively modulate, and / or mitigate RAGE ligand-independent activation by specific activated coexisting GPCRs. Inhibition of RAGE ligand-independent RAGE activation by specific activated coexisting GPCRs by inhibitors appropriately reduces or inhibits its biological effects, including the production of pro-inflammatory mediators, including pro-inflammatory cytokines, by cells, or the modulation of other cellular elements associated with abnormal RAGE ligand-independent activation and the symptoms of RAGE disease. It should be noted that partial agonists can act as inhibitors because they do not produce the maximum effect even when exhibiting agonism. Therefore, by competing with or modulating the agonist activity of more effective agonists, such as endogenous agonists, they reduce signal output compared to their absence, thus effectively acting as inhibitors of receptor polypeptides and / or their signaling pathways. Consequently, partial agonists can act as inhibitors in therapeutic settings. Inhibitors do not necessarily inhibit all aspects of the activity, activation, or function of another molecule; in fact, they may inhibit some aspects while activating others and / or failing to modulate further aspects. Therefore, inhibitors may exhibit ligand bias.

[0542] The term "ligand bias" refers to the phenomenon where distinct ligand-stabilized receptor states can exist for the same receptor, selectively promoting or inhibiting the activation of different signaling pathways (Mustafa et al., 2010). This phenomenon includes, but is not limited to, ligand-biased signaling, ligand-induced biased signaling, receptor signal agonist transport, cell-based functional selectivity, receptor activity state-based selectivity, stimulus transport, biased agonism, secondary efficacy, and ligand-induced selective signaling (Mustafa et al., 2010). For example, this involves the concept that not all agonists activate all signaling pathways normally activated by a reference agonist, which is often an endogenous agonist. An agonist may activate some pathways while not activating others compared to the reference, thereby exhibiting bias. Furthermore, antagonists, inverse agonists, or inhibitors may inhibit only some pathways while leaving others unaffected, and may act at orthosteric ligand-binding sites and / or allosteric binding sites. Orthosteric and allosteric binding sites are defined as commonly known in the art, and allosterism can occur across complexes, from one receptor to another, such that the binding of a ligand to one receptor may cause allosteric modulation of another receptor on the same macromolecular complex. Allosteric modulators may also exhibit ligand bias, modulating some signaling pathways but not others. It is also known in the art that a ligand may, for example, act as an agonist in one signaling pathway while acting as an inhibitor in another, and / or not affect a third signaling pathway. In fact, multiple variations and combinations of signaling modulatory effects can occur. Ligand bias is also not absolute, for example, in that a ligand may reduce signaling through one pathway without completely inhibiting it, and / or incompletely activate another signaling pathway. The path can be adjusted to different degrees, which can be measured by multiple parameters.This includes, but is not limited to, differences in the potency and / or effectiveness and / or temporal aspects of signal transduction and / or spatial aspects of signal transduction.

[0543] The term “functional substitute” is used in its broadest sense to include any compound, including proteins, polypeptides, peptides, antibodies, antibody fragments, high molecular weights, or low molecular weights (less than 10 kDa), that can mimic or enhance at least one aspect of the activity, activation, or function of another molecule in place of that molecule. For example, a functional substitute for RAGE can replicate the activity, activation, or function of RAGE and / or specific coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, and / or appropriately replicate RAGE ligand-independent RAGE activation by activated specific coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, in a system that otherwise lacks RAGE expression. Thus, a “functional substitute” for RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR refers to a drug that can substantially increase, enhance, agonize, and / or positively modulate RAGE ligand-independent RAGE activation by a specific activated coexisting GPCR. Restoring the signaling capacity of RAGE ligand-independent RAGE activation by specific activated coexisting GPCRs using functional substitutes appropriately restores or enhances its biological effects, including the production of pro-inflammatory mediators, including pro-inflammatory cytokines, by cells or the modulation of other cellular elements associated with RAGE ligand-independent activation of RAGE disease symptoms. Functional substitutes do not necessarily mimic all aspects of the activity, activation, or function of another molecule; in fact, they may inhibit some aspects while activating others and / or failing to modulate further aspects. Therefore, functional substitutes can also exhibit ligand bias.

[0544] The term “non-functional substitute” is used in its broadest sense to include any compound, including proteins, polypeptides, peptides, antibodies, antibody fragments, high molecular weights, or low molecular weights (less than 10 kDa), that can inhibit, antagonistize, or reduce at least one aspect of the activity, activation, or function of another molecule in place of that molecule. For example, a non-functional substitute for RAGE may inhibit the activity, activation, or function of RAGE and / or specific coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2, and / or appropriately inhibit RAGE ligand-independent activation by specific activated coexisting GPCRs, such as angiotensin receptors like AT1R or specific chemokine receptors like CCR2. Thus, a “non-functional substitute” for RAGE ligand-independent activation by specific activated coexisting GPCRs refers to a drug that can substantially reduce, inhibit, antagonize, and / or negatively modulate RAGE ligand-independent activation by specific activated coexisting GPCRs. The reduction in the signaling capacity of RAGE ligand-independent RAGE activation by specific activated coexisting GPCRs using non-functional substitutes appropriately reduces or inhibits its biological effects, including the production of pro-inflammatory mediators, including pro-inflammatory cytokines, by cells, or the modulation of other cellular elements associated with RAGE ligand-independent activation of RAGE disease symptoms. It should be noted that partial agonists can also act as inhibitors, as they do not produce the maximum effect even when exhibiting agonism. Therefore, they can effectively act as inhibitors of receptor polypeptides and / or their signaling pathways by reducing signal output compared to their absence, by competing with or modulating the agonist activity of more effective agonists, such as endogenous agonists. As a result, partial agonists can act as inhibitors in therapeutic settings. Non-functional substitutes do not necessarily reduce all aspects of the activity, activation, or function of another molecule; in fact, they may inhibit some aspects while promoting others and / or failing to modulate further aspects. Therefore, non-functional substitutes can also exhibit ligand bias.

[0545] The term "bonding" and its grammatical equivalents refer to the physical association of molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions under physiological conditions, and include interactions such as salt bridges and water bridges, as well as all other conventional bonding means. Bonding can occur directly or through interactions with one or more other intermediate molecules.

[0546] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “contains” will be understood to mean the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. Therefore, the use of terms such as “contains” indicates that the enumerated elements are necessary or essential, while other elements may or may not be present, at the discretion of the user. “Consists of” means the phrase “consists of.” related It means to include and be limited to things. Therefore, the phrase "consists of" means that the listed elements are necessary or essential, and other elements exist. Must not This indicates that "essentially becomes" the phrase before It means that it includes any of the elements listed, but is limited to other elements that do not interfere with or contribute to the activities or actions specified in the disclosure of the listed elements. Thus, the phrase "essentially consists of" indicates that the listed elements are necessary or essential, but other elements may be present or abs...

Claims

1. (i) As shown in sequence numbers 5, 6, 8, 9, 10, 11, 12 or 13; (ii) Residues 22 to 390 of wild-type RAGE in Sequence ID No. 14; (iii) Residues 380 to 404 of wild-type RAGE in Sequence ID No. 14; (iv) Residues 370 to 390 of wild-type RAGE in Sequence ID No. 14, where residue 388 is A; (v) Residues 370 to 390 of wild-type RAGE in Sequence ID No. 14, where residue 380 is A; (vii) Residues 370 to 390 of wild-type RAGE in Sequence ID No. 14, where residue 382 is A; (viiii) residues 370 to 390 of wild-type RAGE of SEQ ID NO: 14, where residue 384 is A; or (ix) Residues 374 to 390 of wild-type RAGE in SEQ ID NO: 14 An isolated or purified peptide consisting of the following amino acid sequence that inhibits RAGE ligand-independent signaling.

2. The isolated or purified peptide according to claim 1, wherein the amino acid sequence is the amino acid sequence shown in SEQ ID NO: 5, 6, or 8.

3. (i) As shown in Sequence ID No. 2 or 7; (ii) Residues 362 to 404 of wild-type RAGE in Sequence ID No. 14, where residue 391 is Y, V, R, N, K, H, G, F, E, or D; (iii) residues 22 to 404 of wild-type RAGE of Sequence ID No. 14, where residue 391 is A or C; or (iv) Residues 342 to 404 of wild-type RAGE in Sequence ID No. 14, where residue 391 is A. An isolated or purified peptide consisting of the following amino acid sequence that inhibits RAGE ligand-independent signaling.

4. A nucleic acid comprising a nucleotide sequence encoding an isolated or purified peptide according to any one of claims 1 to 3.

5. A pharmaceutical composition comprising a therapeutically effective amount of an isolated or purified peptide according to any one of claims 1 to 3, or a nucleic acid according to claim 4.

6. The pharmaceutical composition according to claim 5, further comprising one or more excipients.

7. A pharmaceutical composition according to claim 5 or 6 for treating, preventing or managing RAGE-related disorders in patients requiring such treatment, prevention or management.

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