Methods and compositions for treatment of inflammatory diseases

Abstract

Disclosed herein are compositions and methods for treating inflammatory diseases, comprising inhibiting or blocking the interaction between apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-cytokines.

Description

Atty Docket No.58434-0038WO1 METHODS AND COMPOSITIONS FOR TREATMENT OF INFLAMMATORY DISEASES CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application 63 / 635,440 filed April 17, 2024, which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named “58434-0038WO1_ST26_SL.XML.” The XML file, created on April 17, 2025, is 7,670 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. BACKGROUND

[0003] Inflammasomes are cytosolic innate immune complexes that activate caspase-1 following detection of pathogenic and endogenous dangers.

[0004] Inflammasomes process the pro-IL-1β and pro-IL-18 (non-inflammatory and biologically inactive) cytokine into mature IL-1β and mature-IL-18 (inflammatory and biologically active form) upon sensing pathogenic and sterile signatures. Current therapies explicitly target extracellular mature IL-1β which includes canakinumab, anakinra, and rilonacept (anti-inflammatory therapies). Notably, there are no FDA-approved drugs targeting IL-18, although IL-18 targeting therapies are in clinical trials. However, the intracellular targeting of both pro-IL-1β & pro-IL-18 for therapeutic intervention has not been described. This novel intracellular targeting could be more direct, cost-effective, and less invasive than blocking extracellular cytokines.

[0005] Elevated level of mature IL-1β and mature-IL-18 (an inflammatory cytokine) is deleterious to the host and tissue. These unchecked levels of mature IL-1β and mature- IL-18 exacerbate inflammatory signaling and have been reported in many inflammatory diseases. So, far mature IL-1β, an inflammatory version of cytokine has been therapeutically targeted. Notably, these targeting agents acts extracellularly and required repeated dosing of patients as these interventions cannot halt the processing of pro-IL-1β and mature-IL-18 into a mature IL-1β and mature-IL-18 which occurs intracellularly by the action of caspase-1.Atty Docket No.58434-0038WO1 SUMMARY

[0006] In one embodiment provided herein is a method to restrict the amount of pro-IL-1β converted to mature IL-1β.

[0007] In one embodiment provided herein is a method to restrict the amount of pro--IL- 18 converted to mature-IL-18.

[0008] In one embodiment provided herein is a method to restrict the amount of pro-IL-1β and pro--IL-18 converted to mature IL-1β and mature-IL-18, respectively.

[0009] In one embodiment provided herein is a method to restrict inflammatory outcome, the method comprising restricting the amount of pro-IL-1β and pro--IL-18 converted to mature IL-1β and mature-IL-18, respectively.

[0010] In one embodiment provided herein is a method to selectively enhance inflammatory cell death, comprising restricting the amount of pro-IL-1β and pro--IL-18 converted to mature IL-1β and mature-IL-18, respectively.

[0011] In one embodiment the present methods target the intracellular molecule that sequesters pro-IL-1β and pro-IL-18 molecule. The intracellular molecule serves as a druggable interface that offers a novel target to regulate the production of mature-IL-1β and pro-IL-18 in cells.

[0012] The methods herein can be useful not only in restricting elevated mature IL-1β and mature-IL-18 production in chronic inflammatory diseases but also in potentially fostering the inflammasome pathway towards inflammatory cell death (pyroptosis, without IL-1β & IL-18 release) as inflammatory cell death can be harnessed to generate antitumor immunity by killing the cancerous cells (blocking inflammatory cytokines release can check the undesirable effects in cancer immunotherapy). BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows the dissection of recruitment step – a novel role of ASC in pro- cytokine recruitment, a prerequisite step necessary for inflammasome-dependent cytokine maturation.

[0014] FIG. 2 shows the decoding inflammasome activity at the speck. BMDMs were primed with LPS and stimulated nigericin to activate NLRP3 inflammasome. (A) The speck serves as the primary site for caspase-1 processing. Immortalized mouse bone marrow- derived macrophages (iBMDMs) primed with LPS and stimulated with nigericin to activate the NLRP3 inflammasome express mTuruqoise-pro-Caspase-1-mNeongreen FRETAtty Docket No.58434-0038WO1 reporter, forming specks at the microtubule organizing center (MTOC), with SiR Tubulin serving as the live-cell marker of MTOC. (puncta) indicates caspase-1 cleavage. reporter form specks at the MTOC. (B) Detection of loss of FRET within specks (puncta) indicates caspase-1 cleavage. (C) The speck is identified as the primary site for IL-1β processing using iBMDMs expressing Turuqoise-pro-IL-1β-mNeongreen (D) FRET Loss of FRET within specks (puncta) reflects IL-1β cleavage. (E) NanoBRET analysis, a proximity-based bioluminescence resonance energy transfer (BRET) assay, illustrates the interaction between ASC / pro-IL-1β (row 1), and between ASC / pro-IL-18 is depicted in 3 (left to right). Row 2 and 4 are halo-tag alone controls (left to right). Scale bars: (A & C) 10μM; White arrowheads indicate specks co-localizing with specks.

[0015] Fig. 3A-B shows human ASC and human ASC-pro-IL-1β interaction analysis by nanoBRET assay (A), and by Halo-Tag pull-down assay (B). Red asterisk indicates ASC and black asterisk indicates pulled down pro-IL-1β.

[0016] Figs.4 shows ASC sequestering pro-IL-1β. (A) Single expression patterns of ASC and pro-IL-1β (B) Co-expression of ASC with pro-IL-1β completely redistributes expression of pro-IL-1β. Pro-IL-1β redistributes from diffusely labelling to a speck-like distribution upon co-transfection with ASC within cells (scale 10 µm).

[0017] Fig.5A-B shows mapping of the human ASC domains involved in binding human pro-IL-1β. Among the tested domains, ASCINTdemonstrated robust binding to human pro- IL-1β. In the figure, ITLD, INT denotes an interlinker.

[0018] Fig. 6 shows the ASC-pro-IL-1β interaction, analyzed using ASCINTdomain mutants. The ASCINTmutants disrupted interaction with human pro-IL-1β.

[0019] Fig. 7A-B (A) Single transfection of ASC (wiltype) and pro-IL-1β, and (B) ASC interlinker domain mutants (scale 5 µm).

[0020] Fig.8 shows the interaction of ASCINTmutants with pro-IL-1β. ASCINTmutants co- localized with pro-IL-1β shown by the analysis of ASC speck formation and its co- localization with pro-IL-1β (scale 5 µm).

[0021] Fig. 9 shows the mapping of the human pro-IL-1β domain involved in binding human ASC. The pro-domain of human IL-1β exhibited high affinity for human ASC.

[0022] Fig. 10A-B shows the structure of the pro- and mature-domains of human IL-1β predicted by Alphafold2.

[0023] Fig.11A-B shows the structure function analysis of the mature and pro-domains of human IL-1β using the nanoBRET assay. Pro-domain residues are highlighted yellow.Atty Docket No.58434-0038WO1

[0024] Fig.12A-D shows the interaction of human ASC with human pro-IL-18. (Top, Left) Cartoon showing the domain architecture. (Bottom, Left) NanoBRET analysis. (Top, Right) Expression pattern of ASC and pro-IL-18. (Bottom, Right) ASC co-localized with pro-IL-18 in transfected 293T cells. (scale 5 µm).

[0025] Fig. 13A-B shows the mapping of the domain of human IL-18 responsible for binding with human ASC. Both the pro-domain and mature-domain of human IL-18 are capable of binding human ASC (FIG. 13A). Among the tested domains, ASCINT demonstrated robust binding to human pro-IL-18 (FIG.13B). Note : Interlinker (short) lacks end lysine (K) at the c-terminus of interlinker domain.

[0026] Fig. 14A-C shows structure-based analysis of ASC-pro-IL-18 interactions using ASCPYDdomain mutants.

[0027] Fig. 15A-C shows structure-based analysis of ASC-pro-IL-18 interactions using ASCCARDdomain mutants.

[0028] Fig. 16A-C shows the analysis of ASCINT domain mutants to investigate the interaction between ASC and pro-IL-18.

[0029] Fig. 17A-B shows the relevant residues of the human pro-domain of IL-18 (FIG. 17A) and (mature domain of human IL-18 (Fig.17B).

[0030] Fig. 18 shows the results of the structure-based analysis of the ASC-pro-IL-18 interaction. The mutations in the pro-IL-18 mature domain abolished its interaction with ASC.

[0031] Fig.19A-B shows the results of competition assays. Increasing concentrations of pro-IL-1β can effectively compete out the interaction between ASC and pro-IL-18 (FIG. 19A). Increasing concentrations of pro-IL-18 do not effectively compete out the interaction between ASC and pro-IL-1β (FIG. 19B). Thus, binding of pro-IL-1β and pro-IL-18 are mutually exclusive.

[0032] Fig.20A-B shows an analysis of the binding of ASC-pro-IL-1β and ASC / pro-IL-18 in the presence of pro-caspase-1 and NLRP3. Pro-cytokine bound ASC can still recruit upstream NLRP3 and downstream pro-caspase -1 in heterologous 293T cells. As pro- cytokine bound ASC interacted with upstream NLRP3 (via ASCPYD-NLRP3PYDinteractions) and downstream pro-caspase-1 (via ASCCARD-capsas-1CARDinteractions).

[0033] Fig. 21 (A) shows that cell-permeant peptide perturbs inflammatory cytokine production analyzed in cell culture supernatant using human IL-1β ELISA Human monocytic THP-1 cells exhibit a significantly decrease in IL-1β production when the interaction between ASC and pro-cytokine are perturbed using cell permeant peptides. (B)Atty Docket No.58434-0038WO1 Peptides do not perturb NF-κB dependent priming step, as we similar levels of pro-IL-1β levels were observed.

[0034] Fig. 22 shows the ASC-pro-IL-1β interaction, analyzed using construct lacking interlinker domain, (ASCΔINT).

[0035] Fig.23 shows the analysis of ASC-pro-IL-1β interaction (using ASCINT domain mutants). The ASCINTmutant showed disruption of the interaction with human pro-IL-1β.

[0036] Fig. 24 shows the mapping of the human pro-IL-1β domains involved in binding human ASC. Both domain binds, but Mature-domain (MD) of human IL-1β exhibit a high affinity for human ASC.

[0037] Fig.25 shows validation of a structure-based analysis of ASC-pro-IL-1β interaction (using pro-1L-1β structure).

[0038] Fig.26 shows mapping of the human ASC domains involved in for binding human pro-IL-18. Among the tested domains, ASCITLDdemonstrated robust binding to human pro-IL-18

[0039] Fig. 27 shows a structure-based analysis of ASC-pro-IL-18 interactions. Recruitment-deficient pro-IL-18 mutants have impairment in processing.

[0040] Fig. 28 shows the perturbing of the recruitment of inflammatory cytokines using membrane permeable peptides (THP-1 monocytic cells differentiated to macrophages). Blocking ASC / pro-cytokine interactions in THP-1 macrophages by cell-permeant peptides led to significantly reduced IL-1β and IL-18 secretion. Phorbol 12-myristate 13-acetat (PMA) differentiates monocytes to macrophages.

[0041] Fig. 29 shows the perturbing of the recruitment of inflammatory cytokines using membrane permeable peptides in primary human cells (human monocyte-derived macrophages; hMDMs). Blocking ASC / pro-cytokine interactions in in hMDMs by cell- permeant peptides led to significantly reduced IL-1β and IL-18 secretion..

[0042] Fig.30 shows that ASC speck formation is unperturbed by experimental peptide in transfected 293T cells (qualitative analysis). Cell permeant peptide blocks pro-cytokine recruitment by ASC whereas ASC speck formation in unperturbed. (Scale 100 µm). DETAILED DESCRIPTION

[0043] The protein ASC (apoptosis-associated speck-like protein containing a CARD, PYCARD; UniProt Accession No. Q9ULZ3 ^ ASC_HUMAN) serves as a crucial adaptor, connecting upstream inflammasome sensors with downstream inflammatory caspase-1.Atty Docket No.58434-0038WO1 Notably, most canonical and non-canonical inflammasomes (triggered by intracellular LPS and oxidized lipids) rely on ASC for their biological function. ASC Isoform 1 (UniProt Accession No. Q9ULZ3-1 ^ ASC_HUMAN; SEQ ID NO:1) MGRARDAILD ALENLTAEEL KKFKLKLLSV PLREGYGRIP RGALLSMDAL DLTDKLVSFY LETYGAELTA NVLRDMGLQE MAGQLQAATH QGSGAAPAGI QAPPQSAAKP GLHFIDQHRA ALIARVTNVE WLLDALYGKV LTDEQYQAVR AEPTNPSKMR KLFSFTPAWN WTCKDLLLQA LRESQSYLVE DLERS ASC Isoform 2 (UniProt Accession No. Q9ULZ3-2 ^ ASC_HUMAN; SEQ ID NO:2) MGRARDAILD ALENLTAEEL KKFKLKLLSV PLREGYGRIP RGALLSMDAL DLTDKLVSFY LETYGAELTA NVLRDMGLQE MAGQLQAATH QGLHFIDQHR AALIARVTNV EWLLDALYGK VLTDEQYQAV RAEPTNPSKM RKLFSFTPAW NWTCKDLLLQ ALRESQSYLV EDLERS ASC Isoform 3 (UniProt Accession No. Q9ULZ3-3 ^ ASC_HUMAN; SEQ ID NO:3) MGRARDAILD ALENLTAEEL KKFKLQAATH QGSGAAPAGI QAPPQSAAKP GLHFIDQHRA ALIARVTNVE WLLDALYGKV LTDEQYQAVR AEPTNPSKMR KLFSFTPAWN WTCKDLLLQA LRESQSYLVE DLERS

[0044] The sequences of the cell permeant peptides herein are as follows: (SEQ ID No.4) ASAAAAAAIQ AAAQSAARGG GGSRQIKIWF QNRRMKWKK (SEQ ID No.5) IGAAQKPGAG ASPQAPSAGG GGSRQIKIWF QNRRMKWKK (SEQ ID No.6) GSGAAPAGIQ APPQSAAKGG GGSRQIKIWF QNRRMKWKK

[0045] The sequences of the control peptides herein are as follows: (SEQ ID No.7) ESQSYLVEDG GGGSRQIKIW FQNRRMKWKK

[0046] One striking aspect of inflammasome activation is the formation of a single ASC speck, a micron-sized structure comprised of densely packed oligomeric filaments derived from the ASC protein. These ASC specks are widely recognized as instigators of chronic inflammation, as they can be released during inflammasome-triggered cell death (pyroptosis) and subsequently activate fresh waves of inflammasome activity in neighboring cells upon endocytosis. Furthermore, extracellular ASC specks have been shown to amplify inflammatory cytokine production and have been implicated in the cross- seeding of amyloid-β which exacerbate Alzheimer’s disease progression.Atty Docket No.58434-0038WO1

[0047] Targeting the ASC protein is of paramount importance in clinical settings to regulate inflammation triggered by a broad spectrum of pathogenic and sterile stimuli, as ASC serves as a common adaptor for a diverse array of inflammasomes including NLRP3, NLRP1, AIM2, NLRP6, NLRP9, NLRP12, NLRP11. Current strategies predominantly center around inhibiting ASC polymerization to mitigate its role in exacerbating inflammasome activity. However, this approach may pose challenges, since ASC is integral to various inflammasomes, and inhibiting its polymerization might impair the functions of multiple inflammasomes, hindering the host's ability to respond effectively to different triggers through regulated inflammasome signaling.

[0048] The only know function of oligomerized ASC in speck is to bind pro-caspse-1, facilitating its dimerization and autoproteolysis to generate active caspase-1. Active caspase-1 plays two primary roles: maturation of pro-cytokines (pro-IL-18, pro-IL-1β) to inflammatory cytokines (mature-IL-1β, mature-IL-18), and cleavage of gasdermin D (GSDMD), resulting in pyroptosis, an inflammatory cell death.

[0049] Our data indicate the existence of a potential pre-step step, termed "recruitment," preceding caspase-1-dependent cleavage, which facilitates the proximity of pro-cytokines to active caspase-1 for maturation (FIG.1).

[0050] We have identified ASC protein that binds and recruits pro-cytokines to the inflammasome complex, and this recruitment is essential for caspase-1-dependent maturation. This newly discovered recruitment step not only provides novel biological insights but also reveals an intracellular target for IL-1β and IL-18 at the pre-maturation stage, offering potential therapeutic avenues for persistent inflammatory conditions.

[0051] While the maturation of cytokines mediated by inflammasomes is critical for host survival by ensuring pathogen clearance and resolution of inflammation, unregulated cytokine production can lead to hyperinflammation and potentially fatal outcomes. and has been observed in COVID-19 and various chronic inflammatory conditions, including adult- onset Still’s disease, pyogenic arthritis, cryopyrin-associated periodic syndrome, familial Mediterranean fever, systemic juvenile idiopathic arthritis and macrophage activation syndrome.

[0052] The recognition of both pathogenic and sterile triggers by the inflammasome, along with their clear association with a wide range of inflammatory diseases, has drawn significant attention in clinical settings. Despite the pivotal roles played by inflammasome- derived IL-18 and IL-1β in both normal physiology and disease pathology, there is a notable absence of FDA-approved therapies targeting IL-18. Additionally, while therapies exist for mature IL-1β, such as anakinra, canakinumab, and rilonacept, they are limited toAtty Docket No.58434-0038WO1 extracellular IL-1β and require multiple subcutaneous doses, leading to adverse reactions, particularly problematic in pediatric patients. Consequently, there remains a significant unmet clinical need for novel inhibitors of IL-18 and IL-1β targeting distinct biological pathways, particularly in the treatment of inflammatory diseases like non-alcoholic steatohepatitis, diabetes, neurodegenerative diseases, and sepsis, where current treatment options are severely limited.

[0053] A novel target, the ASC protein, serves as a central adaptor for both canonical and non-canonical inflammasomes (K+efflux in non-canonical inflammasomes activates secondary NLRP3 activation). Previous studies have emphasized the crucial role of the ASC speck in facilitating efficient pro-IL-1β maturation. This process involves the oligomerization of ASCPYDinto filaments, followed by crosslinking by ASCCARD. Mutations affecting ASC oligomerization (ASCPYDmutants) and filament cross-linking (ASCCARDmutants) have been demonstrated to hinder speck formation and pro-IL-1β maturation.

[0054] Upon activation of the NLRP3 inflammasome, caspase-1 processing and pro-IL- 1β conversion predominantly occur at the single micron-sized speck formed at the microtubule-organizing center (MTOC). Remarkably, the majority of pro-IL-1β conversion occurs specifically at the micron-sized speck, despite the presence of pro-IL-1β throughout the volume of macrophage cells. ASC specks act as danger signals; upon release from pyroptotic cells, they accumulate in the extracellular space and are taken up by recipient cells, further promoting IL-1β maturation. Additionally, ASC specks derived from microglia can interact with amyloid-β protein, exacerbating the production of inflammatory cytokines and the progression of Alzheimer’s disease. Thus, blocking the recruitment of pro- cytokines to the ASC speck offers not only a novel target to inhibit cytokine production but also a pioneering class of drugs derived from a unique mechanistic perspective, providing potential therapeutic avenues for persistent inflammatory conditions.

[0055] Selectively dislodging the ASC-pro-cytokine interactions are specific and this target holds greater promise than completely inhibiting the ASC protein, which could increase the host's susceptibility to various infections and sterile injuries, given that ASC serves as a converging adaptor for both canonical and non-canonical inflammasomes.

[0056] In one embodiment provided herein is a method to restrict the amount of pro-IL-1β converted to mature IL-1β.

[0057] In one embodiment provided herein is a method to restrict the amount of pro--IL- 18 converted to mature-IL-18.

[0058] In one embodiment provided herein is a method to restrict the amount of pro-IL-1β and pro--IL-18 converted to mature IL-1β and mature-IL-18, respectively.Atty Docket No.58434-0038WO1

[0059] In one embodiment provided herein is a method to restrict inflammatory outcome, the method comprising restricting the amount of pro-IL-1β converted to mature IL-1β.

[0060] In one embodiment provided herein is a method to restrict inflammatory outcome, the method comprising restricting the amount of pro--IL-18 converted to mature-IL-18.

[0061] In one embodiment provided herein is a method to restrict inflammatory outcome, the method comprising restricting the amount of pro-IL-1β and pro--IL-18 converted to mature IL-1β and mature-IL-18, respectively.

[0062] In one embodiment provided herein is a method to selectively enhance inflammatory cell death, comprising restricting the amount of pro-IL-1β converted to mature IL-1β.

[0063] In one embodiment provided herein is a method to selectively enhance inflammatory cell death, comprising restricting the amount of pro--IL-18 converted to mature-IL-18.

[0064] In one embodiment provided herein is a method to selectively enhance inflammatory cell death, comprising restricting the amount of pro-IL-1β and pro--IL-18 converted to mature IL-1β and mature-IL-18, respectively.

[0065] In one embodiment the present methods target the intracellular molecule that sequesters pro-IL-1β and pro-IL-18 molecule. The intracellular molecule serves as a druggable interface that offers a novel target to regulate the production of mature-IL-1β and / or pro-IL-18 in cells.

[0066] The methods herein can be useful not only in restricting elevated mature IL-1β and mature-IL-18 production in chronic inflammatory diseases but also in potentially fostering the inflammasome pathway towards inflammatory cell death (pyroptosis, without IL-1β & IL-18 release) as inflammatory cell death can be harnessed to generate antitumor immunity by killing the cancerous cells (blocking inflammatory cytokines release can check the undesirable effects in cancer immunotherapy).

[0067] There is no current mechanism or drug available to restrict pro-IL-1β or pro-IL-18 (non-inflammatory cytokine) from being processed into mature IL-1β, or mature-IL-18 respectively, (inflammatory cytokine).

[0068] Therapies are available to block the mature IL-1β (inflammatory cytokine) in the serum as mature IL-1β is secreted outside the cells once it’s being converted from its pro- IL-1β form by action of caspase-1. The current therapies to block action of extracellular mature-IL-1β includes anakinra, canakinumab, and rilonacept.Atty Docket No.58434-0038WO1

[0069] This methods target the intracellular molecule that sequesters pro-IL-1β and pro- IL-18 molecule (a novel druggable interface). This interface offers novel target to regulate the production of mature-IL-1β and pro-IL-18 in cells.

[0070] An inflammatory disorder is where the immune system causes inflammation by mistakenly attacking your body’s own cells or tissues. There are a number of ways that our immune system can go wrong and cause inflammation.

[0071] Inflammation refers to a biological response to stimuli interpreted by the body to have a potentially harmful effect. Inflammation is a normal, healthy response to injury, infection, or certain other medical conditions. In autoimmunity, antibodies and immune cells are directed against autologous cells.

[0072] A healthy immune system helps the body fight disease. It makes proteins called antibodies that identify and attack substances that may harm a person. Some antigens, such as viruses and certain bacteria, may threaten a person’s health. The immune system also attacks cells in the body that have become abnormal, such as cancer cells. Unfortunately, sometimes the immune system goes wrong, and antibodies attack own normal cells. The systemic inflammatory responses associated with such diseases can result in chronic pain, redness, swelling, stiffness and damage to otherwise healthy body tissues.

[0073] In some aspects, disclosed herein are methods of treating inflammatory disorders by inhibiting the interaction of ASC and pro-IL-1β and / or pro-IL-18 (e.g., in the inflammasome). For example, the methods disclosed herein can involve targeting one or more isoforms of ASC (e.g., isoforms 1, 2, or 3). In some embodiments, the methods involve targeting ASC Isoform 1 (e.g., certain amino acids in ASC Isoform 1). In some embodiments, the inhibition is by targeting amino acids 92-109 of ASC SEQ ID NO:1. The methods disclosed herein can include inhibiting or blocking the interaction of ASC with one or both of pro-IL-1β and pro-IL-18. In some embodiments, the disclosed methods include administration of an effective amount of a cell permeant peptide. Cell permeant peptides useful for the methods disclosed herein can have a molecular weight from about 2000 to about 8000 (e.g., about 3000 to about 6000, about 4000 to about 5000). In some embodiments, the methods disclosed herein include administering a cell permeant peptide that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the methods disclosed herein include administering a cell permeant peptide that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5. In someAtty Docket No.58434-0038WO1 embodiments, the methods disclosed herein include administering a cell permeant peptide that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the methods disclosed herein include administering a cell permeant peptide comprising 9 or fewer (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) substitutions, insertions, or deletions in the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the methods disclosed herein include administering a cell permeant peptide comprising 9 or fewer (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) substitutions, insertions, or deletions in the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the methods disclosed herein include administering a cell permeant peptide comprising 9 or fewer (e.g., 8, 7, 6, 5, 4, 3, 2, or 1) substitutions, insertions, or deletions in the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the cell permeant peptides disclosed herein include a flexible linker (e.g., at the C-terminus). Suitable linkers are known in the art.

[0074] The cell permeant peptides can be administered at a dose suitable to treat a condition described herein. For example, in some instances, the cell permeant peptides can be administered at a concentration of about 5-500 micromolar (e.g., about 10-200 micromolar, about 30-100 micromolar, about 40-80 micromolar, about 50-70 micromolar, or about 60 micromolar).

[0075] Exemplary inflammatory diseases include autoimmune diseases (e.g., rheumatoid arthritis), cardiovascular disease (e.g., high blood pressure, heart disease), gastrointestinal disorders (e.g., inflammatory bowel disease, Crohn’s disease, ulcerative colitis), lung diseases (chronic obstructive pulmonary disease (COPD), asthma, mental illnesses (depression, schizophrenia), metabolic diseases, neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease), gout, cancer, fatty liver disease, endometriosis, diabetes (type 2 diabetes mellitus, type 1 diabetes mellitus), infections, and obesity.

[0076] Autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis (ADEM), Addison's disease, allergy, allergic rhinitis, anti-phospholipid antibody syndrome (APS), an arthritis, asthma, acquired immunodeficiency syndrome (AIDS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, celiac disease, Chagas disease, chronic obstructive pulmonary disease (COPD), diabetes mellitus type 1 (IDDM), endometriosis, a gastrointestinal disorder, a glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial nephritis, interstitial cystitis, a lupus, morphea, multiple sclerosis (MS), myasthenia gravis, a myopathy, myositis, narcolepsy,Atty Docket No.58434-0038WO1 neuromyotonia, pemphigus vulgaris, pernicious anemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, a pulmonary fibrosis, recurrent disseminated encephalomyelitis, rheumatic fever, schizophrenia, scleroderma, Sjögren's syndrome, a skin disorder, tenosynovitis, uveitis, a vasculitis, or vitiligo.

[0077] In certain embodiments, the arthritis is monoarthritis, oligoarthritis, polyarthritis, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, septic arthritis, spondyloarthropathy, gout, pseudogout, psoriatic arthritis, or Still's disease.

[0078] In some embodiments, the gastrointestinal disorder is an irritable bowel disease or an inflammatory bowel disease. In other embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0079] In some embodiments, the lupus is discoid lupus erythematosus, drug-induced lupus erythematosus, lupus nephritis, neonatal lupus, subacute cutaneous lupus erythematosus, or systemic lupus erythematosus.

[0080] In some embodiments, the myopathy is dermatomyositis, inclusion body myositis, or polymyositis.

[0081] In some embodiments, the skin disorder is dermatitis, eczema, statis dermatitis, hidradenitis suppurativa, psoriasis, rosacea, or scleroderma.

[0082] In some embodiments, the vasculitis is Buerger's disease, cerebral vasculitis, Churg-Strauss arteritis, cryoglobulinemia, essential cryoglobulinemic vasculitis, giant cell arteritis, Golfer's vasculitis, Henoch-Schonlein purpura, hypersensitivity vasculitis, Kawasaki disease, microscopic polyarteritis / polyangiitis, polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatoid vasculitis, Takayasu arteritis, or Wegener's granulomatosis.

[0083] In some embodiments, the inflammatory disease is inflammation associated with an infectious agent such as, but not limited to, COVID-19. In some embodiments, the inflammatory disease is a chronic inflammatory condition, including, but not limited to, adult- onset Still’s disease, pyogenic arthritis, cryopyrin-associated periodic syndrome, familial Mediterranean fever, systemic juvenile idiopathic arthritis and macrophage activation syndrome.

[0084] Pharmaceutical compositions

[0085] The cell permeant peptides described herein can be formulated as a pharmaceutical composition for administration to a subject, e.g., to treat a disease or condition described herein. Typically, a pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial andAtty Docket No.58434-0038WO1 antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci.66:1-19).

[0086] Pharmaceutical formulation is a well-established art, and is further described, e.g., in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20thed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7thEd., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rded. (2000) (ISBN: 091733096X).

[0087] The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form can depend on the intended mode of administration and therapeutic application. Typically compositions for the agents described herein are in the form of injectable or infusible solutions.

[0088] Such compositions can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). In one embodiment, the composition comprising the cell permeant peptide is administered intravenously. In another embodiment, the composition is administered subcutaneously. The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0089] The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating an agent described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying that yield a powderAtty Docket No.58434-0038WO1 of an agent described herein plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0090] Administration

[0091] The cell permeant peptides described herein can be administered to a subject, e.g., a human subject in need thereof, for example, by a variety of methods. For many applications, the route of administration is one of: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection. It is also possible to use intra-articular delivery. Other modes of parenteral administration can also be used. Examples of such modes include: intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injection. In some cases, administration can be oral.

[0092] The route and / or mode of administration of the peptides can also be tailored for the individual case.

[0093] If a subject is at risk for developing a disease or condition described herein, the peptide can be administered before the full onset of the disease or condition, e.g., as a preventative measure. The duration of such preventative treatment can be a single dosage of the peptides or the treatment may continue (e.g., multiple dosages). For example, a subject at risk for the disease or who has a predisposition for the disease may be treated with the cell permeant peptides for days, weeks, months, or even years so as to prevent the disease from occurring or fulminating.

[0094] A pharmaceutical composition may include a “therapeutically effective amount” of an agent described herein. Such effective amounts can be determined based on the effect of the administered agent, or the combinatorial effect of agents if more than one agent is used. A therapeutically effective amount of an agent may also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual, e.g., amelioration of at least one disease or condition parameter or amelioration of at least one symptom of the disease or condition. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. EXAMPLES

[0095] NanoBRET Assay We have used the nanoBRET assay (Promega), a proximity-based bioluminescence resonance energy transfer (BRET) technique capable of detecting protein interactions in live cells. ThisAtty Docket No.58434-0038WO1 assay measures energy transfer from a bioluminescent protein donor (NanoLuc, NL) to a fluorescent protein acceptor (618 fluorescent ligand bound to HaloTag, HT), serving as a robust readout for identifying binding between ASC and pro-cytokines and characterizing the interaction interface in living cells. We have successfully identified and validated the interface on both ASC and pro-cytokines. Based on these findings, the nanoBRET assay offers a quantitative high-throughput readout for assessing various functional aspects of ASC / pro- cytokine interactions. It facilitates the identification of small molecules binding to the ASCINTdomain, thereby modulating the overall conformation, and signaling activity, of ASC- dependent inflammasomes. We have validated the identified interface using cell-permeable peptides, demonstrating a significant reduction in the secretion of mature IL-1β and mature- IL18 in the human monocytic THP-1 cell line and in human primary monocyte derivatives. FIG.2. Decoding inflammasome activity at the speck. BMDMs were primed with LPS and stimulated nigericin to activate NLRP3 inflammasome. (A) The speck serves as the primary site for caspase-1 processing. Immortalized mouse bone marrow-derived macrophages (iBMDMs) primed with LPS and stimulated with nigericin to activate the NLRP3 inflammasome express mTuruqoise-pro-Caspase-1-mNeongreen FRET reporter, forming specks at the microtubule organizing center (MTOC), with SiR Tubulin serving as the live-cell marker of MTOC. (puncta) indicates caspase-1 cleavage. reporter form specks at the MTOC. (B) Detection of loss of FRET within specks (puncta) indicates caspase-1 cleavage. (C) The speck is identified as the primary site for IL-1β processing using iBMDMs expressing Turuqoise-pro- IL-1β-mNeongreen (D) FRET Loss of FRET within specks (puncta) reflects IL-1β cleavage. (E) NanoBRET analysis, a proximity-based bioluminescence resonance energy transfer (BRET) assay, illustrates the interaction between ASC / pro-IL-1β (row 1), and between ASC / pro-IL-18 is depicted in 3. Row 2 and 4 are halo-tag alone controls. Scale bars: (A & C) 10μM; White arrowheads indicate specks co-localizing with specks.

[0096] Human ASC and human ASC-pro-IL-1b interaction analysis by nanoBRET assay are shown in (FIG.3A-B)

[0097] ASC sequesters pro-IL-1β and alters its distribution within cells. Pro-IL-1β co- localized with ASC in transfected 293T cells is shown in FIG.4A-B, scale 10 µm.

[0098] Mapping the human ASC domains involved in binding human pro-IL-1β is shown in FIG 5A-B. Among the tested domains, ASCINTdemonstrated robust binding to human pro-IL-1β.

[0099] The ASC-pro-IL-1β interaction was analyzed using ASCINTdomain mutants (FIG. 6). The ASCINTmutants disrupted interaction with human pro-IL-1β.Atty Docket No.58434-0038WO1

[0100] The interaction of ASCINTmutants with IL-1β was evaluated. ASCINTmutants co- localized with IL-1β (FIG. 7A-B, scale 5 µm). Analysis of speck formation and co- localization was also evaluated (FIG.8, scale 5 µm).

[0101] Furthermore, the human pro-IL-1β domain involved in binding human ASC was mapped (FIG.9). The pro-domain of human IL-1β exhibited high affinity for human ASC.

[0102] We also conducted structure-based analysis of ASC-pro-IL-1β interaction (suing the pro-IL-1β structure). The structure of the mature and pro-domains of human IL-1β is presented in FIG.10A-B. The structure function analysis using the nanoBRET assay is presented in FIG.11A-B.

[0103] Human ASC also interacts with human pro-IL-18 (FIG.12A-D).

[0104] We then mapped the domain of human IL-18 responsible for binding with human ASC (FIG.13A-B). Both the pro-domain and mature-domain of human IL-18 are capable of binding human ASC (FIG. 13A). Among the tested domains, ASCINT demonstrated robust binding to human pro-IL-18 (FIG.13B).

[0105] We conducted structure-based analysis of ASC-pro-IL-18 interactions using ASCPYD(FIG.14) and ASCCARD(FIG.15) domain mutants.

[0106] None of the tested ASCPYDmutants showed evidence of disrupting binding to human pro-IL-18. These ASCPYDmutants are known to generate monomeric ASC to different extents. Interestingly, these ASCPYDmutants bind pro-IL-18 more strongly than controls. Monomeric ASC has an enhanced affinity for human pro-IL-18.

[0107] None of the examined ASCCARDmutants displayed any indication of interfering with the binding to pro-IL-18. These ASCCARDmutants are known for their varying degrees of producing monomeric ASC. Intriguingly, these ASCCARDmutants bind pro-IL-18 more strongly than controls. Monomeric ASC has an enhanced affinity for human pro-IL-18.

[0108] Next, we analyzed ASCINT domain mutants to investigate the interaction between ASC and pro-IL-18 (FIG.16A-C). The ASCINT mutants displayed evidence of disrupting the binding to pro-IL-18.

[0109] The relevant residues of the human pro domain of IL-18 are depicted in FIG.17A (Pro domain of human IL-18) and 17B (mature domain of human IL-18).

[0110] The results of the structure-based analysis of the ASC-pro-IL-18 interaction are depicted in FIG. 18. The mutations in the pro-IL-18MATURE domain abolished its interaction with ASC.Atty Docket No.58434-0038WO1

[0111] We next conducted competition assays. Increasing concentrations of pro-IL-1β can effectively compete out the interaction between ASC and pro-IL-18 (FIG. 19A). Increasing concentrations of pro-IL-18 do not effectively compete out the interaction between ASC and pro-IL-1β (FIG. 19B). Thus, binding of pro-IL-1b and pro-IL-18 are mutually exclusive.

[0112] We analyzed the binding of ASC-pro-IL-1β and ASC / pro-IL-18 in the presence of pro-caspase-1 and NLRP3 (FIG.20A-B).

[0113] The processing of inflammatory cytokines was perturbed using membrane permeable peptides (FIG. 21A-B). Human monocytic THP-1 cells exhibit a significantly decrease in IL-1β production when the interaction between ASC and pro-cytokine are perturbed using cell permeant peptides. Fig.22 shows the ASC-pro-IL-1β interaction, analyzed using ASCINTdomain mutants. Fig.23 shows the analysis of ASC-pro-IL-1β interaction (using ASCINT domain mutants). The ASCINTmutant showed disruption of the interaction with human pro-IL-1β. Fig.24 shows the mapping of the human pro-IL-1β domains involved in binding human ASC. Both domain binds, but Mature-domain (MD) of human IL-1β exhibit a high affinity for human ASC. Fig.25 shows validation of a structure-based analysis of ASC-pro-IL-1β interaction (using pro- 1L-1β structure). Fig.26 shows mapping of the human ASC domains involved in for binding human pro-IL-18. Among the tested domains, ASCITLDdemonstrated robust binding to human pro-IL-18 Fig. 27 shows structure-based analysis of ASC-pro-IL-18 interaction (using ASCPYDdomain mutants). Identified ASCPYDmutants perturbed ASC / pro-IL-18 interactions Fig. 28 shows the perturbing of the recruitment of inflammatory cytokines using membrane permeable peptides (THP-1 monocytic cells differentiated to macrophages). Blocking ASC / pro-cytokine interactions in THP-1 macrophages by cell-permeant peptides led to significantly reduced IL-1β and IL-18 secretion. Phorbol 12-myristate 13-acetat (PMA) differentiates monocytes to macrophages. Fig. 29 shows the perturbing of the recruitment of inflammatory cytokines using membrane permeable peptides in primary human cells (human monocyte-derived macrophages; hMDMs). Blocking ASC / pro-cytokine interactions in in hMDMs by cell-permeant peptides led to significantly reduced IL-1β and IL-18 secretion..Atty Docket No.58434-0038WO1 Fig.30 shows that ASC speck formation is unperturbed by experimental peptide in transfected 293T cells (qualitative analysis). Cell permeant peptide blocks pro-cytokine recruitment by ASC whereas ASC speck formation in unperturbed. (Scale 100 µm).

[0114] Peptides do not interfere with NF-κB-dependent upregulation of pro-cytokine synthesis. In some embodiments, a cell permeant peptide has a molecular weight from about 4000 to 5000, such as about 4000 to 4500, such as about 4100 to about 4200, such as about 4114.770, 4122.790, or about 4122.790.

[0115] In some embodiments, a cell permeant peptide comprises a flexible linker attached to peptide (antennapedia protein, third helix) at the C-terminus.

[0116] In some embodiments, the cell permeant peptide is administered in a concentration of about 5-500 micromolar, such as about 10-200 micromolar, such as about 30-100 micromolar, such as about 40-80 micromolar, such as about 50-70 micromolar, such as 60 micromolar.

[0117] In a representative example, a cell was pretreated with the cell permeant peptide for about 3 hours. Then the cells were challenged for 3 hours with LPS and 1 hour with nigericin to activate inflammation.

[0118] In some embodiments, the cell permeant peptide has 90% identity with (SEQ ID No.4).

[0119] In some embodiments, the cell permeant peptide has 90% identity with (SEQ ID No.5).

[0120] In some embodiments, the cell permeant peptide has 90% identity with (SEQ ID No.6).

[0121] In some embodiments, the cell permeant peptide has 95% identity with (SEQ ID No.4).

[0122] In some embodiments, the cell permeant peptide has 95% identity with (SEQ ID No.5).

[0123] In some embodiments, the cell permeant peptide has 95% identity with (SEQ ID No.6).

[0124] In some embodiments, the cell permeant peptide is selected from the group consisting of (SEQ ID No.4), (SEQ ID No.5) and (SEQ ID No.6).

[0125] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by theAtty Docket No.58434-0038WO1 term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0126] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0127] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0128] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art uponAtty Docket No.58434-0038WO1 reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0129] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0130] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0131] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

Atty Docket No.58434-0038WO1 What is claimed is:

1. A method of treating an inflammatory disease, comprising inhibiting or blocking the interaction between apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-cytokines.

2. The method of claim 1, wherein the inhibition or blocking targets amino acids 92-109 of ASC (SEQ ID NO:1).

3. The method of claim 1, wherein the pro-cytokine is pro-IL-1β.

4. The method of claim 1, wherein the pro-cytokine is pro-IL-18.

5. The method of claim 1, wherein the method inhibits or blocks the interaction of ASC with both pro-IL-1β and pro-IL-18.

6. The method of any one of claims 1-5, wherein the inflammatory disease is an autoimmune disease.

7. The method of any one of claims 1-5, wherein the inflammatory disease is caused by an infectious agent.

8. The method of any one of claims 1-5, wherein the inflammatory disease is a chronic inflammatory disease.

9. The method of any one of the preceding claims, wherein inhibiting or blocking the interaction between apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and pro-cytokines in a subject comprises administering to the subject an effective amount of a cell permeant peptide.

10. The method of claim 9, wherein the cell permeant peptide has a molecular weight from about 4000 to 5000.

11. The method of claim 9, wherein the cell permeant peptide is selected from the group consisting of (SEQ ID No.4), (SEQ ID No.5) and (SEQ ID No.6).

12. The method of claim 9, wherein the cell permeant peptide comprises a flexible linker attached to peptide (antennapedia protein, third helix) at the C-terminus.

13. The method of claim 9, wherein the cell permeant peptide is administered in a concentration of about 5-500 micromolar, such as about 10-200 micromolar, such as about 30-100 micromolar, such as about 40-80 micromolar, such as about 50-70 micromolar, such as 60 micromolar.

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