Transient receptor potential melastatin 2 (TRPM2) modulators and uses thereof

Abstract

The present invention relates to modulators that inhibit the interaction between protein kinase C y (PKCy) and transient receptor potential melastatin 2 (TRPM2), including peptides, polypeptides, and small molecules, and their use in treatment of neurological and cardiovascular diseases.

Description

Attorney Docket No. 98121.00392 TRANSIENT RECEPTOR POTENTIAL MELASTATIN 2 (TRPM2) MODULATORS AND USES THEREOF RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 615,815, filed on December 29, 2023. The entire contents of the foregoing application are incorporated herein by reference. GOVERNMENT SUPPORT

[0002] This invention was made with government support under HL143750 and NS131661 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE DISCLOSURE

[0003] Described herein are transient receptor potential melastatin 2 (TRPM2) modulators and methods for use of such modulators for treatment of neurological disorders and vascular disorders. BACKGROUND

[0004] Ischemic stroke is a leading cause of death and disability globally, with an increasing prevalence in developing countries. N-methyl-D-aspartate-receptor (NMDAR)-mediated glutamate excitotoxicity has been known to be the major cause of ischemic neuronal death for decades. However, NMDAR antagonists all unfortunately failed to show protective effects in human patients, which was due to the limited understanding of the pathological regulation of NMDAR during ischemic stroke. Based on their location, NMDARs are divided into synaptic NMDARs (sNMDARs) and extrasynaptic NMDARs (esNMDARs). During ischemic stroke, sNMDAR promotes neuron-survival, while esNMDAR causes neuronal death. Thus, the indiscriminable blockade of both sNMDAR and esNMDAR is one of the major reasons for the failure of NMDAR antagonists. However, due to the significant similarities between sNMDARs and esNMDARs, developing an esNMDAR-specific antagonist is a significant challenge, even if it is not impossible. Therefore, there is an unmet need to develop an alternate therapy for ischemic stroke that overcomes the drawbacks of existing NMDAR antagonists.Attorney Docket No. 98121.00392 SUMMARY

[0005] During ischemic stroke, there is an abnormal enhancement of TRPM2 and PKC activity, further potentiating NMDAR-mediated excitotoxicity. Consequently, the dissociation of the TRPM2-PKC binding can be a protective mechanism against ischemic stroke by attenuating excitotoxicity-induced neuronal death. The present invention is based, at least in part, on the discovery of an interaction motif between transient receptor potential melastatin 2 (TRPM2) and protein kinase C γ (PKCγ). The examples herein demonstrate that TRPM2-PKCγ uncoupling is a novel strategy for attenuating NMDARs-mediated excitotoxicity in a neurological disorder (e.g., ischemic stroke). It is demonstrated herein that the TRPM2-PKCγ interaction allows TRPM2- mediated Ca2+influx to promote PKCγ activation, which subsequently enhances TRPM2-induced potentiation of extrasynaptic NMDAR (esNMDAR) activity. The present disclosure has identified the binding motif for PKCγ on TRPM2 (M2PBM), which can directly associate with the C2 domain of PKCγ. Furthermore, the present disclosure has developed a modulator (e.g., M2PBM or TAT-M2PBM) to disrupt TRPM2-PKCγ interaction without compromising PKCγ function.

[0006] In addition, what sets agents that inhibit the TRPM2-PKCγ interaction (e.g., TAT- M2PBM or M2PBM) apart from agents that inhibit the TRPM2- NMDAR interaction (e.g., TAT- EE3) is the broader application of TRPM2-PKCγ inhibitors. While NMDAR is predominantly expressed in neuronal cells, TRPM2 and PKCγ are prevalent in almost all types of cells in the brain, including endothelial cells, microglia, and infiltrated leukocytes during ischemic injury. Therefore, the agents disclosed herein that inhibit the TRPM2-PKCγ interaction (e.g., TAT- M2PBM or M2PBM) can produce broader protective effects in neuron, microglia, blood brain barrier thereby providing better protective effects in treating ischemic stroke, as it will not only be protective during the acutely injury phase, but also will be beneficial for long-term recovery phase after ischemic injury. The agents disclosed herein that inhibit the TRPM2-PKCγ interaction (e.g., TAT-M2PBM or M2PBM) may inhibit degradation of blood brain barrier (BBB), and inhibit inflammatory responses, thereby providing better protective effects by inhibiting all the detrimental effects of TRPM2 in different cells during ischemic stroke.

[0007] Accordingly, in some aspects, disclosed herein is a method for treating or preventing neurological injury or neurological disorder (e.g., ischemic stroke) in a subject comprisingAttorney Docket No. 98121.00392 administering to the subject an agent that inhibits the interaction between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2).

[0008] Moreover, overactivation of TRPM2 and PKCγ activity is closely associated with a myriad of diseases, including atherosclerosis, aneurysm, heart failure, myocardial infarction, obesity, diabetes, and various types of cancers. Thus, inventors have also identified that targeting TRPM2-PKCγ association may also benefit patients with the diseases related to TRPM2 / PKC overactivation.

[0009] Accordingly, in some aspects, disclosed herein is a method for treating or preventing a vascular disorder (e.g., atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, or diabetic angiopathy) in a subject in need thereof, the method comprising administering to the subject an agent that inhibits the interaction between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2).

[0010] In some embodiments, the agent targets a PKCγ-binding site on TRPM2.

[0011] In some embodiments, the PKCγ-binding site comprises residues 151-200 or residues 162-178 of TRPM2.

[0012] In some embodiments, the PKCγ-binding site comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0013] In some embodiments, the agent comprises a small molecule.

[0014] In some embodiments, the small molecule is selected from the group consisting of N- (p-amylcinnamoyl)anthranilic acid (ACA) and 2-Aminoethoxydiphenyl borate (2-APB).

[0015] In some embodiments, the agent comprises a peptide comprising an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0016] In some embodiments, the peptide comprises an amino acid sequence differing by 1, 2, 3, 4, or 5 residues from the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0017] In some embodiments, the peptide comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0018] In some embodiments, the peptide does not comprise the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0019] In some embodiments, the peptide is further conjugated to a cell-penetrating peptide (CPP).Attorney Docket No. 98121.00392

[0020] In some embodiments, the CPP comprises an amino acid sequence with at least 75%, 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 4-19.

[0021] In some embodiments, the CPP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.

[0022] In some embodiments, the CPP consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.

[0023] In some embodiments, the CPP comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0024] In some embodiments, the CPP consists of an amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0025] In some embodiments, the agent comprises an antagonist anti-TRPM2 antibody that binds to the PKCγ-binding site.

[0026] In some embodiments, the agent comprises a mutant TRPM2 protein, wherein the mutant TRPM2 protein comprises a deletion of the PKCγ-binding site.

[0027] In some embodiments, the agent targets a TRPM2-binding site on PKCγ.

[0028] In some embodiments, the TRPM2-binding site on PKCγ is the C2-domain of PKCγ.

[0029] In some embodiments, the agent comprises a small molecule that binds to the TRPM2- binding site on PKCγ.

[0030] In some embodiments, the agent comprises an antagonist anti-PKCγ antibody that binds to the TRPM2-binding site.

[0031] In some embodiments, the agent comprises a mutant PKCγ protein, wherein the mutant PKCγ protein comprises a deletion of the TRPM2-binding site.

[0032] In some embodiments, the neurological injury results from a brain injury.

[0033] In some embodiments, the brain injury comprises stroke, traumatic brain injury, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, brain aneurysm, coma, or any combination thereof.

[0034] In some embodiments, the brain injury is ischemic stroke, hemorrhagic stroke, or transient ischemic attack.

[0035] In some embodiments, the brain injury is ischemic stroke.

[0036] In some embodiments, wherein administering to the subject the agent decreases surface expression of N-methyl-D-aspartate receptor (NMDAR) in a neuronal cell.Attorney Docket No. 98121.00392

[0037] In some embodiments, administering to the subject the agent decreases the amplitude of NMDAR current in a neuronal cell.

[0038] In some embodiments, administering to the subject the agent inhibits an increase of intracellular Ca2+concentration in a neuronal cell.

[0039] In some embodiments, administering to the subject the agent decreases neuronal death.

[0040] In some embodiments, administering to the subject the agent decreases infarct volume in brain.

[0041] In some embodiments, administering to the subject the agent decreases neurological deficit score.

[0042] In some embodiments, administering to the subject the agent ameliorates at least one symptom of ischemic stroke.

[0043] In some embodiments, the symptom is selected from the group consisting of difficulty walking, numbness, weakness and / or paralysis in the face, arm and / or leg, headache, confusion, difficulty speaking and / or understanding speech, and difficulty seeing in one or both eyes.

[0044] In some embodiments, the agent is administered to the subject at a dose of about 10 nmol / kg to about 1000 nmol / kg.

[0045] In some embodiments, the agent is administered to the subject at a dose of about 100 nmol / kg.

[0046] In some embodiments, the agent is administered to the subject every 2 to 24 hours.

[0047] In some embodiments, the agent is administered to the subject every 10 to 12 hours.

[0048] In some embodiments, the agent is administered to the subject less than 6 hours after one or more symptoms of ischemic stroke start.

[0049] In some embodiments, the agent is administered to the subject less than 4 hours after one or more symptoms of ischemic stroke start.

[0050] In some embodiments, the method disclosed herein further comprise administering an additional therapeutic to the subject.

[0051] In some embodiments, the additional therapeutic comprises an anticoagulant or clot- dissolving medicine (e.g., aspirin), clopidogrel, a tissue plasminogen activator (tPA) (e.g., alteplase), thrombectomy, carotid endarterectomy, a medication for antihypertension (e.g., nicardipine, labetalol, or lisinopril), an antipyretic therapy, warfarin, heparin, apixaban, atorvastatin, rosuvastatin, irbesartan, alteplase, or any combination thereof.Attorney Docket No. 98121.00392

[0052] In some aspects, disclosed herein is a method of treating or preventing a vascular disorder in a subject in need thereof, the method comprising administering to the subject the agent disclosed herein that inhibits the interaction between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2).

[0053] In some embodiments, the vascular disorder is a cardiovascular disease.

[0054] In some embodiments, the cardiovascular disease comprises atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, diabetic angiopathy, or any combination thereof.

[0055] In some embodiments, administering to the subject the agent inhibits the loss of one or more contractile proteins in a vascular smooth muscle cell.

[0056] In some embodiments, the contractile protein is selected from the group consisting of α-SMA and SM22α.

[0057] In some aspects, disclosed herein is a peptide comprising an amino acid sequence with at least 80% or 85% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1), or a multimer, derivative, or variant thereof.

[0058] In some embodiments, the peptide comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0059] In some embodiments, the peptide comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0060] In some embodiments, the peptide comprises an amino acid sequence differing by 1, 2, 3, 4, or 5 residues from the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0061] In some embodiments, the peptide comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0062] In some embodiments, the peptide is a synthetic peptide.

[0063] In some embodiments, the peptide inhibits interaction between PKCγ and TRPM2.

[0064] In some embodiments, the peptide inhibits interaction between esNMDAR and TRPM2.

[0065] In some embodiments, the peptide inhibits phosphorylation of TRPM2 at S38.

[0066] In some embodiments, the peptide binds to the C2-domain of PKCγ.

[0067] In some embodiments, the peptide decreases the surface expression of N-methyl-D- aspartate receptor (NMDAR) in a neuronal cell.Attorney Docket No. 98121.00392

[0068] In some embodiments, the peptide decreases the amplitude of NMDAR current in a neuronal cell.

[0069] In some embodiments, the peptide reduces mitochondrial membrane depolarization induced by ischemic injury in neurons.

[0070] In some aspects, disclosed herein is a conjugate comprising the peptide disclosed herein and a cell-penetrating peptide (CPP).

[0071] In some embodiments, the CPP comprises an amino acid sequence with at least 75%, 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 4-19.

[0072] In some embodiments, the CPP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.

[0073] In some embodiments, the CPP consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.

[0074] In some embodiments, the CPP comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0075] In some embodiments, the CPP consists of an amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0076] In some embodiments, the peptide is conjugated to the CPP via a linker.

[0077] In some embodiments, the peptide comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1) and the CPP comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0078] In some embodiments, the conjugate comprises an amino acid sequence with at least 75%, 80%, 85%, 90% or 95% sequence identity YGRKKRRQRRRWGLDVPNLLISVTGGA (SEQ ID NO: 20).

[0079] In some embodiments, the conjugate comprises the amino acid sequence of YGRKKRRQRRRWGLDVPNLLISVTGGA (SEQ ID NO: 20).

[0080] In some embodiments, the conjugate consists of the amino acid sequence of YGRKKRRQRRRWGLDVPNLLISVTGGA (SEQ ID NO: 20).

[0081] In some aspects, disclosed herein is a nucleic acid molecule encoding the peptide or the conjugate disclosed herein.Attorney Docket No. 98121.00392

[0082] In some aspects, disclosed herein is an expression vector comprising the nucleic acid molecule disclosed herein operably linked to a control sequence for the expression of the peptide disclosed herein or the conjugate disclosed herein.

[0083] In some aspects, disclosed herein is a host cell comprising the expression vector disclosed herein.

[0084] In some aspects, disclosed herein is a pharmaceutical composition comprising the peptide disclosed herein or the conjugate disclosed herein, and at least one pharmaceutically acceptable excipient.

[0085] In some aspects, disclosed herein is a method for treating or preventing neurological injury or neurological disorder in a subject comprising administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein.

[0086] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits the interaction between TRPM2 and PKCγ.

[0087] In some embodiments, administering to the subject the agent, the peptide or the pharmaceutical composition decreases surface expression of N-methyl-D-aspartate receptor (NMDAR) in a neuronal cell.

[0088] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition decreases the amplitude of NMDAR current in a neuronal cell.

[0089] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits an increase of intracellular Ca2+concentration in a neuronal cell.

[0090] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition reduces mitochondrial membrane depolarization induced by ischemic injury in neurons.

[0091] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition decreases neuronal death.

[0092] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition reduces infarct volume and / or improves neurological behavior score.

[0093] In some embodiments, the neurological injury results from a brain injury.Attorney Docket No. 98121.00392

[0094] In some embodiments, the brain injury comprises stroke, traumatic brain injury, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, brain aneurysm, coma, or any combination thereof.

[0095] In some embodiments, the conjugate, the peptide, or the pharmaceutical composition is administered to the subject at a dose of about 10 nmol / kg to about 1000 nmol / kg. In some embodiments, the conjugate, the peptide, or the pharmaceutical composition is administered to the subject at a dose of about 100 nmol / kg.

[0096] In some embodiments, the conjugate, the peptide, or the pharmaceutical composition is administered to the subject every 2 to 24 hours.

[0097] In some embodiments, the conjugate, the peptide, or the pharmaceutical composition is administered to the subject every 10 to 12 hours.

[0098] In some embodiments, the conjugate, the peptide, or the pharmaceutical composition is administered to the subject less than 6 hours after one or more symptoms of ischemic stroke start.

[0099] In some embodiments, the conjugate, the peptide, or the pharmaceutical composition is administered to the subject less than 4 hours after one or more symptoms of ischemic stroke start.

[0100] In some embodiments, the method further comprises administering an additional therapeutic to the subject.

[0101] In some aspects, disclosed herein is a method of treating or preventing a vascular disorder in a subject in need thereof, the method comprising administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein.

[0102] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits the interaction between TRPM2 and PKCγ.

[0103] In some embodiments, the vascular disorder is a cardiovascular disease.

[0104] In some embodiments, the cardiovascular disease comprises atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, diabetic angiopathy, or any combination thereof.

[0105] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits the loss of one or more contractile proteins in a vascular smooth muscle cell.Attorney Docket No. 98121.00392

[0106] In some embodiments, the contractile protein is selected from the group consisting of α-SMA and SM22α.

[0107] In some aspects, disclosed herein is a method for identifying a compound useful for treating or preventing neurological injury or neurological disorder in a subject in need thereof, comprising providing a test compound; determining the effect of the test compound on the interaction between transient receptor potential melastatin 2 (TRPM2) and protein kinase C γ (PKCγ); and selecting a compound that inhibits the interaction between TRPM2 and PKCγ, thereby identifying a compound useful for treating or preventing neurological injury or neurological disorder in the subject.

[0108] In some embodiments, the compound binds to a PKCγ-binding site on TRPM2.

[0109] In some embodiments, the PKCγ-binding site comprises residues 151-200 or residues 162-178 of TRPM2.

[0110] In some embodiments, the PKCγ-binding site comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0111] In some embodiments, the compound binds to a TRPM2-binding site on PKCγ.

[0112] In some embodiments, the TRPM2-binding site on PKCγ is the C2-domain of PKCγ.

[0113] In some aspects, disclosed herein is a kit for treating or preventing neurological injury, neurological disorder, or vascular disorder in a subject, wherein the kit comprises the peptide, the conjugate, or the pharmaceutical composition disclosed herein.

[0114] In some embodiments, the kit further comprises: instructions for use; one or more therapeutic agents; one or more reagents; one or more materials for preparing composition for administration to the subject; one or more pharmaceutically acceptable carriers; one or more devices; and / or one or more materials for administration to the subject.

[0115] In some embodiments, the one or more therapeutic agents are for combination therapy and / or emergency therapy of stroke.

[0116] In some embodiments, the one or more reagents comprise one or more diluents.

[0117] In some embodiments, the instructions for use comprise dosages and / or modes of administration to the subject.

[0118] In some aspects, disclosed herein is a kit for diagnosing neurological injury, neurological disorder, or vascular disorder, wherein the kit comprises the peptide, the conjugate, or the pharmaceutical composition disclosed herein.Attorney Docket No. 98121.00392

[0119] In some aspects, disclosed herein is a kit for use in an imaging assay, wherein the kit comprises the peptide, the conjugate, or the pharmaceutical composition disclosed herein.

[0120] In some aspects, disclosed herein is a kit for use in an in vitro assay, wherein the kit comprises the peptide, the conjugate, or the pharmaceutical composition disclosed herein.

[0121] These and other aspects and embodiments of the disclosure are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0123] FIGS. 1A-1P show that PKCγ phosphorylates TRPM2 and enhances TRPM2 activation. FIGS. 1A-1C show whole-cell current recording of TRPM2 in HEK293T cells transfected with TRPM2 / PKCγ without (FIG. 1A) or with (FIG. 1B) H2O2 preincubation for 3 minutes. FIGS. 1A and 1B show time-dependent activation of TRPM2 as indicated by the inward currents plotted against time. TRPM2 currents were elicited by a ramp protocol with pipette solution containing 1 µM ADPR and 500 nM Ca2+. PMA (1 µM) was used to induce TRPM2 activation, NMDG to test seal tightness, and ACA to block TRPM2 current. FIG. 1C shows quantification of current amplitude (n = 9,9). FIGS.1D and 1E show whole-cell current recording of TRPM2 in HEK293T cells transfected with TRPM2 / GFP (FIG. 1D) and TRPM2 / PKCγ-DN (FIG. 1E). FIG. 1F shows mutation of S11 to S11A / D and S38 to S38A / D. FIGS. 1G-1K show whole-cell current recording of TRPM2 in HEK293T cells transfected with TRPM2-S11A / PKCγ (FIG. 1G), TRPM2-S38A / PKCγ (FIG. 1H), TRPM2-S38D / PKCγ (FIG. 1I) and TRPM2- S11D / PKCγ (FIG. 1J). FIGS. 1G-1J show representative traces. (FIG. 1K), Quantification of current amplitude (n = 9,9). FIG. 1L shows internal solution for recording TRPM2 and NMDAR currents. FIG. 1M shows quantification of TRPM2 current amplitude under maximum activation recording conditions (S38: wild-type TRPM2; S38A: TRPM2-S38A; S38D: TRPM2-S38D) (n=10,10,10,10,11,8; original recordings not shown). FIG. 1N shows quantification of TRPM2 current amplitude under maximum activation recording conditions (S11: wild-type TRPM2; S11A: TRPM2-S11A; S11D: TRPM2-S11D) (n=9,10,9; original recordings not shown). FIGS.Attorney Docket No. 98121.00392 1O and 1P show whole-cell current recordings of NMDARs induced by 100 µM NMDA in HEK293T cells transfected with NMDAR / PKCγ and EGFP control, TRPM2-WT, TRPM2-S11A, TRPM2-S11D, TRPM2-S38A and TRPM2-S38D (FIG. 1O), and Quantification of current amplitude (FIG.1P) (n=7,11,10,10,9,10). (ns, no statistical significance, *, p < 0.05, **, p < 0.01, ***, p < 0.001; unpaired t test; mean ± SEM).

[0124] FIGS.2A-2H show that TRPM2-mediated Ca2+influx promotes PKCγ activation. FIG. 2A shows that FRET sensor CKAR consists of a CFP, a YFP and a linker. Without PKC activity, CFP and YFP are in close proximity (<10 nm) for FRET (emission of CFP by 440 nm excitation excites YFP and yield a 527 emission). PKC phosphorylates CKAR, which changes the conformation of the linker and eliminates FRET (490 nm emission of CFP cannot excite YFP). FIGS. 2B-2D show data of CKAR real-time imaging in HEK293T cells transfected with TRPM2 / PKCγ and EGFP / PKCγ. FIG. 2B shows representative FRET intensity view before and 10 minutes after 100 µM H2O2perfusion. FIG. 2C shows averaged representative traces from 5 randomly chosen cells. FIG.2D shows quantification of FRET changes (n=10-20). FIG.2E shows graphic illustration showing where EGTA, ACA and BAPTA-AM target. FIGS.2F-2H show data of CKAR real-time imaging in HEK293T cells transfected with TRPM2 / PKCγ pre-treated with ACA (10 µM) or BAPTA-AM (10 µM) for 30 minutes, and perfused with Ca2+free extracellular solution buffered by 2 mM EGTA. FIG. 2F shows representative imaging of FRET intensity before and 10 minutes after 100 µM H2O2 perfusion. (FIG. 2G), Averaged representative traces from 5 randomly chosen cells. FIG. 2H shows quantification of FRET changes (n=10-20). (***, p < 0.001; unpaired t test; mean ± SEM).

[0125] FIGS. 3A-3K show that PKCγ binding motif in TRPM2 is required for TRPM2- PKCγ coupling. FIG.3A shows alignment of the PKC binding sequence in RACK and annexin1 with human TRPM2. FIG. 3B shows co-immunoprecipitation of PKCγ by TRPM2 in HEK293T cells co-expressed with PKCγ and TRPM2, N tail of TRPM2 (TRPM2-NT) and PKC binding motif deleted TRPM2 (TRPM2-ΔPBM). FIG. 3C shows graphic illustration showing the structure of TRPM2 and the position of PKC binding motif. FIG.3D shows data of in vitro binding assay. The C2 domain of PKCγ was conjugated with a GST tag, while the MHR1 / 2 domain of TRPM2 was conjugated with a His tag. Anti-GST antibody was used for immunoprecipitation, and GST was used as a negative control (Labeled as # 2). Pull-down efficiency was evaluated using Coomassie Blue staining (Left) and immunoblotting by anti-His antibody (Right). FIGS. 3E and 3F showAttorney Docket No. 98121.00392 whole-cell current recording of TRPM2 in HEK293T cells transfected with PKCγ and wild-type TRPM2 (TRPM2-WT) (green) or PKC binding motif deleted TRPM2 (TRPM2-ΔPBM) (red). FIG. 3E shows representative traces. PMA (10 µM) was used to induce TRPM2 activation, NMDG to test seal tightness, and ACA to block TRPM2 current. FIG.3F shows quantification of current amplitude (n=9,8). FIGS. 3G-3I show CKAR real-time imaging in HEK293T cells transfected with PKCγ and TRPM2-WT (green) or TRPM2-ΔPBM (red). FIG. 3G shows representative imaging of FRET intensity before and 10 minutes after 100 µM H2O2 perfusion. FIG. 3H shows averaged representative traces from 5 randomly chosen cells. FIG. 3I shows quantification of FRET changes (n = 10-20). FIGS.3J and 3K shows whole-cell current recording of NMDAR in HEK293T cells transfected with NMDAR / PKCγ and EGFP control (blue), TRPM2-WT (green) and TRPM2-ΔPBM (red). FIG.3J shows representative traces of NMDARs elicited by 100 µM NMDA. FIG.3K shows quantification of current amplitude (n=9, 10, 11). (ns, no statistical significance, *, p < 0.05, **, p < 0.01, ***, p < 0.001; unpaired t test; mean ± SEM).

[0126] FIGS. 4A-4O show disruption of TRPM2-PKCγ coupling by TAT-M2PBM. FIG. 4A shows graphic illustration showing how TAT-M2PBM works. The PKCγ binding motif of TRPM2 (M2PBM) in conjugation with the cell-penetrating peptide TAT was synthesized. TAT- M2PBM binds to the TRPM2 binding site for PKCγ thus achieving the competitive inhibition on the binding of TRPM2 to PKCγ. FIG. 4B shows co-immunoprecipitation of PKCγ by TRPM2 in HEK293T cells co-expressed with PKCγ and TRPM2 treated with TAT-SC (scramble) or TAT- M2PBM at 1 µM for 2 hours. FIGS. 4C and 4D show whole-cell current recording of TRPM2 in HEK293T cells transfected with PKCγ and TRPM2 treated with TAT-SC (green) or TAT-M2PBM (red). FIG. 4C shows representative traces. PMA (10 µM) was used to induce TRPM2 activation, NMDG to test seal tightness, and ACA to block TRPM2 current. FIG.4D shows quantification of current amplitude (n=9,9). FIGS. 4E and 4F show CKAR real-time imaging in HEK293T cells transfected with PKCγ and TRPM2 treated with TAT-SC (green) or TAT-M2PBM (red). FIG.4E shows averaged representative traces from 5 randomly chosen cells. FIG.4F shows quantification of FRET changes (n=10-20). FIGS. 4G and 4H show whole-cell current recording of NMDARs in HEK293T cells transfected with NMDARs / PKCγ and TRPM2 treated with TAT-SC (green) or TAT-M2PBM (red). FIG. 4G shows representative traces. NMDA (100 µM) was used to induce NMDAR activation. FIG. 4H shows quantification of current amplitude (n=11,10,11). FIGS. 4IAttorney Docket No. 98121.00392 and 4J show whole-cell current recording of NMDARs in neurons isolated from the wild-type (WT) and Trpm2 deletion (M2KO) mice treated with TAT-SC or TAT-M2PBM. FIG. 4I shows representative traces. FIG.4J shows quantification of current amplitude (n=15,11,9,6). FIGS.4K and 4L show surface expression of GluN2a and GluN2b in HEK293T cells transfected with NMDAR / PKCγ and TRPM2 treated with TAT-SC or TAT-M2PBM. FIG. 4K shows representative WB bands. FIG.4L shows quantification of relative expression normalized to pan- cadherin (n=6 / group). FIGS. 4M-4O show whole-cell current recording of NMDARs in neurons isolated from the WT and M2KO mice treated with TAT-SC or TAT-M2PBM. FIG. 4M shows representative traces. PMA at 100 µM was used to enhance NMDAR’s activity for 60 seconds. FIG. 4N shows quantification of current amplitude before and after PMA perfusion. FIG. 4O shows quantification of current increases after PMA perfusion (n=6-10 / group). (ns, no statistical significance, *, p < 0.05, **, p < 0.01, ***, p < 0.001; unpaired t test; mean ± SEM).

[0127] FIGS.5A-5J show that TAT-M2PBM attenuates ischemic neuronal death in vitro. FIGS. 5A-5D show ratio Ca2+imaging of neurons isolated from wild-type (WT) and Trpm2 deletion (M2KO) mice subjected to OGD. Neurons were treated with MK801 / AP5 (10 µM), TAT- SC (1 µM) or TAT-M2PBM (1 µM). FIG.5A shows representative images showing Ca2+overload and neuronal death (indicated by white arrows). FIG.5B shows averaged traces from 10 randomly chosen neurons, FIG. 5C shows quantification of Ca2+increases 30 minutes after OGD (n=10- 20 / group). FIG. 5D shows quantification of neuronal death at 30 and 60 minutes of OGD (n=5 / group). FIGS.5E and 5F show DAF-FM nitric oxide (NO) imaging of neurons isolated from WT and M2KO mice subjected to OGD. Neurons were treated with MK801 / AP5, TAT-SC or TAT-M2PBM. FIG. 5E shows representative images showing nitric oxide production. FIG. 5F shows quantification of NO increase 30 minutes after OGD (n=10-20 / group). FIGS. 5G and 5H show MitoSOX reactive oxygen species (ROS) imaging of neurons isolated from WT and M2KO mice subjected to OGD. Neurons were treated with MK801 / AP5, TAT-SC or TAT-M2PBM. FIG. 5G depicts representative images showing ROS generation. FIG.5H shows quantification of ROS increase 30 minutes after OGD (n=10-20 / group). FIGS. 5I and 5J show Rhodamine123 (R123) mitochondrial membrane potential imaging of neurons isolated from WT and M2KO mice subjected to OGD. Neurons were treated with MK801 / AP5, TAT-SC or TAT-M2PBM. FIG. 5I depicts representative images showing mitochondrial depolarization. FIG. 5J showsAttorney Docket No. 98121.00392 quantification of R123 increase 30 minutes after OGD (n=10-20 / group). (ns, no statistical significance, *, p < 0.05, **, p < 0.01, ***, p < 0.001; unpaired t test; mean ± SEM).

[0128] FIGS.6A-6K show that TAT-M2PBM protects mice against ischemic stroke. FIG. 6A shows co-immunoprecipitation of PKCγ, GluN2a and GluN2b by TRPM2 in the brain from wild-type mice 2 hours, 12 hours and 24 hours after injection with TAT-SC (scramble) or TAT- M2PBM at 100 nmol / kg. FIG. 6B depicts a schematic showing exemplary injection strategy for evaluating short-term protective effects. FIG.6C and 6D show brain injury in wild-type (WT) and Trpm2 deletion (M2KO) mice injected with TAT-SC or TAT-M2PBM (100 nmol / kg) 24 hours after MCAO (n = 8,7,5,5). FIG. 6C depicts representative imaging of Triphenyl tetrazolium chloride (TTC) staining showing infract area (white). FIG. 6D shows quantification of brain infarction and neurological deficit score. FIGS.6E and 6F show brain injury in WT mice injected with TAT-SC, TAT-EE3 or TAT-M2PBM 24 hours after MCAO (n = 7,9,9). FIG. 6E depicts representative imaging of TTC staining showing infract area (white). FIG. 6F shows quantification of brain infarction and neurological deficit score. FIG. 6G depicts shematic showing exemplary injection strategy for evaluating short-term protective effects. FIGS. 6H-6K show brain injury in WT and M2KO mice injected with TAT-SC or TAT-M2PBM 7 days after MCAO (n = 8,9). FIG. 6H depicts representative imaging of TTC staining showing infract area (white). FIGS. 6I and 6J show quantification of brain infarction and neurological deficit score. (FIG. 6K), Quantification of rotarod test. (ns, no statistical significance, *, p < 0.05, **, p < 0.01, ***, p < 0.001; unpaired t test; mean ± SEM).

[0129] FIGS. 7A-7D show mass spectrometry of TRPM2 phosphorylation by PKCγ. FIGS.7A and 7B show Western blot analysis of PKCγ expression in total lysates from HEK293T cells and mouse brains. FIGS. 7C and 7D show representative mass spectrometry peaks showing phosphorylation of S10 (FIG. 7C) and S28 (FIG. 7D) after PMA treatment for 5 min.

[0130] FIGS. 8A-8B show alignments of S11 and S28 of TRPM2. FIG. 8A shows alignments of S11 and S28 of TRPM2 in all the TRPM channels. FIG. 8B shows alignments of S11 and S28 of TRPM2 in different species.

[0131] FIGS. 9A-9G show alignments of PKCγ binding motif of TRPM2. FIG. 9A shows alignments of the PKCγ binding motif of TRPM2 in all the TRPM channels. FIG. 9B shows alignments of the PKCγ binding motif of TRPM2 in different species. FIGS.9C-9D show Western blot analysis of TRPM2 and TRPM2-ΔPBM expression in total lysates from transfected HEK293TAttorney Docket No. 98121.00392 cells. FIGS. 9E-9F show purification of MHR1 / 2 domain from human TRPM2 and C2 domain from human PKCγ. FIG. 9G shows in vitro PKCγ activity assay. PKC inhibitor Go 69863 and staurosporine were used as negative controls.

[0132] FIGS.10A-10D show that TAT-M2PBM protects against H2O2-induced neuronal death. FIGS. 10A-10D show ratio Ca2+imaging of neurons isolated from wild-type (mice subjected to H2O2 perfusion at 100 μM. Neurons were treated with TAT-SC (1 µM) or TAT- M2PBM (1 µM). FIG. 10A shows representative images showing Ca2+overload and neuronal death (indicated by white arrows). FIG. 10B shows quantification of neuronal death at 30 and 60 minutes of OGD (n=5 / group). FIG. 10C shows averaged traces from 6 randomly chosen neurons Quantification of Ca2+increases 30 minutes after OGD (n=22 / group). FIG. 10D shows quantification of Ca2+increases 30 minutes after OGD (n=22 / group).

[0133] FIGS. 11A-11B show that TAT-M2PBM inhibits the post-stroke increase of NMDAR surface expression. FIGS. 11A-11B show surface expression of GluN2a and GluN2b in WT and M2KO mice 24 hours after MCAO treated with TAT-SC or TAT-M2PBM.

[0134] FIGS. 12A-12C show that TAT-M2PBM inhibits Ang II-induced loss of contractile proteins α-SMA and SM22α in vascular smooth muscle cells (VSMCs). FIGS. 12A-12C show expression of α-SMA (FIGS. 12A and 12B) and SM22α (FIGS.12A and 12C) in VSMCs. DETAILED DESCRIPTION

[0135] N-methyl-D-aspartate-receptor (NMDAR)-mediated glutamate excitotoxicity is a major cause of neurological injury or neurological disorder (e.g., ischemic neuronal death and / or post-recanalization infarction expansion). However, targeting NMDARs has not been successful in attenuating brain injury in clinical trials. The present disclosure is predicated, at least in part, on the discovery of an interaction motif between transient receptor potential melastatin 2 (TRPM2) and protein kinase C γ (PKCγ) and demonstrates that TRPM2-PKCγ uncoupling is a novel strategy for attenuating NMDARs-mediated excitotoxicity in a neurological injury or neurological disorder (e.g., ischemic stroke). It is demonstrated herein that the TRPM2-PKCγ interaction allows TRPM2-mediated Ca2+influx to promote PKCγ activation, which subsequently enhances TRPM2- induced potentiation of extrasynaptic NMDAR (esNMDAR) activity. Inventors identified the binding motif for PKCγ on TRPM2 (M2PBM), which can directly associate with the C2 domainAttorney Docket No. 98121.00392 of PKCγ. Also disclosed herein is an interfering peptide (e.g., M2PBM) or a conjugate thereof (e.g., TAT- M2PBM) to disrupt TRPM2-PKCγ interaction without compromising PKCγ function. Deleting M2PBM or disrupting the TRPM2-PKCγ association (e.g., using M2PBM or TAT- M2PBM) not only abolishes the functional TRPM2-PKCγ coupling, but also the functional coupling of TRPM2-esNMDAR, leading to reduced ischemic excitotoxicity and neuronal death. These findings provide a novel therapeutic strategy for mitigating the post-recanalization infarction expansion after ischemic stroke by keeping the “accomplices” in the TRPM2-PKCγ- esNMDAR complex apart.

[0136] Accordingly, disclosed herein an agent that inhibits the interaction between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2) (e.g., M2PBM or TAT- M2PBM) and methods for using such agents for treating or preventing neurological injury or neurological disorder (e.g., ischemic stroke) in a subject.

[0137] The inventors of the present invention have also surprisingly discovered that disruption of TRPM2-PKCγ interaction (e.g., using M2PBM or TAT-M2PBM) inhibits loss of one or more contractile proteins (e.g., α-SMA or SM22α) in a vascular smooth muscle cell, indicating the protection against VSMC reprogramming. VSMC reprogramming is critical in the progression of many cardiovascular diseases (e.g., atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, or diabetic angiopathy). Targeting TRPM2 and its coupling with TRPM2-PKCγ can provide a new therapeutic strategy to prevent and / or treat cardiovascular diseases.

[0138] Accordingly, also disclosed herein an agent that inhibits the interaction between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2) (e.g., M2PBM or TAT- M2PBM) and methods for using such agents for treating or preventing a vascular disease (e.g., atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, or diabetic angiopathy) in a subject. I. Definitions

[0139] Throughout the present specification and the accompanying claims the words “comprise,” “include,” and “have” and variations thereof such as “comprises,” “comprising,” “includes,” “including,” “has,” and “having” are to be interpreted inclusively. That is, these wordsAttorney Docket No. 98121.00392 are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0140] The terms “a,” “an,” and “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.

[0141] Recitation of ranges of values herein are 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. Ranges may be expressed herein as from “about” (or “approximately”) one particular value, and / or to “about” (or “approximately”) another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about” or “approximately” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are disclosed both in relation to the other endpoint, and independently of the other endpoint.

[0142] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Further, all methods described herein and having more than one step can be performed by more than one person or entity. Thus, a person or an entity can perform step (a) of a method, another person or another entity can perform step (b) of the method, and a yet another person or a yet another entity can perform step (c) of the method, etc. 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.

[0143] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0144] 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,Attorney Docket No. 98121.00392 a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0145] The headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description or the claims, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0146] Illustrations are for the purpose of describing a preferred embodiment of the invention and are not intended to limit the invention thereto.

[0147] As used herein, the term “about” refers to a range of values of plus or minus 10% of a specified value. For example, the phrase “about 200” includes plus or minus 10% of 200, or from 180 to 220, unless clearly contradicted by context.

[0148] As used herein, the term “TRPM2”, also known as “transient receptor potential melastatin 2”, “transient receptor potential channel 7”, “TRPC7”, “long transient receptor potential channel 2”, “LTRPC-2”, “estrogen-responsive element-associated gene 1 protein”, “EREG1”, “NUDT9L1”, “NUDT9H”, or” KNP3”, refers to a Ca2+-permeable cation channel. TRPM2 is the most abundant transient receptor potential (TRP) channel. TRPM2 is highly distributed in the central nervous system and is activated by hydrogen peroxide and agents that produce reactive oxygen / nitrogen species (ROS / RNS), increasing the Ca2+concentration. In response to oxidative stress stimuli, TRPM2-mediated Ca2+influx leads to cell death of a various cell types including neuron. The sequence of a human TRPM2 mRNA can be found, for example, NCBI Reference Sequences (RefSeq) NM_003307.4 (SEQ ID NO: 21), NM_001320350.2 (SEQ ID NO: 22), or NM_001320351.2 (SEQ ID NO: 23). In some embodiments, the sequence of a human TRPM2 mRNA can be found, for example, NCBI Reference Sequences (RefSeq) NM_001320350.2 (SEQ ID NO: 23). The sequence of a human TRPM2 polypeptide sequence can be found, for example, at GenBank Accession No. GI: 1934153297 (NP_001307279.2; SEQ ID NO: 24).

[0149] As used herein, the term “PKCγ”, also known as “protein kinase C γ” or “PKCG”, refers to a centrally restricted protein kinase involved in Ca2+-dependent intracellular signal transduction. PKCγ is highly enriched in brain and plays a major role in regulating both pre- and postsynaptic aspects of neurotransmission, including neuronal excitability, neurotransmitter release, and long-term alterations in gene expression and plasticity. PKC isozymes share similarAttorney Docket No. 98121.00392 domain composition, including a regulatory N-terminal region, a hinge region, and a C-terminal kinase domain. Contained within the regulatory N-terminal moiety, the pseudosubstrate region binds within the kinase domain active site pocket and prevents signaling in the absence of appropriate second messengers. The regulatory C1 domains bind diacylglycerol (DG). The C2 domain packs against the kinase domain to keep the pseudosubstrate in the active site pocket until, in conventional PKC isozymes, it binds Ca2+and allows PKC to engage with PIP2 at the plasma membrane (Pilo CA, Newton AC. Front Cell Dev Biol. 2022 Jun 21;10:929510). The sequence of a human PKCγ mRNA can be found, for example, at NCBI Reference Sequences (RefSeq) NM_001316329.2 (SEQ ID NO: 25) or NM_002739.5 (SEQ ID NO: 26). The sequence of a human PKCγ polypeptide sequence can be found, for example, at NCBI Reference Sequences (RefSeq) NP_001303258.1 (SEQ ID NO: 27) or NP_002730.1 (SEQ ID NO: 28).

[0150] As used herein, the term “NMDAR”, also known as N-methyl-D-aspartate receptor, NMDA receptor, refers to an ionotropic glutamate receptor and ion channel found in neurons. Ionotropic glutamate receptors are ligand-gated ion channels that allow rapid ion influx in response to glutamate and comprise the gateway to excitotoxicity. They contains both an extracellular glutamate binding site and a transmembrane ion channel. The two main subtypes of ionotropic glutamate receptors are NMDARs and AMPA (α-amino-3-hydroxy-5-methylisoxazole-4- propionic acid) receptors (AMPARs). At the resting state, the channel pores of NMDARs are normally blocked by Mg2+. When glutamate is released from pre-synaptic sites, activated AMPARs cause a partial depolarization in the post-synaptic membrane sufficient to remove the Mg2+block from NMDARs. Once NMDARs are activated, they flux Na+and Ca2+into the cell. The Ca2+influx through NMDARs is not only critical for the normal physiological processes in neurons, but also plays a major role in initiating ischemic cell death (Choi DW. Neuron. 1988;1(8):623–34). In excitotoxicity, excess glutamate release results in over-activation of NMDARs and leads to calcium overload inside the neurons. Calcium overload caused by excitotoxic mechanisms through NMDAR activation triggers a range of downstream pro-death signaling events such as calpain activation, reactive oxygen species (ROS) generation, and mitochondrial damage, resulting in cell necrosis or apoptosis (Xu J, et al., J Neurosci. 2009;29(29):9330–43; Kristian T, Siesjo BK. Stroke. 1998;29(3):705–18; Fujimura M, et al., J Cereb Blood Flow Metab. 1998;18(11):1239–47).Attorney Docket No. 98121.00392

[0151] NMDARs exist as multiple subtypes that differ in their molecular (subunit) composition. They are assembled as tetramers composed of two obligatory GluN1 subunits along with two GluN2 or GluN3 subunits, of which there are four (GluN2A–GluN2D) and two subtypes (GluN3A and GluN3B) respectively.

[0152] Each subunit has a typical modular architecture with two large clamshell-like extracellular domains (the N-terminal domain (NTD) involved in assembly and channel modulation and the agonist-binding domain (ABD)), a transmembrane domain (TMD) and a C- terminal domain (CTD) involved in receptor trafficking and signaling. The NTD and CTD regions are the most divergent and account for much of the functional diversity of NMDARs. Each subunit endows the receptor with distinct biophysical, pharmacological and signaling properties. The large extracellular region of the receptor harbors an array of binding sites for small-molecule ligands acting as endogenous or exogenous allosteric modulators. NMDAR subunit composition is plastic, changing during development and according to neuronal activity. Long-term synaptic plasticity of NMDARs also occurs at mature (adult) synapses and has profound consequences on cell firing and subsequent plasticity.

[0153] As used herein, the term “excitotoxicity” describes the process in which excess quantities of the excitatory neurotransmitter glutamate over-activates NMDARs and induces neuronal toxicity. This process is widely attributed to Ca2+influx, leading to superoxide and nitric oxide production, which together generate the cytotoxic reactive oxygen species.

[0154] As used herein, an “agent” or a “modulator” refers to any compound or molecule that affects the interaction between TRPM2 and its binding protein, e.g., PKCγ. In some embodiments, the agent modulates the biological activity of TRPM2, either directly or indirectly. An agent or a modulator of TRPM2 can act directly on TRPM2, e.g., a small molecule or an antibody which binds to TRPM2 and inhibits or activates its activity and / or function. In another embodiment, the agent or the modulator of TRPM2 can act indirectly, e.g., through another molecule, e.g., a binding partner of TRPM2, e.g., PKCγ, resulting in an increase or a decrease in the activity of TRPM2. Exemplary agents suitable for use in the methods of the invention include proteins, antibodies, peptides, peptidomimetics, small molecules, nucleic acids (e.g., DNA and RNA, e.g., antisense RNAs, sdRNAs, and siRNAs), carbohydrates, lipids, and other drugs.

[0155] As used herein, the terms “modulate,” “modulation,” or “modulating” are art- recognized and refer to up-regulation (i.e., activation, stimulation, increase), or down-regulationAttorney Docket No. 98121.00392 (i.e., inhibition, suppression, reduction, or decrease) of a response, or the two in combination or apart.

[0156] As used herein, the term “inhibit” refers to a decrease in the level of interaction between TRPM2 and its binding protein, e.g., PKCγ, for example, by preventing, reducing or reversing the interaction. In some embodiments, the term “inhibit" refers to the ability to decrease, reduce, suppress, reverse, or down-regulate the activity of TRPM2. In some embodiments, the term “inhibit" refers to the ability to decrease, reduce, suppress, reverse, or down-regulate the activity of TRPM2’s binding protein, e.g., PKCγ.

[0157] As used herein, the term “stimulate” refers to an increase in the level of interaction between TRPM2 and its binding protein, e.g., PKCγ. In some embodiments, the term “stimulate" refers to the ability to increase, promote, enhance, or up-regulate the activity of TRPM2. In some embodiments, the term “stimulate" refers to the ability to increase, promote, enhance, or up- regulate the activity of TRPM2’s binding protein, e.g., PKCγ.

[0158] As used herein, the term “administering” means the actual physical introduction of a composition into or onto (as appropriate) a subject, a host or cell. Any and all methods of introducing the composition into the subject, host or cell are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art, and also are exemplified herein.

[0159] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0160] As used herein, the term “pharmaceutically acceptable” refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a subject, preferably a human subject. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0161] As used herein, the term “substantially decreased”, “decreased”, “decrease”, and grammatical equivalents thereof refer to a level, amount, concentration of a parameter, such as a chemical compound, a metabolite, a nucleic acid, a polypeptide or a physical parameter (pH,Attorney Docket No. 98121.00392 temperature, viscosity, etc.) measured in a sample that has a decrease of at least 10%, preferably about 20%, more preferable about 40%, even more preferable about 50% and still more preferably a decrease of more than 75% when compared to the level, amount, or concentration of the same chemical compound, nucleic acid, polypeptide or physical parameter in a control sample.

[0162] As used herein, the term “substantially increased”, “increased”, “increase”, and grammatical equivalents thereof refer to a level, amount, concentration of a parameter, such as a chemical compound, a metabolite, a nucleic acid, a polypeptide or a physical parameter (pH, temperature, viscosity, etc.) measured in a sample that has an increase of at least 30%, preferably about 50%, more preferable about 75%, and still more preferably an increase of more than 100% when compared to the level, amount, or concentration of the same chemical compound, nucleic acid, polypeptide, or physical parameter in a control sample.

[0163] As used herein, the terms “treat,” “treating,” and “treatment” include inhibiting the pathological condition, disorder, or disease, e.g., arresting or reducing the development of the pathological condition, disorder, or disease or its clinical symptoms; or relieving the pathological condition, disorder, or disease, e.g., causing regression of the pathological condition, disorder, or disease or its clinical symptoms. These terms also encompass therapy and cure. Treatment means any way the symptoms of a pathological condition, disorder, or disease are ameliorated or otherwise beneficially altered. Preferably, the subject in need of such treatment is a mammal, preferably a human.

[0164] As used herein, the term "effective amount" refers to the amount of a therapy, which is sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, inhibit or prevent the advancement of a disorder, cause regression of a disorder, inhibit or prevent the recurrence, development, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy (e.g., prophylactic or therapeutic agent). An effective amount can require more than one dose.

[0165] The term “subject” is used herein to refer to an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g., a duck or a goose), and a shark. In an embodiment, the subject is a human, such as a human being treated or assessed for a disease,Attorney Docket No. 98121.00392 disorder or condition, a human at risk for a disease, disorder or condition, a human having a disease, disorder or condition, and / or human being treated for a disease, disorder or condition as described herein. In some embodiments, the subject does not suffer from an ongoing autoimmune disease. In one embodiment, the subject is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years of age. In another embodiment, the subject is about 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100 years of age. Values and ranges intermediate to the above recited ranges are also intended to be part of this invention. In addition, ranges of values using a combination of any of the above-recited values as upper and / or lower limits are intended to be included. II. Methods

[0166] Previous studies show that the physical and functional coupling between TRPM2 and esNMDAR is important in magnifying ischemic excitotoxicity (Zong P et al., Neuron. 2022 Jun 15;110(12):1944-1958.e8.). TRPM2 is an oxidative stress activated, Ca2+-permeable and nonselective cation channel highly expressed in the brain. TRPM2 knockout was shown to attenuate neuronal death, whereas the underlying molecular mechanisms was elusive. Protein kinase C (PKC) is an important promoter of ischemic brain injury, and PKC inhibition reduces ischemic excitotoxicity. PKCγ, the neuron-specific PKC isoform, potentiates NMDAR activity by increasing the surface trafficking of NMDAR and enhancing the channel activity of NMDAR. PKCγ is rapidly activated after ischemic stroke. However, the mechanisms of the membrane docking and activation of PKCγ in ischemic stroke, and the role of PKCγ in esNMDAR-mediated excitotoxicity remain poorly studied. Furthermore, modulators that can inhibit the interactions between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2), thereby for treating neurological injury or disorder (e.g., ischemic stroke), remains to be explored.

[0167] It is disclosed herein that PKCγ physically associates with the N tail of TRPM2. The recruitment of PKCγ to TRPM2 can be promoted by oxidative stress during ischemic stroke, but the pathological significance of this binding was still unclear. Inventors unexpectedly discovered that the C2 domain of PKCγ directly associates with TRPM2 through a unique interacting motif on TRPM2, the PKC binding motif (M2PBM), which is responsible for physical and functional coupling between TRPM2 and PKC, as well as esNMDAR-mediated excitotoxicity duringAttorney Docket No. 98121.00392 ischemic stroke. Inventors also found that TRPM2-induced potentiation of esNMDAR depends on TRPM2-PKCγ coupling. The examples disclosed herein demonstrate that TRPM2-mediated Ca2+influx promotes PKCγ activation, vice versa, PKCγ phosphorylates TRPM2 and enhances TRPM2 activation. Furthermore, deleting the binding motif M2PBM in TRPM2 or applying M2PBM peptide disrupts the physical and functional coupling of TRPM2-PKCγ. Furthermore, the examples disclosed herein demonstrate that, compared to inhibition of NMDAR-TRPM2 interactions, inhibition of PKCγ-TRPM2 interactions (e.g., using M2PBM or TAT-M2PBM) are unexpectedly more effective to reduce brain damage.

[0168] Moreover, the newly identified PKCγ -binding domain of TRPM2 and the newly identified TRPM2-binding domain of PKCγ can be used as a drug target for developing further therapeutic candidates, such as therapeutic peptides, e.g., M2PBM or TAT-M2PBM, or small molecules, for ischemic stroke and other TRPM2-associated neurodegenerative diseases.

[0169] Accordingly, in one aspect, the present disclosure provides methods and compositions to treat (e.g., alleviate, ameliorate, relieve, stabilize, delay onset of, inhibit progression of, reduce severity of, and / or reduce incidence of) and / or prevent neurological injury or neurological disorders, or one or more symptoms associate with the neurological injury or neurological disorders, in a subject in need thereof.

[0170] In some embodiments, the neurological injury results from a brain injury. In some embodiments, the brain injury comprises stroke (e.g., ischemic stroke, hemorrhagic stroke, or transient ischemic attack), traumatic brain injury (TBI), cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, brain aneurysm, coma, or any combination thereof.

[0171] In some embodiments, the neurological injury results from stroke, e.g., ischemic stroke.

[0172] Additionally, the methods and compositions may also be useful to aide in a subject's recovery from these neurological injuries (e.g., ischemic stroke), for example by improving synaptic function and memory in a patient recovering or rehabilitating following a neurological injury or during an active or prescribed rehabilitation program.

[0173] The data herein shows inhibition of PKCγ-TRPM2 interactions (e.g., using M2PBM or TAT-M2PBM) are unexpectedly more effective to reduce brain damage. Thus, the methods and compositions disclosed herein can be useful to preserve brain function in a subject having a neurological injury or neurological disorder (e.g., ischemic stroke).Attorney Docket No. 98121.00392

[0174] In some embodiments, administering to the subject the agent, the peptide or the pharmaceutical composition disclosed herein decreases surface expression of N-methyl-D- aspartate receptor (NMDAR) by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% in a neuronal cell.

[0175] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein decreases the amplitude of NMDAR current by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% in a neuronal cell.

[0176] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein inhibits an increase of intracellular Ca2+concentration by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% in a neuronal cell.

[0177] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein reduces mitochondrial membrane depolarization induced by ischemic injury by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% in a neuronal cell.

[0178] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein decreases neuronal death by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0179] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein reduces infarct volume by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0180] In some embodiments, administering to the subject the peptide, the conjugate, or the pharmaceutical composition disclosed herein improves neurological behavior score by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0181] Additionally, the inventors of the present invention have also surprisingly discovered that disruption of TRPM2-PKCγ interaction (e.g., using M2PBM or TAT-M2PBM) inhibits loss of one or more contractile proteins (e.g., α-SMA or SM22α) in a vascular smooth muscle cell (VSMC), indicating the protection against VSMC reprogramming. Vascular smooth muscle cells (VSMCs) are the majority of the cells in the aortic wall. VSMCs are not terminally differentiated and retain significant plasticity throughout the lifespan. VSMCs in a subject having vascularAttorney Docket No. 98121.00392 disorder (e.g., atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, or diabetic angiopathy) undergo a variety of heterogeneous differentiations (also termed “VSMC reprogramming”) in response to the microenvironment remodeling, giving rise to a range of cell subpopulations. VSMC reprogramming is crucial for vessel maturation or injury repair in the progression of many cardiovascular diseases (e.g., atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, or diabetic angiopathy). Targeting TRPM2 and its coupling with TRPM2- PKCγ can provide a new therapeutic strategy to prevent and / or treat cardiovascular diseases.

[0182] Accordingly, the methods and compositions disclosed herein may be useful to treat and / or prevent a vascular disease (e.g., atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, or diabetic angiopathy) in a subject.

[0183] In these methods, the subject may be human. The subject may be male or female.

[0184] These treatment methods comprise administering to a subject an agent or a composition comprising the agent that inhibits the activity of TRPM2. Agents suitable for use in the methods of the present invention includes any compound or molecule that can inhibit the biological activity of TRPM2, for example, by inhibiting the interaction between TRPM2 and PKCγ.

[0185] An agent can modulate the activity of TRPM2 either directly or indirectly. In some embodiments, the agent is an inhibitory agent. In some embodiments, the agent can act directly on TRPM2, e.g., an antagonist antibody which binds to TRPM2 and inhibits its activity and / or function. Alternatively, an inhibitory agent of TRPM2 can act indirectly on TRPM2 (e.g., through another molecule, e.g., a binding partner of TRPM2, e.g., PKCγ) resulting in a decreased activity.

[0186] In some embodiments, the agent inhibits the interaction between TRPM2 and PKCγ by targeting the binding site of PKCγ, e.g., a PKCγ-binding site, on TRPM2. An agent may target the PKCγ-binding site by binding to the PKCγ-binding site, thus blocking interaction between TRPM2 and PKCγ. Alternatively, an agent may target the PKCγ-binding site by mimicking or resembling the amino acid sequence of PKCγ-binding site, thus competing with PKCγ for TRPM2 binding.

[0187] In some embodiments, the agent inhibits the interaction between TRPM2 and PKCγ by targeting the binding site of TRPM2, e.g., a TRPM2-binding site, on PKCγ. An agent can target the TRPM2-binding site by binding to the TRPM2-binding site, thus blocking interaction betweenAttorney Docket No. 98121.00392 TRPM2 and PKCγ. Alternatively, an agent can target the TRPM2-binding site by mimicking or resembling the amino acid sequence of TRPM2-binding site, thus competing with TRPM2 for PKCγ binding.

[0188] The inventors of the present invention have successfully identified a specific domain of TRPM2 which is responsible for interaction between TRPM2 and PKCγ. The PKCγ-binding site is located near the N-terminus of TRPM2. In some embodiments, the PKCγ-binding site comprises amino acid residues 151-200 or residues 162-178 of TRPM2. In some embodiments, the PKCγ-binding site comprises the N-terminal amino acid residues 162-178 of TRPM2, i.e., the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1; also referred herein as “M2PBM”). As shown in the working examples, when the M2PBM domain was deleted from full length TRPM2, interaction between TRPM2 and PKCγ was disrupted.

[0189] In some embodiments, TRPM2 modulator such as TAT-M2PBM abolishes the functional TRPM2-PKCγ and the TRPM2-esNMDAR coupling. While not wishing to be bound by this theory, it is believed that TRPM2 serves as an anchor for priming activated PKCγ at the cell membrane during ischemic stroke. In an embodiment, TRPM2-PKCγ uncoupling using the TRPM2 modulator such as TAT-M2PBM attenuates excitotoxicity and neuronal death in vitro and in vivo. Results disclosed herein indicate that the TRPM2-PKCγ-esNMDAR complex plays a critical role in ischemic neuronal death, and that TRPM2-PKCγ uncoupling is a novel therapeutic strategy specifically targeting esNMDARs for ischemic stroke.

[0190] Exemplary agents suitable for use in the methods of the invention include small molecule, peptides, antagonist antibodies, or antigen-binding fragment thereof, recombinant fusion proteins, or interfering nucleic acid molecules (e.g., antisense RNAs, sdRNAs, and siRNAs), etc.

[0191] In some embodiments, the agent comprises a small molecule. In some embodiments, the small molecule is N-(p-amylcinnamoyl)anthranilic acid (ACA), 2-Aminoethoxydiphenyl borate (2-APB), or a combination thereof.

[0192] In some embodiments, the agent comprises a peptide comprising an amino acid sequence with at least 50%, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the agent comprises a peptideAttorney Docket No. 98121.00392 comprising an amino acid sequence differing by 1, 2, 3, 4, or 5 residues from the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the agent comprises a peptide comprising the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the agent comprises a peptide consisting of the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide which does not comprise the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0193] In some embodiments, the peptide is further conjugated to a cell-penetrating peptide (CPP). In some embodiments, the CPP comprises an amino acid sequence with at least 50%, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 4-19. In some embodiments, the CPP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19. In some embodiments, the CPP consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19. In some embodiments, the CPP comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5. In some embodiments, the CPP consists of an amino acid sequence set forth in SEQ ID NO: 4 or 5.

[0194] In some embodiments, the conjugate comprises an amino acid sequence with at least 50%, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20). In some embodiments, the conjugate comprises the amino acid sequence of YGRKKRRQRRR- WGLDVPNLLISVTGGA (SEQ ID NO: 20). In some embodiments, the conjugate consists of the amino acid sequence of YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20).

[0195] In some embodiments, the agent targets a TRPM2-binding site on PKCγ.

[0196] In some embodiments, the TRPM2-binding site on PKCγ is the C2-domain of PKCγ.

[0197] Methods of treating neurological injuries, neurodegenerative diseases, or vascular diseases of this disclosure may include administration of an agent, a peptide, or a pharmaceutical composition of this disclosure via any one of a variety of routes, including intravenous (IV), intramuscular (IM), intraarterial, intramedullary, intrathecal, subcutaneous (SQ), intraventricular, transdermal, interdermal, intradermal, by intratracheal instillation, bronchial instillation, and / or inhalation; as a nasal spray, and / or aerosol, and / or through a portal vein catheter. Any appropriate site of administration may be used. For example, the composition may be administered locally andAttorney Docket No. 98121.00392 directly at the site where action is required or may be attached or otherwise associated, e.g. conjugated, with entities which will facilitate the targeting to an appropriate location in the body.

[0198] In these compositions, any physiologically compatible carrier, excipient, diluent, buffer or stabilizer may be used. Examples of suitable carriers, excipients, diluents, buffers and stabilizers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In some cases, isotonic agents, e.g., sugars, polyalcohols (e.g., mannitol, sorbitol), or sodium chloride may be included. In certain embodiments, the compositions of this disclosure may be formulated so as to provide quick, sustained, or delayed release of the active ingredient (peptides of this disclosure, or variants thereof and / or additional drug(s)) after administration to the subject by employing procedures well known in the art. As described above, in certain embodiments, the composition is in a form suitable for injection and suitable carriers may be present at any appropriate concentration, but exemplary concentrations are from 1% to 20%, or from 5% to 10%.

[0199] Therapeutic compositions typically must be sterile and stable under conditions of manufacture and storage. Appropriate ways of achieving such sterility and stability are well known and described in the art.

[0200] Pharmaceutical compositions are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily (or other) usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically-effective dosage level for any particular subject will depend upon a variety of factors including the activity of the composition employed; the half-life of the composition after administration; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the agent, e.g., the peptide, and (if used) the additional therapeutic agent employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors, well known in the medical arts. Furthermore, effective doses may be extrapolated from dose-response curves derived from in vitro and / or in vivo animal models.

[0201] Thus, suitable doses of the agent of this disclosure and other active ingredients (if included) will vary from patient to patient and will also depend on the severity / stage of the stroke. In some embodiments, said dosages constitute a therapeutically effective amount or aAttorney Docket No. 98121.00392 prophylactically effective amount, depending on the nature of the treatment involved. In some embodiments, the dosages constitute a neuro-restorative- or rehabilitation-enhancing amount. In some embodiments, the dosages constitute an amount effective for treating a vascular disease (e.g., restore cardiovascular function and / or ameliorate a symptoms of a cardiovascular disease). The ability of the agent to elicit a desired response in the individual will also be a factor. Exemplary daily doses are: about 0.1 mg / kg to about 250 mg / kg, or about 0.1 mg / kg to about 200 mg / kg or about 100 mg / kg, or about 0.5 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg or about 1 mg / kg to about 10 mg / kg, of the active ingredient (e.g., M2PBM or TAT-M2PBM). In some embodiments, the agent is administered to the subject at a dose of about 10 nmol / kg to about 1000 nmol / kg (e.g., about 10 nmol / kg to about 100 nmol / kg, about 50 nmol / kg to about 150 nmol / kg, about 50 nmol / kg to about 200 nmol / kg, about 50 nmol / kg to about 300 nmol / kg, about 50 nmol / kg to about 400 nmol / kg, about 50 nmol / kg to about 500 nmol / kg, about 100 nmol / kg to about 500 nmol / kg, about 100 nmol / kg to about 800 nmol / kg, about 100 nmol / kg to about 1000 nmol / kg, about 200 nmol / kg to about 500 nmol / kg, about 200 nmol / kg to about 1000 nmol / kg, about 10 nmol / kg, about 20 nmol / kg, about 30 nmol / kg, about 40 nmol / kg, about 50 nmol / kg, about 60 nmol / kg, about 70 nmol / kg, about 80 nmol / kg, about 90 nmol / kg, about 100 nmol / kg, about 110 nmol / kg, about 120 nmol / kg, about 130 nmol / kg, about 140 nmol / kg, about 150 nmol / kg, about 160 nmol / kg, about 170 nmol / kg, about 180 nmol / kg, about 190 nmol / kg, about 200 nmol / kg, about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, about 400 nmol / kg, about 450 nmol / kg, about 500 nmol / kg, about 550 nmol / kg, about 600 nmol / kg, about 650 nmol / kg, about 700 nmol / kg, about 750 nmol / kg, about 800 nmol / kg, about 850 nmol / kg, about 900 nmol / kg, about 950 nmol / kg, or about 1000 nmol / kg) of the active ingredient (e.g., M2PBM or TAT-M2PBM). In some embodiments, the agent, pharmaceutical composition, peptide, or conjugate disclosed herein is administered to a subject in need about every 2 to 24 hours (e.g., about every 2 to 20 hours, about every 2 to 18 hours, about every 2 to 16 hours, about every 2 to 14 hours, about every 2 to 12 hours, about every 2 to 10 hours, about every 2 to 8 hours, about every 2 to 6 hours, about every 2 to 4 hours, about every 4 to 20 hours, about every 4 to 18 hours, about every 4 to 16 hours, about every 4 to 14 hours, about every 4 to 12 hours, about every 4 to 10 hours, about every 4 to 8 hours, about every 6 to 24 hours, about every 6 to 20 hours, about every 6 to 18 hours, about every 6 to 16 hours, about every 6 to 14 hours, about every 6 to 10 hours, about every 8 to 24 hours, about every 8 to 22 hours, about every 8 to 20 hours, about everyAttorney Docket No. 98121.00392 8 to 18 hours, about every 8 to 16 hours, about every 8 to 14 hours, about every 8 to 12 hours, about every 2 hours, about every 3 hours, about every 4 hours, about every 5 hours, about every 6 hours, about every 7 hours, about every 8 hours, about every 9 hours, about every 10 hours, about every 12 hours, about every 14 hours, about every 16 hours, about every 18 hours, about every 20 hours, about every 22 hours, or about every 24 hours). In some embodiments, the agent, pharmaceutical composition, peptide, or conjugate disclosed herein is administered to a subject in need about less than 24 hours (e.g., less than 20 hours, less than 20 hours, less than 18 hours, less than 16 hours, less than 14 hours, less than 12 hours, less than 10 hours, less than 8 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour) after one or more symptoms of ischemic stroke start. This may be administered as a single unit dose or as multiple unit doses administered more than once a day, for example, subcutaneously, intraperitoneally, or intravenously. It is to be noted, however, that appropriate dosages may vary depending on the patient, and that for any particular subject, specific dosage regimes should be adjusted over time according to the individual needs of the patient. For example, the dosage and administration protocol may be adjusted over time, or with patient advances in rehabilitation to less than once daily, including for example, every other day, three times weekly, or two times weekly, or once weekly, or every other week, etc. Thus, the dosage ranges set forth herein are to be regarded as exemplary and are not intended to limit the scope or practice of the claimed compositions or methods.

[0202] The methods further comprise administering to the subject an additional therapeutic agent or therapy currently known or later discovered to be effective in the prevention and / or treatment of neurological injury or neurological disorder, e.g., stroke, or neurological damage following stroke. In some embodiments, the additional therapeutic comprises an anticoagulant or clot-dissolving medicine (e.g., aspirin), clopidogrel, a tissue plasminogen activator (tPA) (e.g., alteplase), thrombectomy, carotid endarterectomy, a medication for antihypertension (e.g., nicardipine, labetalol, or lisinopril), an antipyretic therapy, warfarin, heparin, apixaban, atorvastatin, rosuvastatin, irbesartan, alteplase, or any combination thereof.

[0203] Contemplated therapies include surgery, such as carotid endarterectomy, or angioplasty, or stent placement. Contemplated therapies may also include physical or mental rehabilitation programs, which have proven particularly efficacious for rehabilitation and recovery following stroke and traumatic brain injury.Attorney Docket No. 98121.00392

[0204] The agents or compositions comprising the agents of this disclosure may be administered prior to, concurrently with, or after the administration of the additional therapeutic agent and / or therapy. These methods may include a step of assessing the efficacy of the therapeutic treatment. Such assessment of efficacy may be based on any number of assessment results. Depending on the level of efficacy assessed, the dosage of the neuroprotective peptides of this disclosure may be adjusted up or down, as needed.

[0205] Thus, by “in combination with,” it is not intended to imply that the agents or compositions of this disclosure and additional agent or therapy must be administered at the same time or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. Furthermore, it will be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent.

[0206] In general, each agent will be administered at a dose and on a time schedule determined for that agent. Additionally, this disclosure encompasses the delivery of the compositions in combination with agents that may improve their bioavailability, reduce or modify their metabolism, inhibit their excretion, or modify their distribution within the body.

[0207] The particular combination of therapies (e.g., therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. III. Agents for Use in the Methods of the Invention

[0208] Accordingly, molecules which modulate, e.g., inhibit, the activity of TRPM2, and / or molecules which modulate, e.g., inhibit, the activity of TRPM2’s binding partner, e.g., PKCγ, are useful in the methods of the present invention. Exemplary agents can modulate, e.g., inhibit, the association between TRPM2 and PKCγ.Attorney Docket No. 98121.00392 Small Molecule Inhibitors

[0209] An inhibitory agent for use in the methods of the present invention can be a small molecule. Small molecules are chemical compounds that inhibit the activity of TRPM2, e.g., by inhibiting the interaction between TRPM2 and PKCγ. Such compounds can be either natural products or members of a combinatorial chemistry library, and can be identified using screening assays, as described in detail below.

[0210] In some embodiments, an inhibitory agent is a small molecule, e.g., a small molecule inhibitor for TRPM2, or a small molecule inhibitor for binding proteins of TRPM2, e.g., PKCγ.

[0211] The small molecule inhibitors of the present invention may block the interaction between TRPM2 and PKCγ. For example, the small molecule inhibitors bind to the PKCγ binding domain of TRPM2. In other embodiments, the small molecule inhibitors of the present invention may bind to the region in PKCγ that interacts with TRPM2, thereby blocking the interaction between TRPM2 and PKCγ.

[0212] In some embodiments, the small molecule inhibitors are selected to bind domains sharing homology to an PKCγ-binding domain of the TRPM2. For example, a small molecule of the present invention may be directed toward a domain which is at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% or 99% identical to the PKCγ-binding domain of the TRPM2. Such a small molecule would be capable of binding to the PKCγ-binding domain of the TRPM2, thus blocking the interaction between TRPM2 and PKCγ.

[0213] In some embodiments, the small molecule binds to specific sequences of the TRPM2 protein, e.g., a PKCγ-binding site, e.g., amino acid residues 151-200 or residues 162-178 of TRPM2. In some embodiments, the PKCγ-binding site comprises an amino acid sequence of WGLDVPNLLISVTGGA.

[0214] In some embodiments, the small molecule inhibitors are selected to bind domains sharing homology to a TRPM2-binding domain of the PKCγ. For example, a small molecule of the present invention may be directed toward a domain which is at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% or at least 99% identical to the TRPM2-binding domain of the PKCγ. Such a small molecule would be capable of binding to the TRPM2-binding domain of the PKCγ, thus blocking the interaction between TRPM2 and PKCγ.Attorney Docket No. 98121.00392

[0215] In some embodiments, the small molecule binds to specific sequences of the PKCΓ protein, e.g., a TRPM2-binding site. The TRPM2-binding site is located in the C-terminus of PKCγ. In some embodiments, the TRPM2-binding site on PKCγ is the C2-domain of PKCγ.

[0216] In an embodiment, the TRPM2 modulator is a small molecule. Any suitable TRPM2 small molecule modulator can be used. In an embodiment, the small molecule is N-(p- amylcinnamoyl)anthranilic acid (ACA), 2-Aminoethoxydiphenyl borate (2-APB), or a combination thereof. In some embodiments, the small molecule is N-(p- amylcinnamoyl)anthranilic acid (ACA). In an embodiment, the small molecule is 2- Aminoethoxydiphenyl borate (2-APB).Mutant Proteins

[0217] Variants of TRPM2 protein or variants of TRPM2’s binding proteins (e.g., PKCγ) that function as antagonists and inhibit the interaction TRPM2 and PKCγ can also be used in the methods of the present invention.

[0218] In some embodiments, the agent is a mutant TRPM2 protein with a deletion of the PKCγ -binding site and has a reduced binding activity for PKCγ. For example, a mutant TRPM2Attorney Docket No. 98121.00392 protein with a deletion of amino acid residues 151-200 or a deletion of amino acid residues 162- 178 of TRPM2 can be used as an inhibitory agent in the present invention. As shown in Example 1, FIG.3, deletion of the TRPM2-PBM domain from the full-length TRPM2 resulted in a complete disruption of the interaction between TRPM2 and PKCγ.

[0219] In some embodiments, variants of TRPM2’s binding proteins (e.g., PKCγ) block the interaction between TRPM2 and PKCγ. In some embodiments, a mutant PKCγ which loses the ability to interact with TRPM2 can function as an inhibitory agent in the present invention, for example, a PKCγ protein without a the C2 domain of PKCγ.

[0220] A recombinant mutant protein for TRPM2 and PKCγ for use in the methods of the present invention may be generated from a recombinant vector according to methods known in the art. The recombinant vectors can comprise a nucleic acid encoding a mutant TRPM2 or a mutant PKCγ protein in a form suitable for expression of the nucleic acid in a host cell.

[0221] In some embodiments, the recombinant vectors may include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed (i.e., a recombinant expression vector). Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Methods in Enzymology: Gene Expression Technology vol.185, Academic Press, San Diego, CA (1991). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including mutant proteins or peptides, encoded by nucleic acids as described herein.

[0222] The recombinant expression vectors of the invention can be designed for expression of a polypeptide, or functional fragment thereof, in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g.,Attorney Docket No. 98121.00392 insect cells using baculovirus expression vectors, yeast cells or mammalian cells). Suitable host cells may include, but not limited to E. coli cells, Bacillus cells, Saccharomyces cells, Pochia cells, NS0 cells, COS cells, Chinese hamster ovary (CHO) cells, myeloma cells, or cells as described herein.

[0223] Another aspect of the invention pertains to host cells into which a recombinant vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0224] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Antagonist Antibodies or Antigen-Binding Portion thereof

[0225] The invention further contemplates methods and compositions comprising an antagonist antibody, or antigen binding portion thereof, which inhibits the activity of TRPM2 and / or its binding protein, e.g., PKCγ.

[0226] In some embodiments, an antagonist antibody of TRPM2 and / or an antagonist antibody of TRPM2’s binding protein, e.g., PKCγ, or an antigen binding portion thereof, block the interaction between TRPM2 and PKCγ.

[0227] The term "antibody," as used herein, is a broad term and is used in its ordinary sense, including, without limitation, to refer to naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavyAttorney Docket No. 98121.00392 chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from N terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0228] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TRPM2, or PKCγ). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab’ fragment, which is essentially an Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al. (1989) Nature 341: 544-546), which consists of a VH domain; (vii) an isolated complementarity determining region (CDR); and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniquesAttorney Docket No. 98121.00392 known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0229] An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0230] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to, e.g., TRPM2 or PKCγ, is substantially free of antibodies that specifically bind antigens other than TRPM2 or PKCγ). Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals. An “isolated antibody” may, however, include polyclonal antibodies, which all bind specifically to, e.g., TRPM2 or PKCγ.

[0231] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0232] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intendedAttorney Docket No. 98121.00392 to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0233] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity, which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma, which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0234] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0235] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.

[0236] The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen.”

[0237] The term “human antibody derivatives” refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody.Attorney Docket No. 98121.00392

[0238] The term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences. It will be appreciated by one of skill in the art that when a sequence is “derived” from a particular species, said sequence may be a protein sequence, such as when variable region amino acids are taken from a murine antibody, or said sequence may be a DNA sequence, such as when variable region encoding nucleic acids are taken from murine DNA. A humanized antibody may also be designed based on the known sequences of human and non-human (e.g., murine or rabbit) antibodies. The designed antibodies, potentially incorporating both human and non-human residues, may be chemically synthesized. The sequences may also be synthesized at the DNA level and expressed in vitro or in vivo to generate the humanized antibodies.

[0239] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0240] The term “antibody mimetic” or “antibody mimic” is intended to refer to molecules capable of mimicking an antibody’s ability to bind an antigen, but which are not limited to native antibody structures. Examples of such antibody mimetics include, but are not limited to, Adnectins (i.e., fibronectin based binding molecules), Affibodies, DARPins, Anticalins, Avimers, and all of which employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms. The embodiments of the instant invention, as they are directed to antibodies, or antigen-binding portions thereof, also apply to the antibody mimetics described above.

[0241] Standard assays to evaluate the binding ability of the antibodies toward TRPM2 are known in the art, including for example, ELISAs, Western blots and RIAs. The binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by ELISA, Scatchard and Biacore analysis.

[0242] Methods for producing antibodies are well-established. One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies, A Laboratory Manual, Ed Harlow and David Lane, Cold Spring HarborAttorney Docket No. 98121.00392 Laboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art will also appreciate that binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (Antibody Engineering: A Practical Approach (Borrebaeck, C., ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920 (1992)). Monoclonal and polyclonal antibodies to molecules, e.g., proteins, and markers also commercially available (R and D Systems, Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc., Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA; Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049 USA; BiosPacific, Emeryville, Calif.).

[0243] In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody.

[0244] Polyclonal antibodies of the present invention can be produced by a variety of techniques that are well known in the art. Polyclonal antibodies are derived from different B-cell lines and thus may recognize multiple epitopes on the same antigen. Polyclonal antibodies are typically produced by immunization of a suitable mammal with the antigen of interest, e.g., TRPM2. Animals often used for production of polyclonal antibodies are chickens, goats, guinea pigs, hamsters, horses, mice, rats, sheep, and, most commonly, rabbit. Standard methods to produce polyclonal antibodies are widely known in the art and can be combined with the methods of the present invention (e.g., U.S. Patent Nos. 4,719,290, 6,335,163, 5,789,208, 2,520,076, 2,543,215, and 3,597,409, the entire contents of which are incorporated herein by reference.

[0245] Monoclonal antibodies of the present invention can be produced by any of a variety of techniques known to those of ordinary skill in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.

[0246] Monoclonal antibodies may be prepared using hybridoma methods, such as the technique of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976), and improvements thereto. These methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity. Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No.4,816,567. DNA encoding antibodies employed in the disclosed methods may be isolated and sequenced using conventional procedures.Attorney Docket No. 98121.00392 Recombinant antibodies, antibody fragments, and / or fusions thereof, can be expressed in vitro or in prokaryotic cells (e.g. bacteria) or eukaryotic cells (e.g. yeast, insect or mammalian cells) and further purified as necessary using well known methods.

[0247] More particularly, monoclonal antibodies may be readily prepared through use of well- known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified expressed protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB / c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

[0248] The animals are injected with antigen as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary. The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant. Booster injections with the same antigen would occur at approximately two-week intervals. Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.

[0249] The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies thatAttorney Docket No. 98121.00392 render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

[0250] The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones may then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma may be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, may then be tapped to provide MAbs in high concentration. The individual cell lines also may be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they may be readily obtained in high concentrations. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

[0251] Large amounts of the monoclonal antibodies of the present invention also may be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into mammals which are histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.

[0252] In accordance with the present invention, fragments of the monoclonal antibody of the invention may be obtained from the monoclonal antibody produced as described above, by methods which include digestion with enzymes such as pepsin or papain and / or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention may be synthesized using an automated peptide synthesizer.

[0253] Antibodies may also be derived from a recombinant antibody library that is based on amino acid sequences that have been designed in silico and encoded by polynucleotides that are synthetically generated. Methods for designing and obtaining in silico-created sequences are known in the art (Knappik et al., J. Mol. Biol. 296:254:57-86, 2000; Krebs et al., J. Immunol. Methods 254:67-84, 2001; U.S. Pat. No. 6,300,064).

[0254] Digestion of antibodies to produce antigen-binding fragments thereof can be performed using techniques well known in the art. For example, the proteolytic enzyme papain preferentiallyAttorney Docket No. 98121.00392 cleaves IgG molecules to yield several fragments, two of which (the "F(ab)" fragments) each comprise a covalent heterodimer that includes an intact antigen-binding site. The enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the "F(ab')2" fragment, which comprises both antigen-binding sites. "Fv" fragments can be produced by preferential proteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, but are more commonly derived using recombinant techniques known in the art. The Fv fragment includes a non-covalent VH::VLheterodimer including an antigen-binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule (Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972); Hochman et al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem. 19:4091-4096 (1980)).

[0255] Antibody fragments that specifically bind to the protein biomarkers disclosed herein can also be isolated from a library of scFvs using known techniques, such as those described in U.S. Pat. No. 5,885,793.

[0256] A wide variety of expression systems are available in the art for the production of antibody fragments, including Fab fragments, scFv, VL and VHs. For example, expression systems of both prokaryotic and eukaryotic origin may be used for the large-scale production of antibody fragments. Particularly advantageous are expression systems that permit the secretion of large amounts of antibody fragments into the culture medium. Eukaryotic expression systems for large- scale production of antibody fragments and antibody fusion proteins have been described that are based on mammalian cells, insect cells, plants, transgenic animals, and lower eukaryotes. For example, the cost-effective, large-scale production of antibody fragments can be achieved in yeast fermentation systems. Large-scale fermentation of these organisms is well known in the art and is currently used for bulk production of several recombinant proteins.

[0257] Following screening and sequencing, antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No.4,816,567, incorporated by reference herein. An isolated nucleic acid encoding, for example, an anti-TRMP2 antibody is used to transform host cells for expression. Such nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g.,Attorney Docket No. 98121.00392 has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0258] For recombinant production of an anti-TRPM2 antibody, a nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0259] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos.5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0260] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0261] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda (Sf9) cells.

[0262] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).Attorney Docket No. 98121.00392

[0263] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243- 251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). TPMR2-Derived or PKCγ-Derived Peptides

[0264] An inhibitory agent for use in the methods of the present invention is a peptidic compound derived from the amino acid sequence of TRPM2 (e.g., the sequence disclosed herein as SEQ ID NO: 1) and / or its binding proteins, e.g., PKCγ. In particular, the inhibitory compound comprises a portion of TRPM2 or PKCγ (or a mimetic thereof) that mediates interaction of TRPM2 with PKCγ, such that contact of TRPM2 or PKCγ with this peptidic compound competitively inhibits the interaction of TRPM2 and PKCγ. For example, a peptide derived from TRPM2 that comprises the amino acid sequence in the PKCγ-binding domain (e.g., M2PBM) may serve as an inhibitory modulator. Alternatively, the peptide resembles a fragment of the PKCγ-binding site of the TRPM2 protein, and can act to specifically block the interaction between TRPM2 and PKCγ. Similarly, a peptide derived from PKCγ that comprises the amino acid sequence in the TRPM2- binding domain may serve as an inhibitory modulator (e.g., the C2 domain of PKCγ). A peptide that resembles a fragment of the TRPM2-binding site of the PKCγ protein can also act to specifically block the interaction between TRPM2 and PKCγ.

[0265] In some aspects, disclosed is a polypeptide / peptide including an amino acid sequence at least 50% (at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,Attorney Docket No. 98121.00392 99%, or 100%) identical to an amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1); wherein the polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following residues: WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide is M2PBM (WGLDVPNLLISVTGGA) (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide is a TRPM2 modulator. In some embodiments, the peptide / polypeptide modulates the functional TRPM2-PKCγ and the TRPM2-esNMDAR coupling.

[0266] In some embodiments, the polypeptide includes an amino acid sequence at least 80% identical to an amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide includes an amino acid sequence at least 90% identical to an amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide includes an amino acid sequence at least 95% identical to an amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide includes an amino acid sequence 100% identical to an amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide includes at least 4, 5, 6, 7, 8, 9, 1, 11, 12, 13, 14, 15, or all 16 of the following residues: WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide includes at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of the following residues: WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide includes at least 10, 11, 12, 13, 14, 15, or all 16 of the following residues: WGLDVPNLLISVTGGA (SEQ ID NO: 1). In some embodiments, the peptide / polypeptide includes at least 12, 13, 14, 15, or all 16 of the following residues: WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0267] In some embodiments, the peptide / polypeptide further includes a heterologous cell- penetrating peptide (CPP) tag, one such non-limiting example of the heterogeneous CPP tag is TAT: YGRKKRRQRRR (SEQ ID NO: 4). Any suitable heterogeneous CPP tag can be used, for example, TAT, P28, ACPPs, a highly charged oligopeptide of human origin, or a combination thereof (for more examples for CPPs that can be used, see Xie et al., Cell-Penetrating Peptides in Diagnosis and Treatment of Human Diseases: From Preclinical Research to Clinical Application, Front. Pharmacol., (2020), 11:697 (doi: 10.3389 / fphar.2020.00697). Some additional non- limiting examples of CPPs are shown in Table 1.Attorney Docket No. 98121.00392

[0268] Table 1: CPPs Cell-penetrating peptide (CPP) Sequence TAT RKKRRQRRR (SEQ ID NO: 5) R9-TAT GRRRRRRRRRPPQ (SEQ ID NO: 6) R8 RRRRRRRR (SEQ ID NO: 7) DPV3 RKKRRRESRKKRRRES (SEQ ID NO: 8) DPV6 GRPRESGKKRKRKRLKP (SEQ ID NO: 9) Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 10) pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 11) ARF RVRVFVVHIPRLT (SEQ ID NO: 12) MPG GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 13) MAP KLALKLALKALKAALKLA (SEQ ID NO: 14) Transportan GWTLNS / AGYLLGKINLKALAALAKKIL (SEQ ID NO: 15) Bip4 VSALK (SEQ ID NO: 16) C105Y CSIPPEVKFNPFVYLI (SEQ ID NO: 17) Melittin GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 18) gH625 HGLASTLTRWAHYNALIRAF (SEQ ID NO: 19)

[0269] In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 50% identical (e.g., have at least 60%, 70%, 80%, 90%, 95% or more sequence identity) to an amino acid sequence of TAT-M2PBM (YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20)); wherein the polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or all 27 of the following residues: YGRKKRRQRRR- WGLDVPNLLISVTGGA (SEQ ID NO: 20). In an embodiment, the peptide / polypeptide is TAT- M2PBM (YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20)). In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 80% identical to an amino acid sequence of YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20). In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 85% identical to an amino acidAttorney Docket No. 98121.00392 sequence of YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20). In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 90% identical to an amino acid sequence of YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20). In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 95% identical to an amino acid sequence of YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20). In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 100% identical to an amino acid sequence of YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20).

[0270] In some embodiments, disclosed herein are peptides that are at least 50% identical (e.g., have at least 60%, 70%, 80%, 90%, 95% or more sequence identity) to WGLDVPNLLISVTGGA (SEQ ID NO: 1) and that retain at least one neuroprotective property thereof. Preferably, a peptide variant of the neuroprotective peptides of this disclosure will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to WGLDVPNLLISVTGGA (SEQ ID NO: 1). Alternatively, the variant peptide(s) will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acid substitutions as compared to WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0271] In some embodiments, the peptide comprises a dimer, a trimer, a tetramer, or a multimer of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

[0272] In some embodiments, disclosed herein are peptides that are at least 50% identical (e.g., have at least 60%, 70%, 80%, 90%, 95% or more sequence identity) to an amino acid sequence of TAT-M2PBM (YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20)) and that retain at least one neuroprotective property thereof. Preferably, a peptide variant of the neuroprotective peptides of this disclosure will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to an amino acid sequence of TAT- M2PBM (YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20)). Alternatively, the variant peptide(s) will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,Attorney Docket No. 98121.00392 19, 20, 21, 22, 23, 24, 25, or 26 amino acid substitutions as compared to an amino acid sequence of TAT-M2PBM (YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20)).

[0273] In some embodiments, the peptide comprises a dimer, a trimer, a tetramer, or a multimer of TAT-M2PBM (YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20)).

[0274] Substituted amino acid residues may be unrelated to the amino acid residue being replaced (e.g., unrelated in terms or hydrophobicity / hydrophilicity, size, charge, polarity, etc.), or the substituted amino acid residues may constitute similar, conservative, or highly conservative amino acid substitutions. As used herein, “similar,” “conservative,” and “highly conservative” amino acid substitutions are defined as shown in the table below. The determination of whether an amino acid residue substitution is similar, conservative, or highly conservative is based exclusively on the side chain of the amino acid residue and not the peptide backbone, which may be modified to increase peptide stability, as discussed below. Amino Acid Similar Amino Acid Conservative Amino Highly Conservative Substitutions Acid Substitutions Amino Acid Substitutions Glycine (G) A, S, N A n / a Alanine (A) S, G, T, V, C, P, Q S, G, T S Serine (S) T, A, N, G, Q T, A, N T, A Threonine (T) S, A, V, N, M S, A V, N S Cysteine (C) A, S, T, V, I A n / a Proline (P) A, S, T A n / a Methionine (M) L, I, V, F L, I, V L, I Valine (V) I, L, M, T, A I, L, M I Leucine (L) M, I, V, F, Τ, Α M, I, V, F M, I Isoleucine (I) V, L, M, F, T, C V, L, M, F, V, L, M Phenylalanine (F) W, L, M, I, V W, L n / a Tyrosine (Y) F, W, H, L, I F, W F Tryptophan (W) F, L, V F n / a Asparagine (N) Q Q Q Glutamine (Q) N N N Aspartic Acid (D) E E E Glutamic Acid € D D D Histidine (H) R, K R, K R, K Lysine (K) R, H R, H R, H Arginine (R) K, H K, H K, HAttorney Docket No. 98121.00392

[0275] Conservative amino acid substitutions in the context of a subject peptide are selected so as to preserve activity of the peptide.

[0276] In some embodiments, the peptide is an isolated peptide. The isolated peptides of the invention can be made intracellularly in cells by introducing into the cells an expression vector encoding the peptide. Such expression vectors can be made by standard techniques. In some embodiments, the expression vector comprises a nucleic acid molecule encoding the peptide of the invention, which is operably linked to a control sequence for the expression of the peptide. The peptide can be expressed in intracellularly as a fusion with another protein or peptide (e.g., a GST fusion).

[0277] Alternative to recombinant synthesis of the peptides in the cells, the peptides can be a synthetic peptide, e.g., made by chemical synthesis using standard peptide synthesis techniques. Synthesized peptides can then be introduced into cells by a variety of means known in the art for introducing peptides into cells (e.g., liposome and the like). Modified Peptides of the Invention

[0278] Also contemplated in the context of the inventive methods and compositions is the modification of any neuroprotective peptides described herein, by chemical or genetic means. Examples of such modification include construction of peptides of partial or complete sequence with non-natural amino acids and / or natural amino acids in L or D enantiomeric forms. For example, any of the peptides disclosed herein, and any variants thereof, could be produced in an all-D form. Furthermore, the peptides may be modified to contain carbohydrate or lipid moieties, such as sugars or fatty acids, covalently linked to the side chains or the N- or C-termini of the amino acids. In addition, the disclosed peptides may be modified by glycosylation and / or phosphorylation.

[0279] In addition, disclosed peptides may be modified to enhance solubility and / or half-life upon being administered. For example, polyethylene glycol (PEG) and related polymers have been used to enhance solubility and the half-life of protein therapeutics in the blood. Accordingly, the disclosed peptides may be modified by PEG polymers and the like. PEG or PEG polymers means a residue containing poly(ethylene glycol) as an essential part. Such a PEG can contain further chemical groups which are necessary for the therapeutic activity of the peptides of this disclosure;Attorney Docket No. 98121.00392 which results from the chemical synthesis of the molecule; or which is a spacer for optimal distance of the parts of the molecule from one another. In addition, such a PEG can consist of one or more PEG sidechains which are linked together. PEG groups with more than one PEG chain are called multiarmed or branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. Branched PEGs usually have 2 to 8 arms and are described in, for example, U.S. Pat. No. 5,932,462. Especially preferred are PEGs with two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., Bioconjugate Chem.6 (1995) 62–69). The term "PEG" is used broadly to encompass any polyethylene glycol molecule, wherein the number of ethylene glycol (EG) units is at least 460, preferably 460 to 2300 and especially preferably 460 to 1840 (230 EG units refers to a molecular weight of about 10 kDa). The upper number of EG units is only limited by solubility of the PEGylated peptides of this disclosure. Usually PEGs which are larger than PEGs containing 2300 units are not used. Preferably, a PEG used in the invention terminates on one end with hydroxy or methoxy (methoxy PEG, mPEG) and is on the other end covalently attached to a linker moiety via an ether oxygen bond. The polymer is either linear or branched. Branched PEGs are e.g., described in Veronese et al., Journal of Bioactive and Compatible Polymers 12 (1997) 196–207. Suitable processes and preferred reagents to produce PEGylated peptides and variants of this disclosure are described in US Patent Pub. No. 2006 / 0154865. It is understood that modifications, for example, based on the methods described by Veronese, F. M., Biomaterials 22 (2001) 405–17, can be made in the procedures so long as the process results in PEGylated peptides of this disclosure. Particularly preferred processes for the preparation of PEGylated peptides of this disclosure are described in US 2008 / 0119409, which is incorporated herein by reference.

[0280] Additionally, the peptides of this disclosure may be is fused to one or more domains of an Fc region of human IgG. Antibodies comprise two functionally independent parts, a variable domain known as “Fab,” that binds an antigen, and a constant domain known as “Fc,” that is involved in effector functions such as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas a Fab is short-lived (Capon et al., 1989, Nature 337:525- 31). When constructed together with a therapeutic protein of this disclosure, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding,Attorney Docket No. 98121.00392 complement fixation, and perhaps even blood-brain barrier, or placental transfer. In one example, a human IgG hinge, CH2, and CH3 region may be fused at either the amino-terminus or carboxyl- terminus of the peptides of this disclosure using methods known to the skilled artisan. The resulting fusion polypeptide may be purified by use of a Protein A affinity column. Peptides and proteins fused to an Fc region have been found to exhibit a substantially greater half-life in vivo than the unfused counterpart. Also, a fusion to an Fc region allows for dimerization / multimerization of the fusion polypeptide. The Fc region may be a naturally occurring Fc region, or may be altered to improve certain qualities, such as therapeutic qualities, circulation time, or reduced aggregation.

[0281] The peptides may also be modified to contain sulfur, phosphorous, halogens, metals, etc. Amino acid mimics may be used to produce polypeptides, and therefore, the polypeptides of this disclosure may include amino acid mimics that have enhanced properties, such as resistance to degradation. For example, the polypeptides may include one or more (e.g., all) peptide monomers. IV. Screening Assays

[0282] The invention further provides methods (also referred herein as “screening assays”) for identifying compounds, e.g., candidate or test compounds (e.g., peptides, small molecules or other drugs) which modulate, e.g., inhibit or stimulate, the activity of TRPM2, e.g., interaction between TRPM2 and PKCγ, and for testing or optimizing the activity of modulators.

[0283] Agents that are capable of modulating the activity of TRMP2 or its binding ligands, e.g., PKCγ, as identified by the methods of the invention, are useful as candidate compounds useful for treating or preventing neurological injury or neurological disorder in a subject in need thereof. For example, in one aspect, the present invention provides methods for identifying a compound useful for treating or preventing neurological injury or neurological disorder. The methods include providing a test compound (or a plurality of test compounds), determining the effect of the test compound on the interaction between TRPM2 and PKCγ, and selecting a compound which modulates, e.g., decreases, the interaction between TRPM2 and PKCγ, thereby identifying a compound useful for treating or preventing neurological injury or neurological disorder.

[0284] In another aspect, agents that are capable of modulating the activity of TRMP2 or its binding ligands, e.g., PKCγ, as identified by the methods of the invention, are useful as candidate compounds useful for treating or preventing vascular disorder in a subject in need thereof. ForAttorney Docket No. 98121.00392 example, in one aspect, the present invention provides methods for identifying a compound useful for treating or preventing vascular disorder. The methods include providing a test compound (or a plurality of test compounds), determining the effect of the test compound on the interaction between TRPM2 and PKCγ, and selecting a compound which modulates, e.g., decreases, the interaction between TRPM2 and PKCγ, thereby identifying a compound useful for treating or preventing vascular disorder.

[0285] In some embodiments, the compound targets a PKCγ -binding site on TRPM2. A compound can target the PKCγ -binding site by binding to the PKCγ -binding site, thus blocking interaction between TRPM2 and PKCγ. A compound can also target the PKCγ -binding site by mimicking the amino acid sequence of PKCγ -binding site, thus competing with PKCγ for TRPM2 binding.

[0286] In some embodiments, the compound targets a TRPM2-binding site on PKCγ. A compound can target the TRPM2-binding site by binding to the TRPM2-binding site, thus blocking interaction between TRPM2 and PKCγ. A compound can also target the TRPM2-binding site by mimicking the amino acid sequence of TRPM2-binding site, thus competing with PKCγ for TRPM2 binding.

[0287] Examples of agents, candidate compounds or test compounds include, but are not limited to, proteins, peptides, peptidomimetics, small molecules, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, and other drugs.

[0288] Compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145; U.S. Patent No. 5,738,996; and U.S. Patent No. 5,807,683, the entire contents of each of the foregoing references are incorporated herein by reference).

[0289] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al.Attorney Docket No. 98121.00392 (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233, the entire contents of each of the foregoing references are incorporated herein by reference. Libraries of compounds may be presented, e.g., presented in solution (e.g., Houghten (1992) Bio / Techniques 13:412-421), or on beads (Lam (1991) Nature 354:82- 84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith (19900 Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310). The entire contents of each of the foregoing references are incorporated herein by reference.

[0290] The test compound can be contacted with a cell that expresses the TRPM2 protein or a molecule with which TRPM2 directly interacts, e.g., PKCγ. For example, the test compound can be contacted with a cell that naturally expresses or has been engineered to express the protein(s) by introducing into the cell an expression vector encoding the protein.

[0291] Alternatively, the test compounds can be subjected to a cell-free composition that includes the protein(s) (e.g., a cell extract or a composition that includes e.g., purified natural or recombinant protein).

[0292] Compounds that modulate the activity of TRPM2, or a binding ligand of TRPM2, e.g., PKCγ, can be identified using various "read-outs." For example, a cell can be transfected with an expression vector, incubated in the presence and in the absence of a test compound, and the effect of the compound on the interaction between TRPM2 and PKCγ or on a biological response regulated by TRPM2 can be determined. The biological activities of TRPM2 include activities determined in vivo, or in vitro, according to standard techniques. Activity can be a direct activity, such as an association with a binding ligand, e.g., PKCγ. Alternatively, the activity is an indirect activity, such as a change in calcium influx in neurons.

[0293] To determine whether a test compound modulates TRPM2 protein expression, in vitro transcriptional assays can be performed. To determine whether a test compound modulates TRPM2 mRNA expression, various methodologies can be performed, such as quantitative or real- time PCR.Attorney Docket No. 98121.00392

[0294] A variety of reporter genes are known in the art and are suitable for use in the screening assays of the invention. Examples of suitable reporter genes include those which encode chloramphenicol acetyltransferase, beta-galactosidase, alkaline phosphatase, green fluorescent protein, or luciferase. Standard methods for measuring the activity of these gene products are known in the art.

[0295] A variety of cell types are suitable for use as an indicator cell in the screening assay. Preferably a cell line is used which expresses low levels of endogenous TRPM2 and is then engineered to express recombinant protein. Cells for use in the subject assays include eukaryotic cells. For example, in one embodiment, a cell is a fungal cell, such as a yeast cell. In another embodiment, a cell is a plant cell. In yet another embodiment, a cell is a vertebrate cell, e.g., an avian cell or a mammalian cell (e.g., a murine cell, or a human cell).

[0296] Recombinant expression vectors that can be used for expression of , e.g., TRPM2, are known in the art. For example, the cDNA is first introduced into a recombinant expression vector using standard molecular biology techniques. A cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library. The nucleotide sequences of cDNAs for or a molecule in a signal transduction pathway involving (e.g., human, murine and yeast) are known in the art and can be used for the design of PCR primers that allow for amplification of a cDNA by standard PCR methods or for the design of a hybridization probe that can be used to screen a cDNA library using standard hybridization methods.

[0297] In another embodiment, the test compounds can be subjected to a cell-free composition that includes the protein(s) (e.g., a cell extract or a composition that includes e.g., either purified natural or recombinant protein). TRPM2 expressed by recombinant methods in a host cells or culture medium can be isolated from the host cells, or cell culture medium using standard methods for protein purification. For example, ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies can be used to produce a purified or semi-purified protein that can be used in a cell free composition. Alternatively, a lysate or an extract of cells expressing the protein of interest can be prepared for use as cell-free composition.

[0298] In one embodiment, compounds that specifically modulate TRPM2 activity or the activity of a binding ligand in a signal transduction pathway involving TRPM2 are identified basedAttorney Docket No. 98121.00392 on their ability to modulate the interaction of TRPM2 with its binding ligand, e.g., PKCγ. Suitable assays are known in the art that allow for the detection of protein-protein interactions (e.g., immunoprecipitations, two-hybrid assays and the like). By performing such assays in the presence and absence of test compounds, these assays can be used to identify compounds that modulate (e.g., inhibit or enhance) the activity of TRPM2 with a binding ligand, e.g., PKCγ.

[0299] Compounds identified in the subject screening assays can be used in methods of modulating one or more of the biological responses regulated by TRPM2. It will be understood that it may be desirable to formulate such compound(s) as pharmaceutical compositions as described herein prior to contacting them with cells.

[0300] Once a test compound is identified that directly or indirectly modulates TRPM2 expression or activity by one of the variety of methods described hereinbefore, the selected test compound (or "compound of interest") can then be further evaluated for its effect on cells, for example by contacting the compound of interest with cells either in vivo (e.g., by administering the compound of interest to an organism) or ex vivo (e.g., by isolating cells from an organism and contacting the isolated cells with the compound of interest or, alternatively, by contacting the compound of interest with a cell line) and determining the effect of the compound of interest on the cells, as compared to an appropriate control (such as untreated cells or cells treated with a control compound, or carrier, that does not modulate the biological response).

[0301] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulator can be identified using a cell-based or a cell-free assay, and the ability of the modulators to increase or decrease the interaction between TRPM2 and PKCγ can be confirmed in vivo, e.g., in an animal, such as, for example, an animal model for, e.g., a mouse model of middle cerebral artery occlusion.

[0302] Moreover, a modulator of TRPM2 can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such a modulator. Alternatively, a modulator identified as described herein can be used in an animal model to determine the mechanism of action of such a modulator.

[0303] In another embodiment, it will be understood that similar screening assays can be used to identify compounds that indirectly modulate the activity of TRPM2, e.g., by performing screening assays such as those described above using molecules with which TRPM2 interacts, e.g., PKCγ, or any molecules that act either upstream or downstream of TRPM2 in the pathway.Attorney Docket No. 98121.00392

[0304] Compounds identified by the screening assays of the present invention are considered as candidate therapeutic compounds useful for treating diseases, e.g., neurological injury or disorders, e.g., stroke, as described herein. Thus, the invention also includes compounds identified in the screening assays, and methods for their administration and use in the treatment, prevention, or delay of development or progression of diseases described herein. V. Pharmaceutical Compositions

[0305] In an aspect, disclosed is a pharmaceutical composition including therapeutically effective amount of at least one of the peptides / polypeptides and / or small molecules disclosed herein. In certain embodiments, the composition further includes a carrier that includes at least one pharmaceutically acceptable excipient. In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 50% identical (e.g., have at least 60%, 70%, 80%, 90%, 95% or more sequence identity) to an amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1). In an embodiment, the peptide / polypeptide includes an amino acid sequence at least 50% identical (e.g., have at least 60%, 70%, 80%, 90%, 95% or more sequence identity) to an amino acid sequence of YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20). In an embodiment, the peptide / polypeptide is M2PBM (WGLDVPNLLISVTGGA (SEQ ID NO: 1)). In an embodiment, the peptide / polypeptide is TAT-M2PBM (YGRKKRRQRRR-WGLDVPNLLISVTGGA (SEQ ID NO: 20)). In an embodiment, the small molecule is N-(p-amylcinnamoyl)anthranilic acid (ACA), 2-Aminoethoxydiphenyl borate (2-APB), or a combination thereof. In an embodiment, the small molecule is N-(p-amylcinnamoyl)anthranilic acid (ACA). In an embodiment, the small molecule is 2-Aminoethoxydiphenyl borate (2-APB).

[0306] The present disclosure includes pharmaceutical compositions comprising a pharmaceutically acceptable salt, in particular, an acid or base addition salt, of a compound, for example, a peptide / polypeptide, as described herein. The disclosed compositions may be prepared in various forms, such as capsules, suppositories, tablets, food / drink and the like. Optionally, the disclosed compositions may include various pharmaceutically acceptable excipients, such as microcrystalline cellulose, mannitol, glucose, defatted milk powder, polyvinylpyrrolidone, starch and combinations thereof.

[0307] Compositions of this disclosure may include a carrier protein, such as serum albumin (e.g., HSA, BSA, and the like). The serum albumin may be purified or recombinantly produced.Attorney Docket No. 98121.00392 By mixing the agents of the invention, e.g., the neuroprotective polypeptide(s), in the pharmaceutical composition with serum album, the agents, e.g., the neuroprotective polypeptides, may be effectively "loaded" onto the serum albumin, allowing a greater amount of the agents to be successfully delivered to a site of neurological injury.

[0308] The therapeutically effective compounds as described herein may, in accordance with the disclosure, be administered in single or divided doses by the oral, parenteral or topical routes. Administration of the active compound may range from continuous (intravenous drip) to several administrations per day (for example, Q.I.D.) and may include administration routes such as oral, topical, parenteral, intramuscular, intravenous, sub-cutaneous, transdermal (which may include a penetration enhancement agent), buccal, sublingual, intranasal, intraocular, intrathecal, vaginal, and suppository administration, among other routes of administration. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Enteric coated oral tablets may be used to enhance bioavailability of the compounds from an oral route of administration. The most effective dosage form will depend upon the pharmacokinetics of the particular agent chosen as well as the type, location and severity of disease, condition or symptom, and the health of the patient. Administration of compounds according to the present disclosure as sprays, mists, or aerosols for intra-nasal, intra-tracheal or pulmonary administration may also be used. The present disclosure therefore also is directed to pharmaceutical compositions comprising an effective amount of compound as described herein or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient. Compounds according to the present disclosure may be administered in immediate release or sustained or controlled release forms. Sustained or controlled release forms are preferably administered orally, but also in suppository and transdermal or other topical forms. Intramuscular injections in liposomal form or in depot formulation may also be used to control or sustain the release of compound at an injection site.

[0001] The compositions as described herein may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in controlled-release formulations. It should also be understood that a specific dosage and treatment regimen for any particular patient will depend on the judgment of the treating physician as based upon a variety of factors, including the activity and bioavailability of the specific compoundAttorney Docket No. 98121.00392 employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the severity of the particular disease or condition being treated.

[0309] A patient or subject in need of therapy using a compound according to the methods described herein can be treated by administering to the patient (subject) an effective amount of the compound according to the present disclosure, either alone, or in combination with another known therapeutic agent. In an embodiment, the patient or subject is a mammal such as human.

[0310] In certain aspects, the compound is conveniently administered in any suitable unit dosage form, including but not limited to a dosage form containing less than 1 milligrams (mg), 1 mg to 3000 mg, or 5 mg to 500 mg of active ingredient per unit dosage form. An oral dosage of about 25 mg-250 mg is often convenient.

[0311] In certain aspects, the active ingredient is preferably administered to achieve peak plasma concentrations of the active compound of about 0.00001-30 millimole (mM), preferably about 0.1-30 micromole (μM). This may be achieved, for example, by the intravenous injection of a solution or formulation of the active ingredient, optionally in saline, or an aqueous medium or administered as a bolus of the active ingredient. Oral administration may also be appropriate to generate effective plasma concentrations of active agent.

[0312] The concentration of active compound in the drug composition will depend on absorption, distribution, metabolism, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the physician administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.

[0313] The composition according to the present invention may be administered to a patient by various routes. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In an embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous,Attorney Docket No. 98121.00392 subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.

[0314] In accordance with any of the embodiments, the composition according to the present invention can be administered orally to a subject in need thereof. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice and include an additive, such as cyclodextrin (e.g., α-, β-, or γ-cyclodextrin, hydroxypropyl cyclodextrin) or polyethylene glycol (e.g., PEG400); (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions and gels. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

[0315] The dose administered to the mammal, particularly human and other mammals, in accordance with the present invention should be sufficient to affect the desired response. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the age, condition or disease state, predisposition to disease, genetic defect or defects, and body weight of the mammal. The size of the dose will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular composition and the desired effect. It will beAttorney Docket No. 98121.00392 appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations. VI. Kits

[0316] In one aspect, this disclosure further provides kits for the treatment of neurological injury, neurological diseases, neurodegenerative diseases, or vascular disorders comprising an agent, e.g., a peptide, of this disclosure, or variants thereof, or a composition comprising the same. Kits may include one or more other elements including, but not limited to, instructions for use; other therapeutic agents (i.e., for combination or emergency therapy of stroke); other reagents, e.g., a diluent, devices or other materials for preparing composition for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject. Instructions for use may include instructions for therapeutic application, including suggested dosages and / or modes of administration, e.g., in a human subject, as described herein. In some embodiments, the kits are for use in the methods and uses as described herein, e.g. therapeutic, diagnostic, or imaging methods, or are for use in in vitro assays or methods.

[0317] In some embodiments, the kits are for diagnosing neurological diseases, disorders or impairments and optionally comprise instructions for use of the kit components to diagnose or evaluate the severity of such neurological diseases, disorders or impairments.

[0318] It is to be understood that this invention is not limited to particular assay methods, or test agents and experimental conditions described, as such methods and agents may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0319] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are hereby incorporated herein by reference. EXAMPLES Example 1. Materials and Methods. AnimalsAttorney Docket No. 98121.00392

[0320] The global TRPM2 knockout (TRPM2-KO, or gM2KO) mice were generated by Dr. Yasuo Mori’s lab at Kyoto University Japan. The deletion of Trpm2 was developed in C57B6J mouse by replacing the third exon (S5–S6 linker in the pore domain) with a neomycin coding region. The knockout mice exhibited no differences in behavior or impairment in breeding, compared to wild type (WT) C57BJ6 mice. TRPM2-KO mice were back-crossed to C57BL / 6 mice for ≥10 generations before being used for experiments. Cortical neuron isolation and culture

[0321] Mice pups at P0 were euthanized based on animal protocol. Whole brain was harvested and immersed in frozen Hank’s Balanced Salt Solution (HBSS). Meninges were peeled. Brain stem and thalamus were removed. Cortical tissues were cut into small pieces and digested with 0.25% trypsin (Thermal Fisher Scientific, 15090–046) in HBSS at 37°C for 15 min. Tissue pellets are washed with HBSS. Cells were resuspended with appropriate amount of Neurobasal Medium (Thermal Fisher Scientific, 21103–049) supplemented with 2% B27 supplement (Thermal Fisher Scientific, 17504–044), 3% horse serum (Thermal Fisher Scientific, 16050114), 0.25% L- glutamine (Thermal Fisher Scientific, 25030–081) and 1% penicillin / streptomycin (Thermal Fisher Scientific, 15140–122). Isolated cells were counted and plated on coverslips pre-coated with poly-L-lysine (Sigma-Aldrich, P4707) at a density of about 500 × 103cells / cm2for OGD and H2O2 treatment, and 100 × 103cells / cm2for current recording. Cytosine arabinoside (Sigma- Aldrich, C1768) was added to maintain a concentration at 1μM to inhibit the proliferation of non- neuronal cells. 24 h after plating, culture medium was changed to Neurobasal Medium supplemented with 2% B27 supplement, 0.25% L-glutamine and 1% penicillin / streptomycin. The concentration of Cytosine arabinoside (araC) was increased to 2 μM. Medium was changed every 3 days. OGD and current recording were performed at 7th day of culture. Cell line culture and transfection

[0322] HEK293T cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Thermal Fisher Scientific, 12100–038) supplemented with 10% BGS (HyClone, SH30541.03) and 0.5% penicillin / streptomycin (Thermal Fisher Scientific, 15140–122) at 37°C and 5% CO2. 2 h prior to transfection, culture medium was replaced with DMEM supplemented with 5% BGS.Attorney Docket No. 98121.00392 Cells were transfected when the confluence was about 60% using Lipofectamine 3000 Transfection Kit (Thermal Fisher Scientific, 2232162) based on the manual. Middle cerebral artery occlusion (MCAO)

[0323] Eight-to nine-week-old male mice (~25 g) were subjected to transient middle cerebral artery occlusion (tMCAO) for 120 min followed by 24 h of reperfusion. The genotype information was blinded to the surgeon who conduct the surgeries. MCAO surgery was performed as previously described. In brief, mice were anesthetized based on animal protocol. The unilateral right middle cerebral artery (MCA) occlusion was achieved by inserting a silicone-coated 6–0 monofilament (Doccol Corporation, Sharon, MA) 10 to 11 mm from internal carotid artery bifurcation via an external carotid artery incision. Mouse body temperature was maintained at ~ 37°C (TCA T-2DF, Physitemp). Cerebral blood flow was monitored before and after occlusion as well as after reperfusion. The bregma was exposed and the skull bone countersunk at two 3 × 3- mm areas over both MCA supply territories for bilateral monitoring of local cortical blood flow. Successful occlusion was confirmed by 85% reduction of cerebral blood flow monitored using laser Doppler blood FlowMeter (Moor-VMS-LDF1, Moor Instrument, Dever, UK). Sham control mice underwent the same procedure but without insertion of filament to occlude the MCA. Neurological deficit score evaluation

[0324] Neurological deficit was scored based on previously reported criteria. In brief, score 0 represents no neurological deficit; score 1 represents failure to extend left paw; Score 2 represents circling to the left; score 3 represents falling to the left; score 4 represents inability of spontaneously walking and decreased level of consciousness; and score 5 represents death due to brain ischemia. The observer to score the neurological deficit was an experienced observer and blinded by the group assignment and genotype information. If the animal score was 0 or 5, it was removed from the study. Rotarod test

[0325] For examining the long-term protective effect of TAT-EE3, motor coordination of mice was evaluated by rotarod test right after the evaluation of neurological deficit score at 3rd and 7th day after MCAO. Briefly, mice were placed on a rotating rod with the speed range of 6–56 roundsAttorney Docket No. 98121.00392 per minute for 5 min. Each mouse was tested for 3 times with two 20-min interval in between. The falling from the rotating rod was recorded and the averaged latency of each mouse was used for quantification. Infarct volume assessment by Triphenyl tetrazolium chloride (TTC) staining

[0326] Tetrazolium chloride (Sigma-Aldrich, T-8877) was dissolved in PBS at a concentration of 2% 30 min prior to use. Post-stroke mice were euthanized, and brains were frozen at -80°C for 5 min. Brains were cut into coronary slices at a thickness of 1 mm. Brain slices were stained with 2% TTC (v / v) for 20 min, and then washed using PBS for 3 times, and fixed in 10% Neutral buffered formalin for later scanning. TTC labels non-injured tissue, leaving the infarct area white. The stained slices were scanned for data analysis using ImageJ software. The infarct volume was calculated and presented as a percentage of total brain volume as previously reported. Antibodies, chemicals and reagents

[0327] Rabbit polyclonal antibodies to TRPM2 (Novus, NB110–81601, 1:50 in protein extraction for IP); Rabbit polyclonal antibodies to GluN2A (Cell Signaling Technology, 4205S, 1:1000 in 5% BSA for WB). Rabbit polyclonal antibodies to GluN2B (Cell Signaling Technology, 4207S, 1:1000 in 5% BSA for WB); Rabbit polyclonal antibodies to PKC-γ (Cell Signaling Technology, 59090S, 1:5000 in 5% BSA for WB); Rabbit polyclonal antibodies to Pan-cadherin (Cell Signaling Technology, 4068S, 1:5000 in 5% BSA for WB); Rabbit monoclonal antibodies to GAPDH (Cell Signaling Technology, 5174S, 1:5000 in 5% BSA for WB); Rabbit monoclonal antibodies to GST-Tag (Cell Signaling Technology, 2622S, 1:100 for IP); Rabbit monoclonal antibodies to His-Tag (Cell Signaling Technology, 12698S, 1:5000 in 5% BSA for WB); Anti- mouse IgG, HRP-linked Antibody (Cell Signaling Technology, 7076, 1:10000 in 5% BSA for WB); Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology, 7074, 1:10000 in 5% BSA for WB); Tetrazolium chloride (Sigma-Aldrich, T-8877); NMDA (Tocris, 0114); AP5 (Cayman chemical, 14539); MK-801 (Sigma-Aldrich, M107); PMA (Sigma-Aldrich, 524400); H2O2 (Thermal Fisher Scientific, 200745); EGTA (Cayman chemical, 11706); BAPTA-AM (Cayman chemical, 15551); NP40 (Thermal Fisher Scientific, 28324); Triton X-100 (Thermal Fisher Scientific, T-9284), Bovine Serum Albumin (Sigma-Aldrich, 9048–46-8). All chemicalsAttorney Docket No. 98121.00392 for making artificial cerebrospinal fluid (aCSF; see below) and recording solution (see below) were purchased from Sigma-Aldrich. Membrane permeable peptide TAT-M2PBM for disrupting TRPM2 and PKCγ coupling and scramble control TAT-SC peptides

[0328] TAT-SC (sequence: YGRKKRRQRRRVILLKDHTLEYPVF (SEQ ID NO: 29)), TAT-M2PBM (sequence: YGRKKRRQRRRWGLDVPNLLISVTGGA (SEQ ID NO: 20)) were synthesized by GenScript Biotech and dissolved in PBS to make a stock concentration at 10 mM. HEK-293T cells or isolated neurons were treated with TAT-SC or TAT-M2PBM at a concentration of 1 μM for at least 2 h prior to use. Mice were intraperitoneal injected (i.p.) with TAT-SC or TAT-M2PBM at a dose of 100 nmol / kg. Detailed injection strategy is described in FIG. 6B. Plasmids and enzymes

[0329] GluN1a (Addgene, 17928), GluN2A (Addgene, 17924), GluN2B (Addgene, 17925), PKC-γ (Addgene, 112266), PKC-γ-DN (Addgene, 21239), CKAR (Addgene, 14860). The pcDNA4 / TO-FLAG-hTRPM2 construct was a kind gift from Dr. Sharenberg AM (University of Washington, Seattle). DpnI (BioLabs, R0176L) and PfuUltra HF (Agilent, 600380–51) were used to generating different deletion or mutation constructs. Subcloning

[0330] For TRPM2, subcloning of N terminus (1–727) was achieved by introducing a stop codon (A2282T) by PCR (F: CAAGGACATGTAGTTTGTGTCTCACG (SEQ ID NO: 30), R: CGTGAGACACAAACTACATGTCCTTG (SEQ ID NO: 31)) using PfuUltra HF. Mutagenesis of S11A (F: TGAGGAAAGCTGGCGCAGAGCAGGAGGAG (SEQ ID NO: 32), R: TCCTCCTGCTCTGCGCCAGCTTTCCTCAG (SEQ ID NO: 33)), S11D (F: TGAGGAAAGCTGGCGAGGAGCAGGAGGAG (SEQ ID NO: 34), R: TCCTCCTGCTCCTCGCCAGCTTTCCTCAG) (SEQ ID NO: 35), S38A (F: GGCGCAGCAACGCCAGCCTCTTCAAG (SEQ ID NO: 36), R: TCTTGAAGAGGCTGGCGTTGCTGCGC (SEQ ID NO: 37)), S38D (F: GGCGCAGCAACGACAGCCTCTTCAAG (SEQ ID NO: 38)), R:Attorney Docket No. 98121.00392 TCTTGAAGAGGCTGTCGTTGCT GCGC (SEQ ID NO: 39)) were achieved by PCR using PfuUltra HF. Deletion of the PBM were achieved by PCR (F: TACCACCTCATGA CCCAGCACAAGAACTTCAACATGAAG (SEQ ID NO: 40), R: TTCATGTTGAAGTTCTTGTGCTGGGTCATGAGGTGGTAG (SEQ ID NO: 41)) using Q5 Site-Directed Mutagenesis Kit (BioLabs, E0554S) based on the manual. Protein expression and purification

[0331] The C2 domain (residues 27–306) of human PKC was cloned into a modified pGEX- 4T3 vector containing a removable tobacco etch virus (TEV) protease recognition site. The GST- tagged C2 domain was expressed in Escherichia coli BL21(DE3) cells grown to an OD600 (optical density at 600 nm) between 0.8 and 1.0 at 37°C followed by induction of protein expression at 21°C overnight using 0.5 mM isopropyl-D-1-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation, resuspended by lysis buffer containing 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1% phenylmethylsulfonyl fluoride (PMSF) and 2 mM dithiothreitol (DTT), and lysed by using high-pressure homogenization (Avestin EmulsiFlex C3). The lysate was clarified by centrifugation at 30,000 rpm at 4°C for 30 min, and the supernatant was applied to a Glutathione Sepharose 4B column (GE Healthcare). After being extensively washed with lysis buffer, the protein was eluted with a buffer containing 25 mM Tris-HCl (pH 8.0), 200 mM NaCl, 15 mM reduced glutathione and 2 mM DTT. The protein was further purified by size exclusion chromatography (SD200; GE Healthcare) in a buffer containing 25 mM Tris-HC (pH 8.0), 150 mM NaCl, 2 mM DTT. The protein was concentrated using an Amicon stirred ultrafiltration cell unit with a 10-kDa cutoff membrane (EMD Millipore) and stored at -80°C until use.

[0332] The MHR1 / 2 domain (residues 127–455) of human TRPM2 was cloned into a modified pET15b vector containing a removable TEV protease recognition site. The His6-tagged MHR1 / 2 domain was expressed in E. coli BL21(DE3) as described above. The cell pellet was resuspended in denaturing buffer containing 25 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1% PMSF and 6 M Urea and lysed by high-pressure homogenization. The lysate was clarified by centrifugation at 30,000 rpm at 4°C for 30 min, and the supernatants were applied to a Ni2+-nitrilotriacetic acid (NTA) column (GE Healthcare). After being extensively washed with 25 mM imidazole in the denaturing buffer, the protein was eluted with a buffer containing 25 mM Tris-HCl (pH 8.0), 300 mM NaCl and 250 mM imidazole. For refolding, the protein was dialyzed at 4° C overnight againstAttorney Docket No. 98121.00392 two changes of a buffer without urea [25 mM Tris-HCl (pH 8.0), 200 mM NaCl and 0.1% Triton X-100]. The refolded protein was concentrated using an Amicon stirred ultrafiltration cell unit with a 10-kDa cutoff membrane and stored at -80°C until use. Live cell imaging

[0333] CKAR experiments was performed based on a well-established protocol. CKAR was transfected into HEK293T cells with other plasmids. 12 h after transfection, cells were split onto 25 mm glass coverslips for FRET imaging. Culture medium was washed using HBSS for 3 times. Fluorescence intensities at 527 nm for YFP and 490 nm for CFP were collected at a rate of 1 Hz using CoolSNAP HQ2 (Photometrics) and data were analyzed using NIS-Elements (Nikon).

[0334] Intracellular Ca2+was measured using ratio Fura2-AM imaging based on a well- established protocol. In brief, Fura2-AM (Thermal Fisher Scientific, F1221) was dissolved in DMSO to make a stock concentration at 1 mM. Pre-warmed Neurobasal Medium (Thermal Fisher Scientific, 21103–049) was used to dilute Fura-2 a.m. to a working concentration at 2.5 mM, and 0.02% Pluronic F-127 (Thermal Fisher Scientific, P3000MP) was added to facilitate loading of Fura-2 a.m. Cortical neurons on 25 mm glass coverslips were washed using pre-warmed PBS for 3 times, and then incubated with 2 mL of Fura-2 a.m. working solution for 20 min at 37°C. Non- incorporated dye was washed away using HBSS. Fluorescence intensities at 510 nm with 340 nm and 380 nm excitation were collected at a rate of 1 Hz using CoolSNAP HQ2 (Photometrics) and data were analyzed using NIS-Elements (Nikon).

[0335] Intracellular nitric oxide content was measured using DAF-FM imaging. DAF-FM (Thermal Fisher Scientific, D23844) was dissolved in DMSO to make a stock concentration at 1 mM. Pre-warmed Neurobasal Medium was used to dilute DAF-FM to a working concentration at 1 μM for loading. Cortical neurons on 25 mm glass coverslips were washed using pre-warmed PBS for 3 times, and then incubated with 2 mL of DAF-FM working solution for 10 min at 37°C. Non-incorporated dye was washed away using HBSS. Fluorescence intensities at 515 nm was collected using CoolSNAP HQ2 and data were analyzed using NIS-Elements.

[0336] Intracellular reactive oxygen species was measured using MitoSOX imaging. MitoSOX-Red (Thermal Fisher Scientific, M36008) was dissolved in DMSO to make a stock concentration at 1 mM. Pre-warmed Neurobasal Medium was used to dilute MitoSOX-Red to a working concentration at 0.5 μM for loading. Cortical neurons on 25 mm glass coverslips wereAttorney Docket No. 98121.00392 washed using pre-warmed PBS for 3 times, and then incubated with 2 mL of MitoSOX-Red working solution for 15 min at room temperature. Non-incorporated dye was washed away using HBSS. Fluorescence intensities at 610 nm was collected using CoolSNAP HQ2 and data were analyzed using NIS-Elements.

[0337] Mitochondrial depolarization was evaluated using Rhodamine-123 imaging based on a well-established protocol. Rhodamine-123 (Thermal Fisher Scientific, R302) dissolved in DMSO to make a stock concentration at 10 mg / mL. Pre-warmed Neurobasal Medium was used to dilute Rhodamine-123 to a working concentration at 2.5 mg / mL for loading. Cortical neurons on 25 mm glass coverslips were washed using pre-warmed PBS for 3 times, and then incubated with 2 mL of Rhodamine-123 working solution for 5 min at room temperature. Non-incorporated dye was washed away using HBSS. Fluorescence intensities at 509 nm was collected using CoolSNAP HQ2 and data were analyzed using NIS-Elements. Mass spectrometry

[0338] Lysis buffer without NP40 (150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH = 8.0) containing proteinase inhibitors (Sigma-Aldrich, 539131–10VL) and phosphatase inhibitors (Thermal Fisher Scientific, 78428) was used to lyse HEK293T cells 24 h after transfection and 5 min after DMSO or PMA (10 μM) treatment. Cell and tissue lysate were lysed by ultrasound using an ultrasonic cleaner filled with ice-cold water for 30 min. After incubated on ice for 1 h, lysate was centrifuged at 13000 g for 30 min and supernatant was collected. Protein samples were sent for mass spectrometry analysis at UConn Proteomics & Metabolomics Center. Co-immunoprecipitation

[0339] Co-immunoprecipitation was performed based on a well-established protocol. NP-40 lysis buffer (10% NP40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH = 8.0) containing proteinase inhibitors (Sigma-Aldrich, 539131–10VL) and phosphatase inhibitors (Thermal Fisher Scientific, 78428) was used to lyse both cultured cells and frozen brain tissue. For transfected cells, proteins were extracted 36 h after transfection. Cell and tissue lysate were lysed by ultrasound using an ultrasonic cleaner (Thermal Fisher Scientific) filled with ice-cold water for 30 min. After incubated on ice for 1 h, lysate was centrifuged at 13000 g for 30 min and supernatant was collected. Protein concentration was measured using Pierce Rapid Gold BCA Protein Assay Kit (Thermal FisherAttorney Docket No. 98121.00392 Scientific, A53225). 300 μg of protein was taken and diluted using NP-40 lysis buffer to make a total volume of 500 μL. Unused protein was allocated and frozen at —80°C for future use. Appropriate amount of antibody was added based on manufacturer instruction. 25 μL of pre- washed Protein A / G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003) was added, and incubated at 4°C for overnight. Then the mixture was centrifuged at 2500g for 1 min to get agarose beads. Agarose beads was washed using NP-40 lysis buffer for 3 times, mixed with same amount of 2x Laemmli Sample Buffer (BIO-RAD, 1610737), and boiled at 95°C for 5 min. Then samples were ready for western blotting analysis. Western blotting

[0340] NP-40 / Triton lysis buffer (10% NP40, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH = 8.0) containing proteinase inhibitors and phosphatase inhibitors was used to lyse both cultured cells and frozen brain tissue. Surface protein was extracted using Pierce Cell Surface Protein Isolation Kit (Thermal Fisher Scientific, 89881) in transfected HEK-293T cells, and using ProteoExtract Native Membrane Protein Extraction Kit (Calbiochem, 444810) in brain tissue based on instructions. For transfected cells, proteins were extracted 36 h after transfection. Cell and tissue lysate were lysed by ultrasound using an ultrasonic cleaner filled with ice-cold water for 30 min. After incubated on ice for 1 h, lysate was centrifuged at 13000 g for 30 min and supernatant was collected. Protein concentration was measured using Pierce Rapid Gold BCA Protein Assay Kit.

[0341] 30–50 μg of total protein was loaded and separated proteins were transferred to Nitrocellulose membranes. Membranes were blocked with 5% BSA and 2.5% goat serum in Tris- buffered saline (TBS, pH = 7.4) at room temperature for 2 h, and incubated with primary antibodies in TBS with 0.05% Tween (TBS-T) at room temperature for 2 h. Then membranes were incubated with secondary antibodies in TBS-T for 1 h at room temperature for 1 h for detection. Blots were developed with ImageQuant LAS 4000 imaging system. Band intensity was quantified using ImageJ software43 and normalized with appropriate loading controls. Electrophysiology

[0342] Whole cell currents were recorded using an Axopatch 200B amplifier. Data were digitized at 10 or 20 kHz and digitally filtered offline at 1 kHz. Patch electrodes were pulled fromAttorney Docket No. 98121.00392 borosilicate glass and fire-polished to a resistance of ~3 MΩ when filled with internal solutions. Series resistance (Rs) was compensated up to 90% to reduce series resistance errors to <5 mV. Cells in which Rs was >10 MΩ were discarded.

[0343] For heterologous expression, transfected HEK-293 cells were identified by GFP fluorescence. TRPM2 current recording in transfected HEK-293T cells was performed as we previously reported. TRPM2 and NMDAR currents recordings from cultured neurons were performed using aCSF as extracellular solution as we previously reported. In brief, for TRPM2 current recordings, voltage stimuli lasting 250 ms were delivered at 1-s intervals, with voltage ramps ranging from -100 to +100 mV at holding potential of 0 mV to elicited currents. For NMDAR current recordings, a gap-free protocol at holding potential of -80 mV was applied to elicit NMDA currents upon agonist stimulation. A fast perfusion system was used to exchange extracellular solutions and to deliver agonists and antagonists to the cells, with a complete solution exchange achieved in about 1–3 s.

[0344] Normal Tyrode solution for current recording in HEK-293 cells contained (mM): 145 NaCl, 5 KCl, 2 CaCl2, 10 HEPES, 10 glucose, osmolarity = 290–320 mOsm / Kg, and pH = 7.4 was adjusted with NaOH. Extracellular solution for current recording in neuron, the aCSF solution contained (mM):124 NaCl, 2.5 KCl, 2 MgSO4, 2 CaCl2, 1.2 NaH2PO4, 24 NaHCO3, 5 HEPES, 12.5 glucose, osmolarity = 300–310 mOsm / Kg, with pH = 7.4 adjusted with NaOH. For oxygen- glucose-deprivation (OGD) solution, glucose was eliminated from extracellular solution, and the solution was saturated with nitrogen (N2) bubbling for 30 min before the experiments.

[0345] The internal pipette solution for whole cell current recordings of TRPM2 overexpressed in HEK293 cells contained (in mM): 135 Cs-methanesulfonate (CsSO3CH3), 8 NaCl, 0.5 CaCl2, 1 EGTA, and 10 HEPES, with pH adjusted to 7.2 with CsOH. Free [Ca2+]i buffered by EGTA was ~100 nM calculated using Max chelator. ADPR 1 μM was included in the pipette solution for the minimal basal activation of TRPM2. Ca2+concentration was increased to 1 mM and EGTA was removed, while ADPR concentration was increased to 500 μM to achieve the maximal activation of TRPM2 current. The intracellular solution for NMDAR current recording contained (mM): 110 K-ASP, 20 KCl, 1 MgSO4, 10 mM BAPTA, 0.1 GTP, 5 ATP-Mg2, 10 HEPES, osmolarity = 275– 285 mOsm / Kg, pH = 7.2 adjusted with KOH. For the experiments using cells pretreated with the disrupting peptides TAT-SC and TAT-M2PBM, 1 μM TAT-SC or TAT-M2PBM was included in the pipette solution, and at least 10 min was allowed for achieving intracellular equilibration ofAttorney Docket No. 98121.00392 TAT-SC or TAT-EE3 before current recording. For current recordings in neurons, tetrodotoxin (0.5 μM) was included in the external solution to block voltage-gated Na+current, and 10 μM nifedipine was used to block voltage-gated Ca2+currents for recording TRPM2 currents.

[0346] The above-mentioned pipette solutions and extracellular solutions were specific for TRPM2 or NMDAR current recordings, without “cross-contamination” for each other or from other channel activation. The omitting of ADPR and including high concentration of potent calcium chelator BAPTA in the pipette solution for NMDAR current recordings eliminated any possibility of TRPM2 channel activation because TRPM2 requires Ca2+and ADPR to be activated. Ca2+-free pipette solution for NMDAR current recording also prevented other Ca2+-activated currents such as TRPM4. Moreover, using CsSO3CH3in the pipette solution for TRPM2 current recordings eliminated contamination from any potassium channels for recordings in neurons and in HEK-293 cells. Table 2. Primers for mutagenesis and subcloning, Related to FIGS. 1, 3, and 4 Primer Name Sequence SEQ ID NO TRPM2- CAAGGACATGTAGTTTGTGTCTCACG 30 A2282T-F TRPM2- CGTGAGACACAAACTACATGTCCTTG 31 A2282T-R TRPM2-S11A-F TGAGGAAAGCTGGCGCAGAGCAGGAGGAG 32 TRPM2-S11A-R TCCTCCTGCTCTGCGCCAGCTTTCCTCAG 33 TRPM2-S11D-F TGAGGAAAGCTGGCGAGGAGCAGGAGGAG 34 TRPM2-S11D-R TCCTCCTGCTCCTCGCCAGCTTTCCTCAG 35 TRPM2-S38A-F GGCGCAGCAACGCCAGCCTCTTCAAG 36 TRPM2-S38A-R TCTTGAAGAGGCTGGCGTTGCTGCGC 37 TRPM2-S38D-F GGCGCAGCAACGACAGCCTCTTCAAG 38 TRPM2-S38D-R TCTTGAAGAGGCTGTCGTTGCTGCGC 39 TACCACCTCATGACCCAGCACAAGAACTTCAACAT PBM Deletion-F 40 GAAG TTCATGTTGAAGTTCTTGTGCTGGGTCATGAGGTG PBM Deletion-R 41 GTAG TRPM2- ATATGCGGCCGCCAGGAGATGCCAACCGATGC 42 MHR1 / 2-F TRPM2- ATATGGTACCTCAGTCTTGGCTCCGTGAGGCTTT 43 MHR1 / 2-R PKCγ-C2-F ATATGCGGCCGCCAGAAGGTGGTCCACGAGGT 44 PKCγ-C2-R ATATGGTACCTCAATTGCAGGCCTCAAACTTCTGG 45Attorney Docket No. 98121.00392 Example 2. Results 2.1. PKCγ phosphorylates TRPM2 and enhances TRPM2 activation

[0347] Protein kinase C (PKC) is an important regulator of ion channel activity. Previously, inventors reported that PKCγ binds to TRPM2, but whether this binding influences TRPM2 activity was not clear. As PKCγ is regarded as a neuron specific PKC isoform, the endogenous PKCγ expression was examined in HEK293T cells using Western blot analysis. The results showed that although very low, there was still detectable PKCγ expression in HEK293T cells (FIGS.7A and 7B). Thus, in all the experiments performed in this study, PKCγ was overexpressed. Here, it was found that the PKCγ activator phorbol 12-myristate 13-acetate (PMA) induced the activation of TRPM2 in HEK293T cells expressed with TRPM2 and PKCγ using the internal pipette solution containing 1 µM ADPR and 500 nM Ca2+(FIG. 1A). Moreover, a 3-minute H2O2 preincubation enhanced the PMA-induced TRPM2 activation (FIGS. 1B, 1C), consistent with the previous findings that oxidative stress promotes the binding of PKCγ to TRPM2. However, PMA failed to induce TRPM2 activation in the TRPM2 / EGFP control (FIG.1D) and the TRPM2 / PKCγ- DN (dominate negative kinase dead PKCγ) (FIG. 1E) groups even after H2O2preincubation, suggesting that PMA regulates TRPM2 activation in a PKCγ-dependent manner.

[0348] PKC regulates ion channel functions through direct phosphorylation or influencing the membrane phosphatidylinositol (4,5) bisphosphate level. Mass spectrometry analysis was performed to detect the potential phosphorylation of TRPM2 by PKCγ, and it was found that PMA induced the phosphorylation of TRPM2 at serine 11 and 38 (S11 and S38) (FIGS. 7C and 7D), both of which match conserved PKC consensus phosphorylation motif (RKxxS, RRxxS). Protein sequence alignments shows that S11 and S38 are unique in TRPM2 among all the human TRPM channels (FIG. 8A), and S38 of TRPM2 is highly conserved in TRPM2 of different species (FIG. 8B). To determine whether S11 or S38 phosphorylation is responsible for the activation of TRPM2 by PMA, serine (S) was mutated to alanine (A) at S11 and S38 to abolish their phosphorylation (FIG. 1F), and it was found that the S11A mutation did not influence the activation of TRPM2 by PMA, whereas the S38A mutation eliminated this activation (FIGS. 1G, 1H, 1K). To further confirm the potentiation of S11 / 38 phosphorylation in TRPM2 activation, serine was mutated to aspartate (D) at S11 and S38 to create the “phosphomimetic” mutants (FIG. 1F). It was found thatAttorney Docket No. 98121.00392 the S38D mutation led to the activation of TRPM2 without perfusion of PMA under the same recording condition, which was not seen in the S11D mutation (FIGS.1I-1K). This result strongly indicates that phosphorylation at S38, but not S11, facilitates the activation of TRPM2 when ADPR and Ca2+concentration is minimal and not enough for inducing TRPM2 activity without extra stimulations.

[0349] The activation of TRPM2 requires both Ca2+and ADP ribose (ADPR). Thus, the concentration of Ca2+and ADPR was titrated in the internal recording solution to 500 nM and 1 µM, respectively, to achieve the minimal baseline activation, but the rapid activation of TRPM2 upon PMA treatment (FIG. 1L, left). To examine the maximal activation of TRPM2, the concentration of Ca2+and ADPR was increased in the internal recording solution to 1 mM and 500 µM, respectively (FIG. 1L, middle). The results of averaged current amplitude (original traces were not shown) showed that the PMA treatment enhanced the maximal activation of wild-type TRPM2 (WT-TRPM2), but not the S38A mutant (FIG. 1M). Moreover, PMA did not further increase the activation of the S38D mutant (FIG. 1M), and mutation of S11 to either A or D did not influence the activation of TRPM2 (FIG. 1N). Thus, it appears that S38 phosphorylation by PMA mediates PKCγ-induced TRPM2 activation. As S38 is conserved in TRPM2 from different species (FIG. 8B), the drastic regulation of TRPM2 activation by PMA through S38 phosphorylation site suggests that the S38 of TRPM2 may play an important role in the evolution.

[0350] Then it was determined whether S11 / 38 phosphorylation influence the potentiation of NMDAR currents by TRPM2. To eliminate any potential contamination from the activation of TRPM2 during NMDAR current recording, a potent Ca2+chelator BAPTA was added into the internal pipette recording solution which does not contain any ADPR (FIG. 1L, right). Consistent with inventors’ previous findings, co-expression with TRPM2 increased the amplitude of NMDAR currents (FIGS. 1O, 1P). However, mutation of S11 / 38 to A and D did not affect the potentiation of TRPM2 on the channel activity of NMDAR (FIGS. 1O, 1P), indicating that phosphorylation of TRPM2 by PMA is not required for the functional coupling between TRPM2 and NMDAR under this recording condition. 2.2 TRPM2-mediated Ca2+influx promotes PKCγ activation

[0351] The activation of PKCγ requires Ca2+15, and TRPM2 is permeable to Ca2+16. Considering the strong physical binding between TRPM2 and PKCγ induced under oxidative stressAttorney Docket No. 98121.00392 conditions, while not wishing to be bound by this theory, it is hypothesized that TRPM2 may be an important Ca2+source for PKCγ activation. The C kinase activation reporter (CKAR) is a sensitive sensor which can be used for real-time detection of PKC activity. PKC activation induces conformational changes of the CKAR, which abolishes the basal fluorescence resonance energy transfer (FRET) inside CKAR, as reflected by the reduced FRET intensity and increased CFP / YFP ratio (FIG. 2A). Inventors found that H2O2 perfusion at 100 µM induced a stronger activation of PKCγ in HEK293T cells co-expressed with both TRPM2 and PKCγ compared to PKCγ single- expressed cells (FIGS.2B-2D), indicating that TRPM2 promotes PKCγ activation under oxidative stress conditions. Inventors further assessed the role of TRPM2-mediated Ca2+influx in PKCγ activation (FIG. 2E) and found that the TRPM2 blocker N-(p-amylcinnamoyl) anthranilic acid (ACA) eliminated the increase of PKCγ activation induced by TRPM2 (FIGS.2F-2H). Moreover, buffering of extracellular Ca2+influx using EGTA or chelating of intracellular Ca2+using BAPTA (AM) completely abolished the activation of PKCγ by H2O2 in TRPM2 / PKCγ co-expressed cells (FIGS. 2F-2H). These results indicate that TRPM2-mediated Ca2+influx promotes PKCγ activation under oxidative stress conditions. 2.3. Physical and functional coupling between TRPM2 and PKCγ

[0352] As Ca2+entered from extracellular environment is quickly chelated by endogenous intracellular Ca2+buffering proteins, including parvalbumins, calbindin and calretinin, to effectively supply PKCγ with Ca2+, TRPM2 need to be located in the close proximity of PKCγ. Although inventors have previously found that TRPM2 binds to PKCγ using co- immunoprecipitation, whether the binding between TRPM2 and PKCγ is a direct association or mediated by an adaptor protein remains unclear. Thus, it was decided to locate the binding site at TRPM2 for PKCγ. Inventors found that the amino acid sequence (WGLDVPNLLISVTGGA (SEQ ID NO: 1)) at an area near the N-terminus of TRPM2 highly resembles several known PKCγ binding sequences, including the ones in RACK1 (DIINALC (SEQ ID NO: 2)) and annexin1 (KGDYEKILVALCGGN (SEQ ID NO: 3)) (FIG.3A), which both bind to the C2-domain of PKC. All other TRPM channels, except TRPM8, share a similar consensus sequence (~50%) in this area (FIG. 9A), which is also highly conserved in the TRPM2 from different species (FIG. 9B). Importantly, after deletion of this sequence area, the physical interaction between TRPM2 andAttorney Docket No. 98121.00392 PKCγ was abolished (FIG. 3B), and this deletion did not affect the expression of TRPM2 in HEK293T cells (FIGS. 9C, 9D). This result indicates a critical role of this sequence motif in the physical coupling of PKCγ and TRPM2. Therefore this sequence area was designated as “TRPM2- PKC binding motif” (M2PBM) (FIG. 3C).

[0353] To further determine whether the association between TRPM2 and PKCγ is a directing binding or requires an additional adapter protein, the MHR1 / 2 domain of TRPM2 was purified and the C2 domain of PKCγ and performed in vitro binding experiments as it was previously reported (FIGS. 9E, 9F). After incubation in binding buffer for 24 hours, compared to the negative control of GST protein, the C2 domain of PKCγ (tagged with GST) was used to immunoprecipitate the MHR1 / 2 domain of TRPM2 (tagged with His) (FIG. 3D), indicating that the PBM of TRPM2 can directly associate with the C2 domain of PKCγ.

[0354] Inventors then attempted to determine whether M2PBM is required for the functional coupling between TRPM2 and PKCγ. Whole cell current recording showed that PMA failed to induce the activation of TRPM2 with PBM deletion (TRPM2-ΔPBM) (FIGS. 3E, 3F). Similarly, deletion of PBM eliminated the increase of PKCγ activation by TRPM2 upon H2O2treatment (FIGS. 3G-3I). These results suggest that the functional coupling between TRPM2 and PKCγ depends on their physical association. More importantly, the potentiation of NMDARs by WT- TRPM2 could not be replicated by TRPM2-ΔPBM (FIGS. 3J, 3K), indicating that the functional coupling between TRPM2 and NMDAR requires the direct binding of PKCγ to TRPM2. 2.4. TAT-M2PBM abolishes TRPM2-PKCγ and TRPM2-NMDAR coupling

[0355] To further confirm the role of TRPM2- PKCγ physical binding via M2PBM in the functional coupling of TRPM2- PKCγ as well as the functional coupling of TRPM2-NMDAR, a membrane permeable peptide TAT-M2PBM was synthesized and used it as a tool to dissociate the binding between TRPM2 and PKCγ (FIG. 4A). It was found that TAT-M2PBM incubation at 1 µM for 2 hours effectively prevented pulling-down of PKCγ by anti-TRPM2, indicating that TAT- M2PBM inhibited the TRPM2-PKCγ interaction (FIG. 4B). Importantly, when used at 1 µM, TAT-M2PBM did not significantly affect PKCγ activity in vitro (FIG. 9G). Moreover, the PMA- induced activation of TRPM2 current in PKCγ / TRPM2 co-expressed cells (FIGS.4C, 4D), as well as the enhancement of PKCγ activity by TRPM2 during H2O2perfusion (FIGS. 4E, 4F), wereAttorney Docket No. 98121.00392 abolished by TAT-M2PBM, indicating that TAT-M2PBM is effective in disrupting the functional coupling of TRPM2- PKCγ. Furthermore, TAT-M2PBM suppressed TRPM2-induced potentiation of NMDAR currents (FIGS.4G, 4H). These results validated a critical role of M2PBM in TRPM2- PKCγ coupling, and the indispensable role of physical TRPM2-PKCγ binding in functional coupling of TRPM2- PKCγ as well as TRPM2-NMDARs.

[0356] Next was examined the effect of TAT-M2PBM on TRPM2-NMDAR coupling in cortical neurons isolated from wild-type (WT) and Trpm2 deletion (M2KO) mice. Compared with WT neurons, smaller NMDAR current was recorded in M2KO neurons as it was observed before (FIGS. 4I, 4J). Importantly, TAT-M2PBM preincubation effectively inhibited NMDAR current amplitude in WT neurons to a level similar to that in M2KO neurons (FIGS. 4I, 4J). Consistent with current recording results obtained in neurons, the increase of NMDAR’s surface expression induced by co-expression with TRPM2 and PKCγ in HEK293 cells was inhibited by TAT- M2PBM preincubation (FIGS. 4K, 4L).

[0357] Since TRPM2-mediated Ca2+influx is a strong promotor of PKCγ activation (FIG. 2), and PKCγ is an important activator of NMDAR, it was evaluated whether TAT-M2PBM affects the regulation of NMDAR activity by PKCγ. A 60-second perfusion of PMA markedly increased NMDAR current amplitude in WT neurons, but not in M2KO neurons (FIGS. 4M, left; 4N, and 4O). Similarly, it was found that TAT-M2PBM effectively inhibited the enhanced NMDAR activity by PMA in WT neurons, and produced a similar effect as TRPM2 knockout (FIGS. 4M, right; 4N, and 4O), suggesting that TRPM2-mediated Ca2+influx promotes PKCγ activation, which subsequently enhances NMDAR activity. 2.5. TRPM2-PKCγuncoupling protects neurons against OGD

[0358] As uncoupling of TRPM2-PKCγ by TAT-M2PBM inhibited the potentiation of NMDARs by TRPM2, it was reasoned that TAT-M2PBM may protect against NMDAR-mediated excitotoxicity. Thus, it was examined whether TAT-M2PBM attenuates oxygen-glucose deprivation (OGD)-induced ischemic neuronal injury.

[0359] As Ca2+overload is a critical cause of ischemic neuronal death, real-time ratio Ca2+imaging was used for detecting Ca2+overload during OGD perfusion. It was found that TAT- M2PBM preincubation for 2 hours at 1 µM effectively inhibited the increase of intracellular Ca2+Attorney Docket No. 98121.00392 concentration (FIGS.5A-5C) as well as neuronal death (FIG.5D) during OGD, a potent protective effect which was similar to that produced by NMDAR antagonists MK801 / AP5 and M2KO (FIGS. 5A-5D), indicating that Ca2+overload as well as NMDAR-mediated excitotoxicity was inhibited by TAT-M2PBM. As TRPM2 is an oxidative stress activated ion channel, and the phosphorylation by PKCγ may enhance the activity of TRPM2 under ischemic conditions, it was also evaluated whether TAT-M2PBM protects H2O2-induced neuronal death. It was found that when perfused continuously at 100 μM, H2O2induced rapid Ca2+overload and neuronal death in WT cortical neurons treated with TAT-SC, while the incubation of TAT-M2PBM effectively attenuated this cytotoxic effect (FIGS. 10A-10D). This result shows that the PKC phosphorylation may be an important contributor for TRPM2 activation under oxidative stress conditions.

[0360] Since nitric oxide (NO) production caused by energy imbalance during ischemic stroke is an important promoter for neuronal death, NO production was measured using DAF-FM in neurons subjected to OGD. It was found that OGD caused a substantial increase of NO concentration in WT neurons, which was inhibited by TAT-M2PBM, MK801 / AP5, and M2KO (FIGS. 5E, 5F), suggesting that TAT-M2PBM attenuated the NO toxicity caused by OGD in WT neurons. As reactive oxygen species (ROS) is an initiator and mediator of ischemic neuronal death, MitoSOX was used to evaluate ROS generation in neurons during OGD. It was found that the increase of MitoSOX in WT neurons induced by OGD was inhibited by TAT-M2PBM, MK801 / AP5 and M2KO (FIGS. 5G, 5H), indicating that ROS production was attenuated by uncoupling of TRPM2 and PKCγ. As shutting down the energy supply caused by mitochondrial dysfunction is an early sign of neuronal death during ischemic stroke, mitochondria function was evaluated using Rhodamine-123 (R123), a heavily positively charged dye which becomes self- dequenched upon attracted to mitochondria, and get released from depolarized mitochondria during mitochondrial stress. It was observed that OGD induced a marked increase of R123 florescence in WT neurons, but not in WT neurons treated with TAT-M2PBM or MK801 / AP5, or in M2KO neurons (FIGS. 5I, 5J), indicating that mitochondrial function was preserved by TAT- M2PBM.

[0361] In summary, the above data indicate that uncoupling of TRPM2-PKCγ attenuates ischemic neuronal dysfunction and death. It is noteworthy to mention that TAT-M2PBM did not produce any additional protective effect in neurons from M2KO mice, indicating the specificity of TAT-M2PBM in disrupting TRPM2-PKCγ association.Attorney Docket No. 98121.00392 2.6. TAT-M2PBM attenuates ischemic stroke

[0362] After confirming the protective effect of TRPM2-PKCγ uncoupling against ischemic neuronal death in vitro, inventors sought to evaluate the therapeutic value of TAT-M2PBM using transient middle cerebral occlusion (tMCAO) in mice. In order to determine the administration dosage and frequency, inventors first examined the in vivo disruption efficiency of TAT-M2PBM. As shown in FIG, 6A, TAT-M2PBM eliminated the physical association between TRPM2 and PKCγ in the mouse brain as early as 2 hours after the intraperitoneal (i.p.) injection at a dose of 100 nmol / kg, and this disruption can be maintained for at least 12 hours (FIG. 6A). Importantly, TAT-M2PBM did not influence the binding between TRPM2 and NMDARs (FIG.6A). Therefore the administration strategy was designed as shown in FIG. 6B to mimic the clinical setting: an injection was given 2 hours after MCAO and right before the reperfusion, and then after 12 hours, another i.p. injection was given to maintain the disruption until the harvest of brains 24 hours after reperfusion (FIG. 6B). It was found that TAT-M2PBM reduced the infarction size and prevented the impairment of neurological deficient (ND) score 24 hours after MCAO reperfusion in WT mice, but did not produce any additional protective effect in M2KO mice (FIGS. 6C, 6D). Moreover, the increased surface expression of NMDARs in WT mice after MCAO was inhibited by TAT-M2PBM (FIGS. 11A, 11B). Furthermore, the in vivo protective effect of TAT-M2PBM was compared with previously reported TAT-EE3, which functions by dissociating TRPM2- NMDAR complex. The results showed that compared to TAT-EE3, TAT-M2PBM treatment reduced brain damage (FIG. 6E and FIG. 6F, left panel), although there was no difference in ND score between two groups (FIG. 6F, right panel). As NMDAR is only expressed in neuronal cells, this data suggests that TRPM2-PKCγ uncoupling may produce a broader protective effect by attenuating ischemia-induced dysfunction and damage in other cell types. The long-term protective effects of TAT-M2PBM was then examined and found that TAT-M2PBM effectively reduced brain injury and preserved brain function (also reflected by rotarod test) 7 days after MCAO (Fig. 6H-6K). The above results indicate that TRPM2-PKCγ coupling plays a pivotal role in mediating NMDARs' excitotoxicity and neuronal death during ischemic stroke.

[0363] In conclusion, during ischemic stroke, ROS promotes the binding of PKCγ to TRPM2- NMDAR complex, which leads to phosphorylation and activation of TRPM2, and enhanced NMDARs’ activity. Meanwhile, TRPM2-mediated Ca2+influx enhances the activation of PKCγ,Attorney Docket No. 98121.00392 which further increases NMDAR-mediated excitotoxicity, neuronal death and brain injury (FIG. 6G, left). In contrast, TAT-M2PBM selectively inhibits the binding of PKCγ to TRPM2, resulting in an effective suppression of the potentiated NMDAR activity and producing a potent protective effect against neuronal death and ischemic brain injury (FIG. 6G, right). This study suggests that uncoupling of TRPM2-PKCγ interaction represents a promising therapeutic strategy for screening effective treatments for ischemic stroke. 2.7. Discussion

[0364] Decades after the demonstration of the critical role of glutamate excitotoxicity in ischemic stroke, there is still no therapy available for attenuating ischemic brain injury through targeting the culprit of excitotoxicity, NMDARs. One of the significant challenges for developing neuroprotectants by targeting NMDARs is to selectively inhibit esNMDARs, which is almost mission impossible using pharmacological approaches. Here, inventors show that, functional coupling of TRPM2-PKCγ is necessary for NMDAR-mediated excitotoxicity in ischemic stroke. More importantly, inventors demonstrate that, a peptide / polypeptide such as TAT-M2PBM, a cell- permeable peptide that disrupts TRPM2-PKCγ coupling, markedly inhibits es-NMDAR mediated excitotoxicity and exhibits potent protective effect against ischemic brain injury. This study provides proof-of-concept that, approaches which keep the associating proteins away from esNMDARs, represent a promising new direction for developing therapeutics for ischemic stroke. TRPM2 and PKCγ functional coupling is necessary for TRPM2-NMDAR coupling mediated excitotoxicity

[0365] Using mass spectrometry analysis, inventors identified the PKCγ phosphorylation sites in TRPM2, and found that PKCγ-mediated phosphorylation regulates TRPM2 channel activity. Phosphorylation of TRPM2 has been shown in previous studies. The first evidence of phosphorylation-mediated regulation of TRPM2 was that tyrosine phosphatase PTPL1 inhibited the phosphorylation of TRPM2 and reduces the increase of intracellular Ca2+concentration following a 5-minute H2O2treatment. In another study, H2O2treatment was shown to increase the phosphorylation of the TRPM2 short variant (TRPM2-S), but not the full length TRPM2, at S39 in endothelial cells, which can be abolished by a specific PKCα inhibitor. It remains unknown why the full-length TRPM2 was not phosphorylated since the TRPM2-S and TRPM2 share the identicalAttorney Docket No. 98121.00392 amino acid sequence near the phosphorylation site. As both previous studies used Ca2+imaging to assess TRPM2 channel activity changes by PKC phosphorylation, inventors applied current recordings of TRPM2 to directly evaluate how phosphorylation of TRPM2 regulate channel activity. It was found that PKCγ-induced phosphorylation, as well as phosphomemetics mutation of TRPM2, drastically potentiated TRPM2 channel activity, suggesting a functional significance of phosphorylation of TRPM2 by PKCγ under physiological or pathological conditions.

[0366] Using mutagenesis approaches, a recent study demonstrated that treatment of PKC activator PMA at 100 nM for 1 hour to 6 hours increased the phosphorylation of TRPM2 at T738, which led to inhibited TRPM2 activation and elevated the temperature threshold for TRPM2 activation. The T738 phosphorylation site was not identified using Mass Spec. In experiments disclosed herein, inventors used a 5-minute PMA treatment to induce phosphorylation as phosphorylation happens within minutes, whereas Kasshio and colleagues induced phosphorylation for 1 to 6 hours by 100 nM PMA. The different duration of PMA treatment could have contributed to the discrepancy of phosphorylation status at T738 in our and their studies (Kashio, M., et al., (2022). Protein kinase C-mediated phosphorylation of transient receptor potential melastatin type 2 Thr738 counteracts the effect of cytosolic Ca(2+) and elevates the temperature threshold. J Physiol 600, 4287-4302). Nonetheless, the potentiation of TRPM2 activity by PKC phosphorylation detected by current recordings in this study is supported by the previous report which showed enhanced TRPM2-mediated Ca2+influx by phosphorylation (Zhang, W., et al., (2007). Regulation of TRP channel TRPM2 by the tyrosine phosphatase PTPL1. Am J Physiol Cell Physiol 292, C1746-1758).

[0367] The activation of PKCγ during ischemic stroke and the potentiation of NMDARs directly by PKCγ have been well documented. During early ischemic injury, PKCγ can be quickly activated and regulates NMDAR activity by directly phosphorylating NMDARs, thereby increasing neuronal death. Previous data generated by inventors suggest that PKCγ recruited to the TRPM2-NMDAR complex may be necessary for TRPM2-mediated increase of NMDAR activity during ischemic stroke. Here, it was found that the physical interaction of PKCγ with TRPM2 is not only required for PKCγ induced potentiation of TRPM2 activity, but also lead to enhanced activity of PKCγ, as the close vicinity of PKCγ to TRPM2 brings itself a reliable Ca2+source for its own activation. Results disclosed herein suggest that during ischemic stroke, uncoupling of TRPM2-PKCγ association will inhibit functional coupling of esNMDAR-TRPM2 and diminishAttorney Docket No. 98121.00392 the esNMDAR-mediated excitotoxicity. Uncoupling of TRPM2 and PKCγ is sufficient to attenuate excitotoxicity in ischemic stroke

[0368] Inventors identified a unique PKCγ binding motif (M2PBM) in the MHR1 domain of the N tail of TRPM2, which particularly resembles the sequence motifs in RACK1 and annexin1 for binding to the C2 domain of PKC. This could explain why an increased binding of PKCγ to TRPM2 was observed after H2O2treatment and ischemic stroke: activation of PKCγ during oxidative stress exposes the C2 domain and allows its binding to TRPM2, which may “lock” the PKCγ in the activated state. Conventional PKCs (α, β and γ) share highly conserved sequences, suggesting that PKCα, PKCβ may also interact with TRPM2 through the same binding motif M2PBM. However, this will not cause non-specific or off-target effects in the peripheral tissues, since TRPM2-NMDARs mediated excitotoxicity only happens in neuronal cells where NMDARs are localized.

[0369] The important discovery of this study is the effects of disrupting peptide such as TAT- M2PBM which effectively interferes PKCγ-TRPM2 coupling as well as TRPM2-NMDAR functional coupling, attenuates excitotoxicity, and produces potent protective effects against ischemic stroke in vitro and in vivo. A New strategy in targeting NMDAR for treatment of ischemic stroke

[0370] NMDAR-mediated glutamate excitotoxicity is the culprit of ischemic neuronal death. One of the major reasons for the past failures of NMDAR antagonists in the treatment of clinical ischemic stroke is their non-selective blockade on both the “beneficial” sNMDAR and the “detrimental” esNMDAR3. Selectively inhibiting the neurotoxic effects of esNMDAR without impairing the critical physiological and pro-survival functions of sNMDAR represents a new strategy of developing more effective NMDAR antagonists for neuronprotectants to treat ischemic stroke. However, sNMDAR and esNMDAR share similar components and structures, and current pharmacological technology is not able to develop a NMDAR antagonist that is selective for esNMDARs. Thus, targeting the “accomplices” of esNMDAR which specifically enhance esNMDAR activity during ischemic stroke is an innovative way to achieve selective inhibition on the excitotoxic esNMDAR.

[0371] Inventors’ previous findings show that TRPM2 is only coupled with NMDARs at theAttorney Docket No. 98121.00392 extrasynaptic sites and specifically enhances the activity of esNMDAR but not sNMDAR. In this study, the results present a functional TRPM2-PKCγ-esNMDAR complex at extrasynaptic sites in ischemic injury (Fig.6G). During ischemic stroke, oxidative stress activates TRPM2 and promotes the binding of PKCγ to TRPM2, leading to enhanced TRPM2 channel activity and TRPM2- mediated Ca2+influx, which in turn becomes a reliable Ca2+source for PKCγ activation. PKCγ phosphorylation and activation of TRPM2 and subsequent activation of PKCγ by TRPM2- mediated Ca2+form a positive feedback loop to perpetuate PKCγ activation. Consequentially, by directly associating with NMDARs, TRPM2 serves as a scaffold to recruit the activated PKCγ to its substrate, NMDARs, in the TRPM2-PKCγ-esNMDARs complex, leading to substantial potentiation of esNMDAR-mediated excitotoxicity (Fig. 6G). Interfering peptide TAT-M2PBM effectively disrupts TRPM2-PKCγ coupling, thereby protecting mice against ischemic stroke.

[0372] In summary, the results disclosed herein provide a proof of concept that targeting TRPM2-PKCγ-esNMDAR complex by keeping the “accomplices” apart is a promising new direction to develop better therapeutics for ischemic stroke. This study also sheds light on future strategies of mitigating neuronal injury by selectively inhibiting the detrimental activity of es- NMDARs in various neurodegenerative diseases, including but not limited to ischemic stroke and hemorrhagic stroke, traumatic brain injury, and neurodegeneration such as Alzheimer’s disease. Example 3. TAT-M2PBM for treatment of vascular disorders.

[0373] Inventors sought to evaluate the therapeutic value of TAT-M2PBM in treatment of vascular disorders. Primary vascular smooth muscle cells (VSMCs) were isolated from the mouse abdominal aorta. After 7 days of culture, Ang II (10 ng / µL) was applied to induce reprogramming. During the Ang II incubation, interfering peptides, either TAT-SC or TAT-M2PBM, were included at a concentration of 1 µM. Disruption of TRPM2-PKC binding by TAT-M2PBM inhibited Ang II-induced loss of contractile proteins α-SMA and SM22α in VSMCs (FIGS.12A-12C), indicating the protection against VSMC reprogramming. Considering VSMC reprogramming is critical in the progression of many cardiovascular diseases, such as atherosclerosis, aneurysm, hypertension, diabetic angiopathy, e.g., TAT-M2PBM can also have promising therapeutic value in these diseases.Attorney Docket No. 98121.00392 Other Embodiments

[0374] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.Attorney Docket No. 98121.00392 SEQUENCES SEQ ID NO: 21; >NM_003307.4 Homo sapiens transient receptor potential cation channel subfamily M member 2 (TRPM2), transcript variant 1, mRNA AGAACCCCAGTGTAGCGAGCTGGAGAGAGGACTGTCCTGAGGGCAGCAGGCCTGGTTGCAGCTG GCGTGGGGGTCTCAGAATGGAGCCCTCAGCCCTGAGGAAAGCTGGCTCGGAGCAGGAGGAGGGC TTTGAGGGGCTGCCCAGAAGGGTCACTGACCTGGGGATGGTCTCCAATCTCCGGCGCAGCAACA GCAGCCTCTTCAAGAGCTGGAGGCTACAGTGCCCCTTCGGCAACAATGACAAGCAAGAAAGCCT CAGTTCGTGGATTCCTGAAAACATCAAGAAGAAAGAATGCGTGTATTTTGTGGAAAGTTCCAAA CTGTCTGATGCTGGGAAGGTGGTGTGTCAGTGTGGCTACACGCATGAGCAGCACTTGGAGGAGG CTACCAAGCCCCACACCTTCCAGGGCACACAGTGGGACCCAAAGAAACATGTCCAGGAGATGCC AACCGATGCCTTTGGCGACATCGTCTTCACGGGCCTGAGCCAGAAGGTGAAAAAGTACGTCCGA GTCTCCCAGGACACGCCCTCCAGCGTGATCTACCACCTCATGACCCAGCACTGGGGGCTGGACG TCCCCAATCTCTTGATCTCGGTGACCGGGGGGGCCAAGAACTTCAACATGAAGCCGCGGCTGAA GAGCATTTTCCGCAGAGGCCTGGTCAAGGTGGCTCAGACCACAGGGGCCTGGATCATCACAGGG GGGTCCCACACCGGCGTCATGAAGCAGGTAGGCGAGGCGGTGCGGGACTTCAGCCTGAGCAGCA GCTACAAGGAAGGCGAGCTCATCACCATCGGAGTCGCCACCTGGGGCACTGTCCACCGCCGCGA GGGCCTGATCCATCCCACGGGCAGCTTCCCCGCCGAGTACATACTGGATGAGGATGGCCAAGGG AACCTGACCTGCCTAGACAGCAACCACTCTCACTTCATCCTCGTGGACGACGGGACCCACGGCC AGTACGGGGTGGAGATTCCTCTGAGGACCAGGCTGGAGAAGTTCATATCGGAGCAGACCAAGGA AAGAGGAGGTGTGGCCATCAAGATCCCCATCGTGTGCGTGGTGCTGGAGGGCGGCCCGGGCACG TTGCACACCATCGACAACGCCACCACCAACGGCACCCCCTGTGTGGTTGTGGAGGGCTCGGGCC GCGTGGCCGACGTCATTGCCCAGGTGGCCAACCTGCCTGTCTCGGACATCACTATCTCCCTGAT CCAGCAGAAACTGAGCGTGTTCTTCCAGGAGATGTTTGAGACCTTCACGGAAAGCAGGATTGTC GAGTGGACCAAAAAGATCCAAGATATCGTCCGGAGGCGGCAGCTGCTGACTGTCTTCCGGGAAG GCAAGGATGGTCAGCAGGACGTGGATGTGGCCATCTTGCAGGCCTTGCTGAAAGCCTCACGGAG CCAAGACCACTTTGGCCACGAGAACTGGGACCACCAGCTGAAACTGGCAGTGGCATGGAATCGC GTGGACATTGCCCGCAGTGAGATCTTCATGGATGAGTGGCAGTGGAAGCCTTCAGATCTGCACC CCACGATGACAGCTGCACTCATCTCCAACAAGCCTGAGTTTGTGAAGCTCTTCCTGGAGAACGG GGTGCAGCTGAAGGAGTTTGTCACCTGGGACACCTTGCTCTACCTGTACGAGAACCTGGACCCC TCCTGCCTGTTCCACAGCAAGCTGCAGAAGGTGCTGGTGGAGGATCCCGAGCGCCCGGCTTGCG CGCCCGCGGCGCCCCGCCTGCAGATGCACCACGTGGCCCAGGTGCTGCGGGAGCTGCTGGGGGA CTTCACGCAGCCGCTTTATCCCCGGCCCCGGCACAACGACCGGCTGCGGCTCCTGCTGCCCGTT CCCCACGTCAAGCTCAACGTGCAGGGAGTGAGCCTCCGGTCCCTCTACAAGCGTTCCTCAGGCC ATGTGACCTTCACCATGGACCCCATCCGTGACCTTCTCATTTGGGCCATTGTCCAGAACCGTCG GGAGCTGGCAGGAATCATCTGGGCTCAGAGCCAGGACTGCATCGCAGCGGCCTTGGCCTGCAGC AAGATCCTGAAGGAACTGTCCAAGGAGGAGGAGGACACGGACAGCTCGGAGGAGATGCTGGCGC TGGCGGAGGAGTATGAGCACAGAGCCATCGGGGTCTTCACCGAGTGCTACCGGAAGGACGAAGA GAGAGCCCAGAAACTGCTCACCCGCGTGTCCGAGGCCTGGGGGAAGACCACCTGCCTGCAGCTC GCCCTGGAGGCCAAGGACATGAAGTTTGTGTCTCACGGGGGCATCCAGGCCTTCCTGACCAAGG TGTGGTGGGGCCAGCTCTCCGTGGACAATGGGCTGTGGCGTGTGACCCTGTGCATGCTGGCCTT CCCGCTGCTCCTCACCGGCCTCATCTCCTTCAGGGAGAAGAGGCTGCAGGATGTGGGCACCCCC GCGGCCCGCGCCCGTGCCTTCTTCACCGCACCCGTGGTGGTCTTCCACCTGAACATCCTCTCCT ACTTCGCCTTCCTCTGCCTGTTCGCCTACGTGCTCATGGTGGACTTCCAGCCTGTGCCCTCCTGAttorney Docket No. 98121.00392 GTGCGAGTGTGCCATCTACCTCTGGCTCTTCTCCTTGGTGTGCGAGGAGATGCGGCAGCTCTTC TATGACCCTGACGAGTGCGGGCTGATGAAGAAGGCAGCCTTGTACTTCAGTGACTTCTGGAATA AGCTGGACGTCGGCGCAATCTTGCTCTTCGTGGCAGGGCTGACCTGCAGGCTCATCCCGGCGAC GCTGTACCCCGGGCGCGTCATCCTCTCTCTGGACTTCATCCTGTTCTGCCTCCGGCTCATGCAC ATTTTTACCATCAGTAAGACGCTGGGGCCCAAGATCATCATTGTGAAGCGGATGATGAAGGACG TCTTCTTCTTCCTCTTCCTGCTGGCTGTGTGGGTGGTGTCCTTCGGGGTGGCCAAGCAGGCCAT CCTCATCCACAACGAGCGCCGGGTGGACTGGCTGTTCCGAGGGGCCGTCTACCACTCCTACCTC ACCATCTTCGGGCAGATCCCGGGCTACATCGACGGTGTGAACTTCAACCCGGAGCACTGCAGCC CCAATGGCACCGACCCCTACAAGCCTAAGTGCCCCGAGAGCGACGCGACGCAGCAGAGGCCGGC CTTCCCTGAGTGGCTGACGGTCCTCCTACTCTGCCTCTACCTGCTCTTCACCAACATCCTGCTG CTCAACCTCCTCATCGCCATGTTCAACTACACCTTCCAGCAGGTGCAGGAGCACACGGACCAGA TTTGGAAGTTCCAGCGCCATGACCTGATCGAGGAGTACCACGGCCGCCCCGCCGCGCCGCCCCC CTTCATCCTCCTCAGCCACCTGCAGCTCTTCATCAAGAGGGTGGTCCTGAAGACTCCGGCCAAG AGGCACAAGCAGCTCAAGAACAAGCTGGAGAAGAACGAGGAGGCGGCCCTGCTATCCTGGGAGA TCTACCTGAAGGAGAACTACCTCCAGAACCGACAGTTCCAGCAAAAGCAGCGGCCCGAGCAGAA GATCGAGGACATCAGCAATAAGGTTGACGCCATGGTGGACCTGCTGGACCTGGACCCACTGAAG AGGTCGGGCTCCATGGAGCAGAGGTTGGCCTCCCTGGAGGAGCAGGTGGCCCAGACAGCCCAAG CCCTGCACTGGATCGTGAGGACGCTGCGGGCCAGCGGCTTCAGCTCGGAGGCGGACGTCCCCAC TCTGGCCTCCCAGAAGGCCGCGGAGGAGCCGGATGCTGAGCCGGGAGGCAGGAAGAAGACGGAG GAGCCGGGCGACAGCTACCACGTGAATGCCCGGCACCTCCTCTACCCCAACTGCCCTGTCACGC GCTTCCCCGTGCCCAACGAGAAGGTGCCCTGGGAGACGGAGTTCCTGATCTATGACCCACCCTT TTACACGGCAGAGAGGAAGGACGCGGCCGCCATGGACCCCATGGGAGACACCCTGGAGCCACTG TCCACGATCCAGTACAACGTGGTGGATGGCCTGAGGGACCGCCGGAGCTTCCACGGGCCGTACA CAGTGCAGGCCGGGTTGCCCCTGAACCCCATGGGCCGCACAGGACTGCGTGGGCGCGGGAGCCT CAGCTGCTTCGGACCCAACCACACGCTGTACCCCATGGTCACGCGGTGGAGGCGGAACGAGGAT GGAGCCATCTGCAGGAAGAGCATAAAGAAGATGCTGGAAGTGCTGGTGGTGAAGCTCCCTCTCT CCGAGCACTGGGCCCTGCCTGGGGGCTCCCGGGAGCCAGGGGAGATGCTACCTCGGAAGCTGAA GCGGATCCTCCGGCAGGAGCACTGGCCGTCTTTTGAAAACTTGCTGAAGTGCGGCATGGAGGTG TACAAAGGCTACATGGATGACCCGAGGAACACGGACAATGCCTGGATCGAGACGGTGGCCGTCA GCGTCCACTTCCAGGACCAGAATGACGTGGAGCTGAACAGGCTGAACTCTAACCTGCACGCCTG CGACTCGGGGGCCTCCATCCGATGGCAGGTGGTGGACAGGCGCATCCCACTCTATGCGAACCAC AAGACCCTCCTCCAGAAGGCAGCCGCTGAGTTCGGGGCTCACTACTGACTGTGCCCTCAGGCTG GGCGGCTCCAGTCCATAGACGTTCCCCCCAGAAACCAGGGCTTCTCTCTCCTGAGCCTGGCCAG GACTCAGGCTGTTCCTGGGCCCTGCACATGATGGGGTTTGGTGGACCCAGTGCCCCTCACGGCT GCCGCAAGTCTGCTGCAGATGACCTCATGAACTGGAAGGGGTCAAGGTGACCCGGGAGGAGAGC TCAAGACAGGGCACAGGCTACTCAGAGCTGAGGGGCCCCTGGGACCCTTGGCCATCAGGCGAGG GGCTGGGCCTGTGCAGCTGGGCCCTTGGCCAGAGTCCACTCCCTTCCTGGCTGTGTCACCCCGA GCAGCTCATCCACCATGGAGGTCATTGGCCTGAGGCAAGTTCCCCGGAGAGTCGGGGTCCCCTG TGGCCCCCTCAGGCCTATGTCTGTGAGGAAGGGGCCCTGCCACTCTCCCCAAGAGGGCCTCCAT GTTTCGAGGTGCCTCAACATGGAGCCTTGCCTGGCCTGGGCTAGGGGCACTGTCTGAACTCCTG ACTGTCAGGATAAACTCCGTGGGGGTACAGGAGCCCAGACAAAGCCCAGGCCTGTCAAGAGACG CAGAGGGCCCCTGCCAGGGTTGGCCCCAGGGACCCTGGGACGAGGCTGCAGAAGCTCTCCCTCC CTACTCCCTGGGAGCCACGTGCTGGCCATGTGGCCAGGGACGGCATGAGCAGGAGGCGGGGACG TGGGGGCCTTCTGGTTTGGTGTCAACAGCTCACAGGAGCGTGAACCATGAGGGCCCTCAGGAGG GGAACGTGGTAAAACCCAAGACATTAAATCTGCCATCTCAGGCAttorney Docket No. 98121.00392 SEQ ID NO: 22; >NM_001320350.2 Homo sapiens transient receptor potential cation channel subfamily M member 2 (TRPM2), transcript variant 3, mRNA AGAACCCCAGTGTAGCGAGCTGGAGAGAGGACTGTCCTGAGGGCAGCAGGCCTGGTTGCAGCTG GCGTGGGGGTCTCAGAATGGAGCCCTCAGCCCTGAGGAAAGCTGGCTCGGAGCAGGAGGAGGGC TTTGAGGGGCTGCCCAGAAGGGTCACTGACCTGGGGATGGTCTCCAATCTCCGGCGCAGCAACA GCAGCCTCTTCAAGAGCTGGAGGCTACAGTGCCCCTTCGGCAACAATGACAAGCAAGAAAGCCT CAGTTCGTGGATTCCTGAAAACATCAAGAAGAAAGAATGCGTGTATTTTGTGGAAAGTTCCAAA CTGTCTGATGCTGGGAAGGTGGTGTGTCAGTGTGGCTACACGCATGAGCAGCACTTGGAGGAGG CTACCAAGCCCCACACCTTCCAGGGCACACAGTGGGACCCAAAGAAACATGTCCAGGAGATGCC AACCGATGCCTTTGGCGACATCGTCTTCACGGGCCTGAGCCAGAAGGTGAAAAAGTACGTCCGA GTCTCCCAGGACACGCCCTCCAGCGTGATCTACCACCTCATGACCCAGCACTGGGGGCTGGACG TCCCCAATCTCTTGATCTCGGTGACCGGGGGGGCCAAGAACTTCAACATGAAGCCGCGGCTGAA GAGCATTTTCCGCAGAGGCCTGGTCAAGGTGGCTCAGACCACAGGGGCCTGGATCATCACAGGG GGGTCCCACACCGGCGTCATGAAGCAGGTAGGCGAGGCGGTGCGGGACTTCAGCCTGAGCAGCA GCTACAAGGAAGGCGAGCTCATCACCATCGGAGTCGCCACCTGGGGCACTGTCCACCGCCGCGA GGGCCTGATCCATCCCACGGGCAGCTTCCCCGCCGAGTACATACTGGATGAGGATGGCCAAGGG AACCTGACCTGCCTAGACAGCAACCACTCTCACTTCATCCTCGTGGACGACGGGACCCACGGCC AGTACGGGGTGGAGATTCCTCTGAGGACCAGGCTGGAGAAGTTCATATCGGAGCAGACCAAGGA AAGAGGAGGTGTGGCCATCAAGATCCCCATCGTGTGCGTGGTGCTGGAGGGCGGCCCGGGCACG TTGCACACCATCGACAACGCCACCACCAACGGCACCCCCTGTGTGGTTGTGGAGGGCTCGGGCC GCGTGGCCGACGTCATTGCCCAGGTGGCCAACCTGCCTGTCTCGGACATCACTATCTCCCTGAT CCAGCAGAAACTGAGCGTGTTCTTCCAGGAGATGTTTGAGACCTTCACGGAAAGCAGGATTGTC GAGTGGACCAAAAAGATCCAAGATATCGTCCGGAGGCGGCAGCTGCTGACTGTCTTCCGGGAAG GCAAGGATGGTCAGCAGGACGTGGATGTGGCCATCTTGCAGGCCTTGCTGAAAGCCTCACGGAG CCAAGACCACTTTGGCCACGAGAACTGGGACCACCAGCTGAAACTGGCAGTGGCATGGAATCGC GTGGACATTGCCCGCAGTGAGATCTTCATGGATGAGTGGCAGTGGAAGCCTTCAGATCTGCACC CCACGATGACAGCTGCACTCATCTCCAACAAGCCTGAGTTTGTGAAGCTCTTCCTGGAGAACGG GGTGCAGCTGAAGGAGTTTGTCACCTGGGACACCTTGCTCTACCTGTACGAGAACCTGGACCCC TCCTGCCTGTTCCACAGCAAGCTGCAGAAGGTGCTGGTGGAGGATCCCGAGCGCCCGGCTTGCG CGCCCGCGGCGCCCCGCCTGCAGATGCACCACGTGGCCCAGGTGCTGCGGGAGCTGCTGGGGGA CTTCACGCAGCCGCTTTATCCCCGGCCCCGGCACAACGACCGGCTGCGGCTCCTGCTGCCCGTT CCCCACGTCAAGCTCAACGTGCAGGGAGTGAGCCTCCGGTCCCTCTACAAGCGTTCCTCAGGCC ATGTGACCTTCACCATGGACCCCATCCGTGACCTTCTCATTTGGGCCATTGTCCAGAACCGTCG GGAGCTGGCAGGAATCATCTGGGCTCAGAGCCAGGACTGCATCGCAGCGGCCTTGGCCTGCAGC AAGATCCTGAAGGAACTGTCCAAGGAGGAGGAGGACACGGACAGCTCGGAGGAGATGCTGGCGC TGGCGGAGGAGTATGAGCACAGAGCCATCGGGGTCTTCACCGAGTGCTACCGGAAGGACGAAGA GAGAGCCCAGAAACTGCTCACCCGCGTGTCCGAGGCCTGGGGGAAGACCACCTGCCTGCAGCTC GCCCTGGAGGCCAAGGACATGAAGTTTGTGTCTCACGGGGGCATCCAGGCCTTCCTGACCAAGG TGTGGTGGGGCCAGCTCTCCGTGGACAATGGGCTGTGGCGTGTGACCCTGTGCATGCTGGCCTT CCCGCTGCTCCTCACCGGCCTCATCTCCTTCAGGGAGAAGAGGCTGCAGGATGTGGGCACCCCC GCGGCCCGCGCCCGTGCCTTCTTCACCGCACCCGTGGTGGTCTTCCACCTGAACATCCTCTCCT ACTTCGCCTTCCTCTGCCTGTTCGCCTACGTGCTCATGGTGGACTTCCAGCCTGTGCCCTCCTG GTGCGAGTGTGCCATCTACCTCTGGCTCTTCTCCTTGGTGTGCGAGGAGATGCGGCAGCTCTTCAttorney Docket No. 98121.00392 TATGACCCTGACGAGTGCGGGCTGATGAAGAAGGCAGCCTTGTACTTCAGTGACTTCTGGAATA AGCTGGACGTCGGCGCAATCTTGCTCTTCGTGGCAGGGCTGACCTGCAGGCTCATCCCGGCGAC GCTGTACCCCGGGCGCGTCATCCTCTCTCTGGACTTCATCCTGTTCTGCCTCCGGCTCATGCAC ATTTTTACCATCAGTAAGACGCTGGGGCCCAAGATCATCATTGTGAAGCGGATGATGAAGGACG TCTTCTTCTTCCTCTTCCTGCTGGCTGTGTGGGTGGTGTCCTTCGGGGTGGCCAAGCAGGCCAT CCTCATCCACAACGAGCGCCGGGTGGACTGGCTGTTCCGAGGGGCCGTCTACCACTCCTACCTC ACCATCTTCGGGCAGATCCCGGGCTACATCGACGGTGTGAACTTCAACCCGGAGCACTGCAGCC CCAATGGCACCGACCCCTACAAGCCTAAGTGCCCCGAGAGCGACGCGACGCAGCAGAGGCCGGC CTTCCCTGAGTGGCTGACGGTCCTCCTACTCTGCCTCTACCTGCTCTTCACCAACATCCTGCTG CTCAACCTCCTCATCGCCATGTTCAACTACACCTTCCAGCAGGTGCAGGAGCACACGGACCAGA TTTGGAAGTTCCAGCGCCATGACCTGATCGAGGAGTACCACGGCCGCCCCGCCGCGCCGCCCCC CTTCATCCTCCTCAGCCACCTGCAGCTCTTCATCAAGAGGGTGGTCCTGAAGACTCCGGCCAAG AGGCACAAGCAGCTCAAGAACAAGCTGGAGAAGAACGAGGAGGCGGCCCTGCTATCCTGGGAGA TCTACCTGAAGGAGAACTACCTCCAGAACCGACAGTTCCAGCAAAAGCAGCGGCCCGAGCAGAA GATCGAGGACATCAGCAATAAGGCAGGGCTGGAGCTTTGGGGGTCAAGGACAAGCTCTGTCTGC AAATCTAGACAGTTCCTGCATCTCACGGTGGTGGAATCACGCGGGTCCGAGAAGGCTCCAGTAA CCACCCTCGCATGTTTGCAAGGCTGTCTGGGAAAACATGGCAGGGTTGACGCCATGGTGGACCT GCTGGACCTGGACCCACTGAAGAGGTCGGGCTCCATGGAGCAGAGGTTGGCCTCCCTGGAGGAG CAGGTGGCCCAGACAGCCCAAGCCCTGCACTGGATCGTGAGGACGCTGCGGGCCAGCGGCTTCA GCTCGGAGGCGGACGTCCCCACTCTGGCCTCCCAGAAGGCCGCGGAGGAGCCGGATGCTGAGCC GGGAGGCAGGAAGAAGACGGAGGAGCCGGGCGACAGCTACCACGTGAATGCCCGGCACCTCCTC TACCCCAACTGCCCTGTCACGCGCTTCCCCGTGCCCAACGAGAAGGTGCCCTGGGAGACGGAGT TCCTGATCTATGACCCACCCTTTTACACGGCAGAGAGGAAGGACGCGGCCGCCATGGACCCCAT GGGAGACACCCTGGAGCCACTGTCCACGATCCAGTACAACGTGGTGGATGGCCTGAGGGACCGC CGGAGCTTCCACGGGCCGTACACAGTGCAGGCCGGGTTGCCCCTGAACCCCATGGGCCGCACAG GACTGCGTGGGCGCGGGAGCCTCAGCTGCTTCGGACCCAACCACACGCTGTACCCCATGGTCAC GCGGTGGAGGCGGAACGAGGATGGAGCCATCTGCAGGAAGAGCATAAAGAAGATGCTGGAAGTG CTGGTGGTGAAGCTCCCTCTCTCCGAGCACTGGGCCCTGCCTGGGGGCTCCCGGGAGCCAGGGG AGATGCTACCTCGGAAGCTGAAGCGGATCCTCCGGCAGGAGCACTGGCCGTCTTTTGAAAACTT GCTGAAGTGCGGCATGGAGGTGTACAAAGGCTACATGGATGACCCGAGGAACACGGACAATGCC TGGATCGAGACGGTGGCCGTCAGCGTCCACTTCCAGGACCAGAATGACGTGGAGCTGAACAGGC TGAACTCTAACCTGCACGCCTGCGACTCGGGGGCCTCCATCCGATGGCAGGTGGTGGACAGGCG CATCCCACTCTATGCGAACCACAAGACCCTCCTCCAGAAGGCAGCCGCTGAGTTCGGGGCTCAC TACTGACTGTGCCCTCAGGCTGGGCGGCTCCAGTCCATAGACGTTCCCCCCAGAAACCAGGGCT TCTCTCTCCTGAGCCTGGCCAGGACTCAGGCTGTTCCTGGGCCCTGCACATGATGGGGTTTGGT GGACCCAGTGCCCCTCACGGCTGCCGCAAGTCTGCTGCAGATGACCTCATGAACTGGAAGGGGT CAAGGTGACCCGGGAGGAGAGCTCAAGACAGGGCACAGGCTACTCAGAGCTGAGGGGCCCCTGG GACCCTTGGCCATCAGGCGAGGGGCTGGGCCTGTGCAGCTGGGCCCTTGGCCAGAGTCCACTCC CTTCCTGGCTGTGTCACCCCGAGCAGCTCATCCACCATGGAGGTCATTGGCCTGAGGCAAGTTC CCCGGAGAGTCGGGGTCCCCTGTGGCCCCCTCAGGCCTATGTCTGTGAGGAAGGGGCCCTGCCA CTCTCCCCAAGAGGGCCTCCATGTTTCGAGGTGCCTCAACATGGAGCCTTGCCTGGCCTGGGCT AGGGGCACTGTCTGAACTCCTGACTGTCAGGATAAACTCCGTGGGGGTACAGGAGCCCAGACAA AGCCCAGGCCTGTCAAGAGACGCAGAGGGCCCCTGCCAGGGTTGGCCCCAGGGACCCTGGGACG AGGCTGCAGAAGCTCTCCCTCCCTACTCCCTGGGAGCCACGTGCTGGCCATGTGGCCAGGGACG GCATGAGCAGGAGGCGGGGACGTGGGGGCCTTCTGGTTTGGTGTCAACAGCTCACAGGAGCGTG AACCATGAGGGCCCTCAGGAGGGGAACGTGGTAAAACCCAAGACATTAAATCTGCCATCTCAGG CAttorney Docket No. 98121.00392 SEQ ID NO: 23; >NM_001320351.2 Homo sapiens transient receptor potential cation channel subfamily M member 2 (TRPM2), transcript variant 4, mRNA AGAACCCCAGTGTAGCGAGCTGGAGAGAGGACTGTCCTGAGGGCAGCAGGCCTGGTTGCAGCTG GCGTGGGGGTCTCAGAATGGAGCCCTCAGCCCTGAGGAAAGCTGGCTCGGAGCAGGAGGAGGGC TTTGAGGGGCTGCCCAGAAGGGTCACTGACCTGGGGATGGTCTCCAATCTCCGGCGCAGCAACA GCAGCCTCTTCAAGAGCTGGAGGCTACAGTGCCCCTTCGGCAACAATGACAAGCAAGAAAGCCT CAGTTCGTGGATTCCTGAAAACATCAAGAAGAAAGAATGCGTGTATTTTGTGGAAAGTTCCAAA CTGTCTGATGCTGGGAAGGTGGTGTGTCAGTGTGGCTACACGCATGAGCAGCACTTGGAGGAGG CTACCAAGCCCCACACCTTCCAGGGCACACAGTGGGACCCAAAGAAACATGTCCAGGAGATGCC AACCGATGCCTTTGGCGACATCGTCTTCACGGGCCTGAGCCAGAAGGTGAAAAAGTACGTCCGA GTCTCCCAGGACACGCCCTCCAGCGTGATCTACCACCTCATGACCCAGCACTGGGGGCTGGACG TCCCCAATCTCTTGATCTCGGTGACCGGGGGGGCCAAGAACTTCAACATGAAGCCGCGGCTGAA GAGCATTTTCCGCAGAGGCCTGGTCAAGGTGGCTCAGACCACAGGGGCCTGGATCATCACAGGG GGGTCCCACACCGGCGTCATGAAGCAGGTAGGCGAGGCGGTGCGGGACTTCAGCCTGAGCAGCA GCTACAAGGAAGGCGAGCTCATCACCATCGGAGTCGCCACCTGGGGCACTGTCCACCGCCGCGA GGGCCTGATCCATCCCACGGGCAGCTTCCCCGCCGAGTACATACTGGATGAGGATGGCCAAGGG AACCTGACCTGCCTAGACAGCAACCACTCTCACTTCATCCTCGTGGACGACGGGACCCACGGCC AGTACGGGGTGGAGATTCCTCTGAGGACCAGGCTGGAGAAGTTCATATCGGAGCAGACCAAGGA AAGAGGAGGTGTGGCCATCAAGATCCCCATCGTGTGCGTGGTGCTGGAGGGCGGCCCGGGCACG TTGCACACCATCGACAACGCCACCACCAACGGCACCCCCTGTGTGGTTGTGGAGGGCTCGGGCC GCGTGGCCGACGTCATTGCCCAGGTGGCCAACCTGCCTGTCTCGGACATCACTATCTCCCTGAT CCAGCAGAAACTGAGCGTGTTCTTCCAGGAGATGTTTGAGACCTTCACGGAAAGCAGGATTGTC GAGTGGACCAAAAAGATCCAAGATATCGTCCGGAGGCGGCAGCTGCTGACTGTCTTCCGGGAAG GCAAGGATGGTCAGCAGGACGTGGATGTGGCCATCTTGCAGGCCTTGCTGAAAGCCTCACGGAG CCAAGACCACTTTGGCCACGAGAACTGGGACCACCAGCTGAAACTGGCAGTGGCATGGAATCGC GTGGACATTGCCCGCAGTGAGATCTTCATGGATGAGTGGCAGTGGAAGCCTTCAGATCTGCACC CCACGATGACAGCTGCACTCATCTCCAACAAGCCTGAGTTTGTGAAGCTCTTCCTGGAGAACGG GGTGCAGCTGAAGGAGTTTGTCACCTGGGACACCTTGCTCTACCTGTACGAGAACCTGGACCCC TCCTGCCTGTTCCACAGCAAGCTGCAGAAGGTGCTGGTGGAGGATCCCGAGCGCCCGGCTTGCG CGCCCGCGGCGCCCCGCCTGCAGATGCACCACGTGGCCCAGGTGCTGCGGGAGCTGCTGGGGGA CTTCACGCAGCCGCTTTATCCCCGGCCCCGGCACAACGACCGGCTGCGGCTCCTGCTGCCCGTT CCCCACGTCAAGCTCAACGTGCAGGGAGTGAGCCTCCGGTCCCTCTACAAGCGTTCCTCAGGCC ATGTGACCTTCACCATGGACCCCATCCGTGACCTTCTCATTTGGGCCATTGTCCAGAACCGTCG GGAGCTGGCAGGAATCATCTGGGCTCAGAGCCAGGACTGCATCGCAGCGGCCTTGGCCTGCAGC AAGATCCTGAAGGAACTGTCCAAGGAGGAGGAGGACACGGACAGCTCGGAGGAGATGCTGGCGC TGGCGGAGGAGTATGAGCACAGAGCCATCGGGGTCTTCACCGAGTGCTACCGGAAGGACGAAGA GAGAGCCCAGAAACTGCTCACCCGCGTGTCCGAGGCCTGGGGGAAGACCACCTGCCTGCAGCTC GCCCTGGAGGCCAAGGACATGAAGTTTGTGTCTCACGGGGGCATCCAGGCCTTCCTGACCAAGG TGTGGTGGGGCCAGCTCTCCGTGGACAATGGGCTGTGGCGTGTGACCCTGTGCATGCTGGCCTT CCCGCTGCTCCTCACCGGCCTCATCTCCTTCAGGGAGAAGAGGCTGCAGGATGTGGGCACCCCC GCGGCCCGCGCCCGTGCCTTCTTCACCGCACCCGTGGTGGTCTTCCACCTGAACATCCTCTCCT ACTTCGCCTTCCTCTGCCTGTTCGCCTACGTGCTCATGGTGGACTTCCAGCCTGTGCCCTCCTGAttorney Docket No. 98121.00392 GTGCGAGTGTGCCATCTACCTCTGGCTCTTCTCCTTGGTGTGCGAGGAGATGCGGCAGCTCTTC TATGACCCTGACGAGTGCGGGCTGATGAAGAAGGCAGCCTTGTACTTCAGTGACTTCTGGAATA AGCTGGACGTCGGCGCAATCTTGCTCTTCGTGGCAGGGCTGACCTGCAGGCTCATCCCGGCGAC GCTGTACCCCGGGCGCGTCATCCTCTCTCTGGACTTCATCCTGTTCTGCCTCCGGCTCATGCAC ATTTTTACCATCAGTAAGACGCTGGGGCCCAAGATCATCATTGTGAAGCGGATGATGAAGGACG TCTTCTTCTTCCTCTTCCTGCTGGCTGTGTGGGTGGTGTCCTTCGGGGTGGCCAAGCAGGCCAT CCTCATCCACAACGAGCGCCGGGTGGACTGGCTGTTCCGAGGGGCCGTCTACCACTCCTACCTC ACCATCTTCGGGCAGATCCCGGGCTACATCGACGGTGTGAACTTCAACCCGGAGCACTGCAGCC CCAATGGCACCGACCCCTACAAGCCTAAGTGCCCCGAGAGCGACGCGACGCAGCAGAGGCCGGC CTTCCCTGAGTGGCTGACGGTCCTCCTACTCTGCCTCTACCTGCTCTTCACCAACATCCTGCTG CTCAACCTCCTCATCGCCATGTTCAACTACACCTTCCAGCAGGTGCAGGAGCACACGGACCAGA TTTGGAAGTTCCAGCGCCATGACCTGATCGAGGAGTACCACGGCCGCCCCGCCGCGCCGCCCCC CTTCATCCTCCTCAGCCACCTGCAGCTCTTCATCAAGAGGGTGGTCCTGAAGACTCCGGCCAAG AGGCACAAGCAGCTCAAGAACAAGCTGGAGAAGAACGAGGAGGCGGCCCTGCTATCCTGGGAGA TCTACCTGAAGGAGAACTACCTCCAGAACCGACAGTTCCAGCAAAAGCAGCGGCCCGAGCAGAA GATCGAGGACATCAGCAATAAGGTTGACGCCATGGTGGACCTGCTGGACCTGGACCCACTGAAG AGGTCGGGCTCCATGGAGCAGAGGTTGGCCTCCCTGGAGGAGCAGGTGGCCCAGACAGCCCAAG CCCTGCACTGGATCGTGAGGACGCTGCGGGCCAGCGGCTTCAGCTCGGAGGCGGACGTCCCCAC TCTGGCCTCCCAGAAGGCCGCGGAGGAGCCGGATGCTGAGCCGGGAGGCAGGAAGAAGACGGAG GAGCCGGGCGACAGCTACCACGTGAATGCCCGGCACCTCCTCTACCCCAACTGCCCTGTCACGC GCTTCCCCGTGCCCAACGAGAAGGTGCCCTGGGAGACGGAGTTCCTGATCTATGACCCACCCTT TTACACGGCAGAGAGGAAGGACGCGGCCGCCATGGACCCCATGGGAGAGAACCCCATGGGCCGC ACAGGACTGCGTGGGCGCGGGAGCCTCAGCTGCTTCGGACCCAACCACACGCTGTACCCCATGG TCACGCGGTGGAGGCGGAACGAGGATGGAGCCATCTGCAGGAAGAGCATAAAGAAGATGCTGGA AGTGCTGGTGGTGAAGCTCCCTCTCTCCGAGCACTGGGCCCTGCCTGGGGGCTCCCGGGAGCCA GGGGAGATGCTACCTCGGAAGCTGAAGCGGATCCTCCGGCAGGAGCACTGGCCGTCTTTTGAAA ACTTGCTGAAGTGCGGCATGGAGGTGTACAAAGGCTACATGGATGACCCGAGGAACACGGACAA TGCCTGGATCGAGACGGTGGCCGTCAGCGTCCACTTCCAGGACCAGAATGACGTGGAGCTGAAC AGGCTGAACTCTAACCTGCACGCCTGCGACTCGGGGGCCTCCATCCGATGGCAGGTGGTGGACA GGCGCATCCCACTCTATGCGAACCACAAGACCCTCCTCCAGAAGGCAGCCGCTGAGTTCGGGGC TCACTACTGACTGTGCCCTCAGGCTGGGCGGCTCCAGTCCATAGACGTTCCCCCCAGAAACCAG GGCTTCTCTCTCCTGAGCCTGGCCAGGACTCAGGCTGTTCCTGGGCCCTGCACATGATGGGGTT TGGTGGACCCAGTGCCCCTCACGGCTGCCGCAAGTCTGCTGCAGATGACCTCATGAACTGGAAG GGGTCAAGGTGACCCGGGAGGAGAGCTCAAGACAGGGCACAGGCTACTCAGAGCTGAGGGGCCC CTGGGACCCTTGGCCATCAGGCGAGGGGCTGGGCCTGTGCAGCTGGGCCCTTGGCCAGAGTCCA CTCCCTTCCTGGCTGTGTCACCCCGAGCAGCTCATCCACCATGGAGGTCATTGGCCTGAGGCAA GTTCCCCGGAGAGTCGGGGTCCCCTGTGGCCCCCTCAGGCCTATGTCTGTGAGGAAGGGGCCCT GCCACTCTCCCCAAGAGGGCCTCCATGTTTCGAGGTGCCTCAACATGGAGCCTTGCCTGGCCTG GGCTAGGGGCACTGTCTGAACTCCTGACTGTCAGGATAAACTCCGTGGGGGTACAGGAGCCCAG ACAAAGCCCAGGCCTGTCAAGAGACGCAGAGGGCCCCTGCCAGGGTTGGCCCCAGGGACCCTGG GACGAGGCTGCAGAAGCTCTCCCTCCCTACTCCCTGGGAGCCACGTGCTGGCCATGTGGCCAGG GACGGCATGAGCAGGAGGCGGGGACGTGGGGGCCTTCTGGTTTGGTGTCAACAGCTCACAGGAG CGTGAACCATGAGGGCCCTCAGGAGGGGAACGTGGTAAAACCCAAGACATTAAATCTGCCATCT CAGGC SEQ ID NO: 24; >NP_001307279.2 transient receptor potential cation channel subfamily M member 2 isoform 2 [Homo sapiens]Attorney Docket No. 98121.00392 MEPSALRKAGSEQEEGFEGLPRRVTDLGMVSNLRRSNSSLFKSWRLQCPFGNNDKQESLSSWIP ENIKKKECVYFVESSKLSDAGKVVCQCGYTHEQHLEEATKPHTFQGTQWDPKKHVQEMPTDAFG DIVFTGLSQKVKKYVRVSQDTPSSVIYHLMTQHWGLDVPNLLISVTGGAKNFNMKPRLKSIFRR GLVKVAQTTGAWIITGGSHTGVMKQVGEAVRDFSLSSSYKEGELITIGVATWGTVHRREGLIHP TGSFPAEYILDEDGQGNLTCLDSNHSHFILVDDGTHGQYGVEIPLRTRLEKFISEQTKERGGVA IKIPIVCVVLEGGPGTLHTIDNATTNGTPCVVVEGSGRVADVIAQVANLPVSDITISLIQQKLS VFFQEMFETFTESRIVEWTKKIQDIVRRRQLLTVFREGKDGQQDVDVAILQALLKASRSQDHFG HENWDHQLKLAVAWNRVDIARSEIFMDEWQWKPSDLHPTMTAALISNKPEFVKLFLENGVQLKE FVTWDTLLYLYENLDPSCLFHSKLQKVLVEDPERPACAPAAPRLQMHHVAQVLRELLGDFTQPL YPRPRHNDRLRLLLPVPHVKLNVQGVSLRSLYKRSSGHVTFTMDPIRDLLIWAIVQNRRELAGI IWAQSQDCIAAALACSKILKELSKEEEDTDSSEEMLALAEEYEHRAIGVFTECYRKDEERAQKL LTRVSEAWGKTTCLQLALEAKDMKFVSHGGIQAFLTKVWWGQLSVDNGLWRVTLCMLAFPLLLT GLISFREKRLQDVGTPAARARAFFTAPVVVFHLNILSYFAFLCLFAYVLMVDFQPVPSWCECAI YLWLFSLVCEEMRQLFYDPDECGLMKKAALYFSDFWNKLDVGAILLFVAGLTCRLIPATLYPGR VILSLDFILFCLRLMHIFTISKTLGPKIIIVKRMMKDVFFFLFLLAVWVVSFGVAKQAILIHNE RRVDWLFRGAVYHSYLTIFGQIPGYIDGVNFNPEHCSPNGTDPYKPKCPESDATQQRPAFPEWL TVLLLCLYLLFTNILLLNLLIAMFNYTFQQVQEHTDQIWKFQRHDLIEEYHGRPAAPPPFILLS HLQLFIKRVVLKTPAKRHKQLKNKLEKNEEAALLSWEIYLKENYLQNRQFQQKQRPEQKIEDIS NKAGLELWGSRTSSVCKSRQFLHLTVVESRGSEKAPVTTLACLQGCLGKHGRVDAMVDLLDLDP LKRSGSMEQRLASLEEQVAQTAQALHWIVRTLRASGFSSEADVPTLASQKAAEEPDAEPGGRKK TEEPGDSYHVNARHLLYPNCPVTRFPVPNEKVPWETEFLIYDPPFYTAERKDAAAMDPMGDTLE PLSTIQYNVVDGLRDRRSFHGPYTVQAGLPLNPMGRTGLRGRGSLSCFGPNHTLYPMVTRWRRN EDGAICRKSIKKMLEVLVVKLPLSEHWALPGGSREPGEMLPRKLKRILRQEHWPSFENLLKCGM EVYKGYMDDPRNTDNAWIETVAVSVHFQDQNDVELNRLNSNLHACDSGASIRWQVVDRRIPLYA NHKTLLQKAAAEFGAHY SEQ ID NO: 25; > NM_001316329.2 Homo sapiens protein kinase C gamma (PRKCG), transcript variant 1, mRNA ACATTTCAGCAGGTGCCGGAGCTGGAGCTCCCACCGCCGCCGCCCGTGCCTCCGGCTGCCGGCG CCCCTGCCTTTGGCTCTTCCTCCCCACTCGCCCGCTCCCCCTGGCGGAGCCGGCGCGCCCGGGG TGCCGCTCCCTGCCTGGCGCGCTCCGCACCTGGAGGTGCCTTGCCCCTCTCCTGCCCACCTCGG AATTTCCCTGTGGCTCCTTTGATCCTTCGAGTCTCCAGCTCCTCTCCCTTCCACCTGTTTCCCC CAAGAAAGGCAGGATCCTGGTCCCTGCTACGTTTCTGGGGCCATGGCTGGTCTGGGCCCCGGCG TAGGCGATTCAGAGGGGGGACCCCGGCCCCTGTTTTGCAGAAAGGGGGCCCTGAGGCAGAAGGT GGTCCACGAAGTCAAGAGCCACAAGTTCACCGCTCGCTTCTTCAAGCAGCCCACCTTCTGCAGC CACTGCACCGACTTCATCTGGGGTATCGGAAAGCAGGGCCTGCAATGTCAAGTCTGCAGCTTTG TGGTTCATCGACGATGCCACGAATTTGTGACCTTCGAGTGTCCAGGCGCTGGGAAGGGCCCCCA GACGGACGACCCCCGGAACAAACACAAGTTCCGCCTGCATAGCTACAGCAGCCCCACCTTCTGC GACCACTGTGGCTCCCTCCTCTACGGGCTTGTGCACCAGGGCATGAAATGCTCCTGCTGCGAGA TGAACGTGCACCGGCGCTGTGTGCGTAGCGTGCCCTCCCTGTGCGGTGTGGACCACACCGAGCG CCGCGGGCGCCTGCAGCTGGAGATCCGGGCTCCCACAGCAGATGAGATCCACGTAACTGTTGGC GAGGCCCGTAACCTAATTCCTATGGACCCCAATGGTCTCTCTGATCCCTATGTGAAACTGAAGC TCATCCCAGACCCTCGGAACCTGACGAAACAGAAGACCCGAACGGTGAAAGCCACGCTAAACCC TGTGTGGAATGAGACCTTTGTGTTCAACCTGAAGCCAGGGGATGTGGAGCGCCGGCTCAGCGTG GAGGTGTGGGACTGGGACCGGACCTCCCGCAACGACTTCATGGGGGCCATGTCCTTTGGCGTCTAttorney Docket No. 98121.00392 CGGAGCTGCTCAAGGCGCCCGTGGATGGCTGGTACAAGTTACTGAACCAGGAGGAGGGCGAGTA TTACAATGTGCCGGTGGCCGATGCTGACAACTGCAGCCTCCTCCAGAAGTTTGAGGCTTGTAAC TACCCCCTGGAATTGTATGAGCGGGTGCGGATGGGCCCCTCTTCCTCTCCCATCCCCTCCCCTT CCCCTAGTCCCACCGACCCCAAGCGCTGCTTCTTCGGGGCGAGTCCAGGACGCCTGCACATCTC CGACTTCAGCTTCCTCATGGTTCTAGGAAAAGGCAGTTTTGGGAAGGTGATGCTGGCCGAGCGC AGGGGCTCTGATGAGCTCTACGCCATCAAGATCTTGAAAAAGGACGTGATCGTCCAGGACGACG ATGTGGACTGCACGCTGGTGGAGAAACGTGTGCTGGCGCTGGGGGGCCGGGGTCCTGGCGGCCG GCCCCACTTCCTCACCCAGCTCCACTCCACCTTCCAGACCCCGGACCGCCTGTATTTCGTGATG GAGTACGTCACCGGGGGAGACTTGATGTACCACATTCAACAGCTGGGCAAGTTTAAGGAGCCCC ATGCAGCGTTCTACGCGGCAGAAATCGCTATCGGCCTCTTCTTCCTTCACAATCAGGGCATCAT CTACAGGGACCTGAAGCTGGACAATGTGATGCTGGATGCTGAGGGACACATCAAGATCACTGAC TTTGGCATGTGTAAGGAGAACGTCTTCCCCGGGACGACAACCCGCACCTTCTGCGGGACCCCGG ACTACATAGCCCCGGAGATCATTGCCTACCAGCCCTATGGGAAGTCTGTCGATTGGTGGTCCTT TGGAGTTCTGCTGTATGAGATGTTGGCAGGACAGCCTCCCTTCGATGGGGAGGACGAGGAGGAG CTGTTTCAGGCCATCATGGAACAAACTGTCACCTACCCCAAGTCGCTTTCCCGGGAAGCCGTGG CCATCTGCAAGGGGTTCCTGACCAAGCACCCAGGGAAGCGCCTGGGCTCAGGGCCTGATGGGGA ACCTACCATCCGTGCACATGGCTTTTTCCGCTGGATTGACTGGGAGCGGCTGGAACGATTGGAG ATCCCGCCTCCTTTCAGACCCCGCCCGTGTGGCCGCAGCGGCGAGAACTTTGACAAGTTCTTCA CGCGGGCGGCGCCAGCGCTGACCCCTCCAGACCGCCTAGTCCTGGCCAGCATCGACCAGGCCGA TTTCCAGGGCTTCACCTACGTGAACCCCGACTTCGTGCACCCGGATGCCCGCAGCCCCACCAGC CCAGTGCCTGTGCCCGTCATATCCTGCACCCCAGCATTCCAGCTCTGCCCCCGCGGGTTCTAGA CGCCCCTCCCAAGCGTTCCTGGCCTTCTGAACTCCATACAGCCTCTACAGCCGTCCCGCGTTCA AGACTTGAGCGGAGCCCGATATTCTCCCTGACCTTAGCGTTCTGGACTCTGCCCCAATCGGGTC CAGAGACCACACCACTAACCATCCCCAACTCCATGGGGTTCGAGACTCCATCTTGGTAGTTCTG TGCCTCCCCCCAGACCCCGCCCCTGGGGAAATAGCCTCACGGGGTTGGCTGTTCCAGACTCAGG TTCCAGAACAGCCCTCGGCCTCCGAGGCTCCCCGCCTCCACTCTAGTTCTAGATGAGTGGGAGG CGTGCCCCCCTCCTCCAGTACGTCCCGCTGCTGTGCTCTGGGGATTTCTGGGATATATGGAGGA TTCTTTCCCCAGAGGCTCCCAATCAGCTTTTGTTCTAGACTTCCCCATCCCGAAGCCATCACTT CTCCCCGCAGCCCGCCTGCCGTGCATGGCTCCTGTCTGGCTCGGACCCACCCCAACTCTCCCCA GTGCCTGCCACTCTCTGGGACTCTCCTCCTCCCCTCCTCTTCCCTTAGCCTCTCCCACCCGGCC ACAGCTGCTGGAGAATAAATTTGGGATGCTGATGCTGAA SEQ ID NO: 26; > NM_002739.5 Homo sapiens protein kinase C gamma (PRKCG), transcript variant 2, mRNA ACATTTCAGCAGGTGCCGGAGCTGGAGCTCCCACCGCCGCCGCCCGTGCCTCCGGCTGCCGGCG CCCCTGCCTTTGGCTCTTCCTCCCCACTCGCCCGCTCCCCCTGGCGGAGCCGGCGCGCCCGGGG TGCCGCTCCCTGCCTGGCGCGCTCCGCACCTGGAGGTGCCTTGCCCCTCTCCTGCCCACCTCGG AATTTCCCTGTGGCTCCTTTGATCCTTCGAGTCTCCAGCTCCTCTCCCTTCCACCTGTTTCCCC CAAGAAAGGCAGGATCCTGGTCCCTGCTACGTTTCTGGGGCCATGGCTGGTCTGGGCCCCGGCG TAGGCGATTCAGAGGGGGGACCCCGGCCCCTGTTTTGCAGAAAGGGGGCCCTGAGGCAGAAGGT GGTCCACGAAGTCAAGAGCCACAAGTTCACCGCTCGCTTCTTCAAGCAGCCCACCTTCTGCAGC CACTGCACCGACTTCATCTGGGGTATCGGAAAGCAGGGCCTGCAATGTCAAGTCTGCAGCTTTG TGGTTCATCGACGATGCCACGAATTTGTGACCTTCGAGTGTCCAGGCGCTGGGAAGGGCCCCCA GACGGACGACCCCCGGAACAAACACAAGTTCCGCCTGCATAGCTACAGCAGCCCCACCTTCTGC GACCACTGTGGCTCCCTCCTCTACGGGCTTGTGCACCAGGGCATGAAATGCTCCTGCTGCGAGA TGAACGTGCACCGGCGCTGTGTGCGTAGCGTGCCCTCCCTGTGCGGTGTGGACCACACCGAGCGAttorney Docket No. 98121.00392 CCGCGGGCGCCTGCAGCTGGAGATCCGGGCTCCCACAGCAGATGAGATCCACGTAACTGTTGGC GAGGCCCGTAACCTAATTCCTATGGACCCCAATGGTCTCTCTGATCCCTATGTGAAACTGAAGC TCATCCCAGACCCTCGGAACCTGACGAAACAGAAGACCCGAACGGTGAAAGCCACGCTAAACCC TGTGTGGAATGAGACCTTTGTGTTCAACCTGAAGCCAGGGGATGTGGAGCGCCGGCTCAGCGTG GAGGTGTGGGACTGGGACCGGACCTCCCGCAACGACTTCATGGGGGCCATGTCCTTTGGCGTCT CGGAGCTGCTCAAGGCGCCCGTGGATGGCTGGTACAAGTTACTGAACCAGGAGGAGGGCGAGTA TTACAATGTGCCGGTGGCCGATGCTGACAACTGCAGCCTCCTCCAGAAGTTTGAGGCTTGTAAC TACCCCCTGGAATTGTATGAGCGGGTGCGGATGGGCCCCTCTTCCTCTCCCATCCCCTCCCCTT CCCCTAGTCCCACCGACCCCAAGCGCTGCTTCTTCGGGGCGAGTCCAGGACGCCTGCACATCTC CGACTTCAGCTTCCTCATGGTTCTAGGAAAAGGCAGTTTTGGGAAGGTGATGCTGGCCGAGCGC AGGGGCTCTGATGAGCTCTACGCCATCAAGATCTTGAAAAAGGACGTGATCGTCCAGGACGACG ATGTGGACTGCACGCTGGTGGAGAAACGTGTGCTGGCGCTGGGGGGCCGGGGTCCTGGCGGCCG GCCCCACTTCCTCACCCAGCTCCACTCCACCTTCCAGACCCCGGACCGCCTGTATTTCGTGATG GAGTACGTCACCGGGGGAGACTTGATGTACCACATTCAACAGCTGGGCAAGTTTAAGGAGCCCC ATGCAGCGTTCTACGCGGCAGAAATCGCTATCGGCCTCTTCTTCCTTCACAATCAGGGCATCAT CTACAGGGACCTGAAGCTGGACAATGTGATGCTGGATGCTGAGGGACACATCAAGATCACTGAC TTTGGCATGTGTAAGGAGAACGTCTTCCCCGGGACGACAACCCGCACCTTCTGCGGGACCCCGG ACTACATAGCCCCGGAGATCATTGCCTACCAGCCCTATGGGAAGTCTGTCGATTGGTGGTCCTT TGGAGTTCTGCTGTATGAGATGTTGGCAGGACAGCCTCCCTTCGATGGGGAGGACGAGGAGGAG CTGTTTCAGGCCATCATGGAACAAACTGTCACCTACCCCAAGTCGCTTTCCCGGGAAGCCGTGG CCATCTGCAAGGGGTTCCTGACCAAGCACCCAGGGAAGCGCCTGGGCTCAGGGCCTGATGGGGA ACCTACCATCCGTGCACATGGCTTTTTCCGCTGGATTGACTGGGAGCGGCTGGAACGATTGGAG ATCCCGCCTCCTTTCAGACCCCGCCCGTGTGGCCGCAGCGGCGAGAACTTTGACAAGTTCTTCA CGCGGGCGGCGCCAGCGCTGACCCCTCCAGACCGCCTAGTCCTGGCCAGCATCGACCAGGCCGA TTTCCAGGGCTTCACCTACGTGAACCCCGACTTCGTGCACCCGGATGCCCGCAGCCCCACCAGC CCAGTGCCTGTGCCCGTCATGTAATCTCACCCGCCGCCACTAGGTGTCCCCAACGTCCCCTCCG CCGTGCCGGCGGCAGCCCCACTTCACCCCCAACTTCACCACCCCCTGTCCCATTCTAGATCCTG CACCCCAGCATTCCAGCTCTGCCCCCGCGGGTTCTAGACGCCCCTCCCAAGCGTTCCTGGCCTT CTGAACTCCATACAGCCTCTACAGCCGTCCCGCGTTCAAGACTTGAGCGGAGCCCGATATTCTC CCTGACCTTAGCGTTCTGGACTCTGCCCCAATCGGGTCCAGAGACCACACCACTAACCATCCCC AACTCCATGGGGTTCGAGACTCCATCTTGGTAGTTCTGTGCCTCCCCCCAGACCCCGCCCCTGG GGAAATAGCCTCACGGGGTTGGCTGTTCCAGACTCAGGTTCCAGAACAGCCCTCGGCCTCCGAG GCTCCCCGCCTCCACTCTAGTTCTAGATGAGTGGGAGGCGTGCCCCCCTCCTCCAGTACGTCCC GCTGCTGTGCTCTGGGGATTTCTGGGATATATGGAGGATTCTTTCCCCAGAGGCTCCCAATCAG CTTTTGTTCTAGACTTCCCCATCCCGAAGCCATCACTTCTCCCCGCAGCCCGCCTGCCGTGCAT GGCTCCTGTCTGGCTCGGACCCACCCCAACTCTCCCCAGTGCCTGCCACTCTCTGGGACTCTCC TCCTCCCCTCCTCTTCCCTTAGCCTCTCCCACCCGGCCACAGCTGCTGGAGAATAAATTTGGGA TGCTGATGCTGAA SEQ ID NO: 27; >NP_001303258.1 protein kinase C gamma type isoform 1 [Homo sapiens] MAGLGPGVGDSEGGPRPLFCRKGALRQKVVHEVKSHKFTARFFKQPTFCSHCTDFIWGIGKQGL QCQVCSFVVHRRCHEFVTFECPGAGKGPQTDDPRNKHKFRLHSYSSPTFCDHCGSLLYGLVHQG MKCSCCEMNVHRRCVRSVPSLCGVDHTERRGRLQLEIRAPTADEIHVTVGEARNLIPMDPNGLS DPYVKLKLIPDPRNLTKQKTRTVKATLNPVWNETFVFNLKPGDVERRLSVEVWDWDRTSRNDFM GAMSFGVSELLKAPVDGWYKLLNQEEGEYYNVPVADADNCSLLQKFEACNYPLELYERVRMGPSAttorney Docket No. 98121.00392 SSPIPSPSPSPTDPKRCFFGASPGRLHISDFSFLMVLGKGSFGKVMLAERRGSDELYAIKILKK DVIVQDDDVDCTLVEKRVLALGGRGPGGRPHFLTQLHSTFQTPDRLYFVMEYVTGGDLMYHIQQ LGKFKEPHAAFYAAEIAIGLFFLHNQGIIYRDLKLDNVMLDAEGHIKITDFGMCKENVFPGTTT RTFCGTPDYIAPEIIAYQPYGKSVDWWSFGVLLYEMLAGQPPFDGEDEEELFQAIMEQTVTYPK SLSREAVAICKGFLTKHPGKRLGSGPDGEPTIRAHGFFRWIDWERLERLEIPPPFRPRPCGRSG ENFDKFFTRAAPALTPPDRLVLASIDQADFQGFTYVNPDFVHPDARSPTSPVPVPVISCTPAFQ LCPRGF SEQ ID NO: 28; >NP_002730.1 protein kinase C gamma type isoform 2 [Homo sapiens] MAGLGPGVGDSEGGPRPLFCRKGALRQKVVHEVKSHKFTARFFKQPTFCSHCTDFIWGIGKQGL QCQVCSFVVHRRCHEFVTFECPGAGKGPQTDDPRNKHKFRLHSYSSPTFCDHCGSLLYGLVHQG MKCSCCEMNVHRRCVRSVPSLCGVDHTERRGRLQLEIRAPTADEIHVTVGEARNLIPMDPNGLS DPYVKLKLIPDPRNLTKQKTRTVKATLNPVWNETFVFNLKPGDVERRLSVEVWDWDRTSRNDFM GAMSFGVSELLKAPVDGWYKLLNQEEGEYYNVPVADADNCSLLQKFEACNYPLELYERVRMGPS SSPIPSPSPSPTDPKRCFFGASPGRLHISDFSFLMVLGKGSFGKVMLAERRGSDELYAIKILKK DVIVQDDDVDCTLVEKRVLALGGRGPGGRPHFLTQLHSTFQTPDRLYFVMEYVTGGDLMYHIQQ LGKFKEPHAAFYAAEIAIGLFFLHNQGIIYRDLKLDNVMLDAEGHIKITDFGMCKENVFPGTTT RTFCGTPDYIAPEIIAYQPYGKSVDWWSFGVLLYEMLAGQPPFDGEDEEELFQAIMEQTVTYPK SLSREAVAICKGFLTKHPGKRLGSGPDGEPTIRAHGFFRWIDWERLERLEIPPPFRPRPCGRSG ENFDKFFTRAAPALTPPDRLVLASIDQADFQGFTYVNPDFVHPDARSPTSPVPVPVM

Claims

Attorney Docket No. 98121.00392 CLAIMS What is claimed is:

1. A method for treating or preventing neurological injury or neurological disorder in a subject comprising administering to the subject an agent that inhibits the interaction between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2).

2. A method of treating or preventing a vascular disorder in a subject in need thereof, the method comprising administering to the subject an agent that inhibits the interaction between protein kinase C γ (PKCγ) and transient receptor potential melastatin 2 (TRPM2).

3. The method of claim 1 or 2, wherein the agent targets a PKCγ-binding site on TRPM2.

4. The method of claim 3, wherein the PKCγ-binding site comprises residues 151-200 or residues 162-178 of TRPM2.

5. The method of claim 3, wherein the PKCγ-binding site comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

6. The method of claim 1, wherein the agent comprises a small molecule.

7. The method of claim 6, wherein the small molecule is selected from the group consisting of N-(p-amylcinnamoyl)anthranilic acid (ACA) and 2- Aminoethoxydiphenyl borate (2-APB).Attorney Docket No. 98121.00392 8. The method of any one of claims 1-5, wherein the agent comprises a peptide comprising an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

9. The method of claim 8, wherein the peptide comprises an amino acid sequence differing by 1, 2, 3, 4, or 5 residues from the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

10. The method of claim 8, wherein the peptide comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

11. The method of claim 8, wherein the peptide does not comprise the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

12. The method of any one of claims 8-11, wherein the peptide is further conjugated to a cell-penetrating peptide (CPP).

13. The method of claim 12, wherein the CPP comprises an amino acid sequence with at least 75%, 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 4- 19.

14. The method of claim 12 or 13, wherein the CPP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.

15. The method of any one of claims 12-14, wherein the CPP consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.

16. The conjugate of any one of claims 12-15, wherein the CPP comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5.

17. The conjugate of any one of claims 12-16, wherein the CPP consists of an amino acid sequence set forth in SEQ ID NO: 4 or 5.Attorney Docket No. 98121.00392 18. The method of any one of claims 1-5, wherein the agent comprises an antagonist anti- TRPM2 antibody that binds to the PKCγ-binding site.

19. The method of any one of claims 1-5, wherein the agent comprises a mutant TRPM2 protein, wherein the mutant TRPM2 protein comprises a deletion of the PKCγ- binding site.

20. The method of claim 1 or 2, wherein the agent targets a TRPM2-binding site on PKCγ.

21. The method of claim 20, wherein the TRPM2-binding site on PKCγ is the C2-domain of PKCγ.

22. The method of any one of claims 20-21, wherein the agent comprises a small molecule that binds to the TRPM2-binding site on PKCγ.

23. The method of any one of claims 20-21, wherein the agent comprises an antagonist anti-PKCγ antibody that binds to the TRPM2-binding site.

24. The method of any one of claims 20-21, wherein the agent comprises a mutant PKCγ protein, wherein the mutant PKCγ protein comprises a deletion of the TRPM2- binding site.

25. The method of any one of claims 1 and 3-24, wherein the neurological injury results from a brain injury.

26. The method of claim 25, wherein the brain injury comprises stroke, traumatic brain injury, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, brain aneurysm, coma, or any combination thereof.

27. The method of claim 26, wherein the brain injury is ischemic stroke, hemorrhagic stroke, or transient ischemic attack.Attorney Docket No. 98121.00392 28. The method of claim 27, wherein the brain injury is ischemic stroke.

29. The method of any one of claims 1 and 3-28, wherein administering to the subject the agent decreases surface expression of N-methyl-D-aspartate receptor (NMDAR) in a neuronal cell.

30. The method of any one of claims 1 and 3-28, wherein administering to the subject the agent decreases the amplitude of NMDAR current in a neuronal cell.

31. The method of any one of claims 1 and 3-28, wherein administering to the subject the agent inhibits an increase of intracellular Ca2+concentration in a neuronal cell.

32. The method of any one of claims 1 and 3-28, wherein administering to the subject the agent decreases neuronal death.

33. The method of any one of claims 1 and 3-28, wherein administering to the subject the agent decreases infarct volume in brain.

34. The method of any one of claims 1 and 3-28, wherein administering to the subject the agent decreases neurological deficit score.

35. The method of any one of claims 1 and 3-34, wherein administering to the subject the agent ameliorates at least one symptom of ischemic stroke.

36. The method of claim 35, wherein the symptom is selected from the group consisting of difficulty walking, numbness, weakness and / or paralysis in the face, arm and / or leg, headache, confusion, difficulty speaking and / or understanding speech, and difficulty seeing in one or both eyes.

37. The method of any one of claims 1 and 3-36, wherein the agent is administered to the subject at a dose of about 10 nmol / kg to about 1000 nmol / kg.Attorney Docket No. 98121.00392 38. The method of claim 37, wherein the agent is administered to the subject at a dose of about 100 nmol / kg.

39. The method of any one of claims 1 and 3-38, wherein the agent is administered to the subject every 2 to 24 hours.

40. The method of claim 39, wherein the agent is administered to the subject every 10 to 12 hours.

41. The method of any one of claims 1 and 3-40, wherein the agent is administered to the subject less than 6 hours after one or more symptoms of ischemic stroke start.

42. The method of claim 41, wherein the agent is administered to the subject less than 4 hours after one or more symptoms of ischemic stroke start.

43. The method of any one of claims 1 and 3-42, further comprising administering an additional therapeutic to the subject.

44. The method of claim 43, wherein the additional therapeutic comprises an anticoagulant or clot-dissolving medicine (e.g., aspirin), clopidogrel, a tissue plasminogen activator (tPA) (e.g., alteplase), thrombectomy, carotid endarterectomy, a medication for antihypertension (e.g., nicardipine, labetalol, or lisinopril), an antipyretic therapy, warfarin, heparin, apixaban, atorvastatin, rosuvastatin, irbesartan, alteplase, or any combination thereof.

45. The method of any one of claims 2-24, wherein the vascular disorder is a cardiovascular disease.

46. The method of claim 45, wherein the cardiovascular disease comprises atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, diabetic angiopathy, or any combination thereof.Attorney Docket No. 98121.00392 47. The method of any one of claims 2-24 and 45-46, wherein administering to the subject the agent inhibits the loss of one or more contractile proteins in a vascular smooth muscle cell.

48. The method of claim 47, wherein the contractile protein is selected from the group consisting of α-SMA and SM22α.

49. The method of any one of claims 1-48, wherein the subject is a human subject.

50. A peptide comprising an amino acid sequence with at least 80% or 85% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1), or a multimer, derivative, or variant thereof.

51. The peptide of claim 50, comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

52. The peptide of claim 50 or 51, comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

53. The peptide of any one of claims 50-52, wherein the peptide comprises an amino acid sequence differing by 1, 2, 3, 4, or 5 residues from the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

54. The peptide of any one of claims 50-52, wherein the peptide comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

55. The peptide of any one of claims 50-54, wherein the peptide is a synthetic peptide.

56. The peptide of any one of claims 50-55, wherein the peptide inhibits interaction between PKCγ and TRPM2.Attorney Docket No. 98121.00392 57. The peptide of any one of claims 50-55, wherein the peptide inhibits interaction between esNMDAR and TRPM2.

58. The peptide of any one of claims 50-55, wherein the peptide inhibits phosphorylation of TRPM2 at S38.

59. The peptide of any one of claims 50-55, wherein the peptide binds to the C2-domain of PKCγ.

60. The peptide of any one of claims 50-59, wherein the peptide decreases the surface expression of N-methyl-D-aspartate receptor (NMDAR) in a neuronal cell.

61. The peptide of any one of claims 50-59, wherein the peptide decreases the amplitude of NMDAR current in a neuronal cell.

62. The peptide of any one of claims 50-59, wherein the peptide reduces mitochondrial membrane depolarization induced by ischemic injury in neurons.

63. The peptide of any one of claims 50-59, wherein the peptide decreases neuronal death induced by ischemic injury.

64. A conjugate comprising the peptide of any one of claims 50-63 and a cell-penetrating peptide (CPP).

65. The conjugate of claim 64, wherein the CPP comprises an amino acid sequence with at least 75%, 80%, 85%, 90% or 95% sequence identity to any one of SEQ ID NOs: 4-19.

66. The conjugate of claim 64 or 65, wherein the CPP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.Attorney Docket No. 98121.00392 67. The conjugate of any one of claims 64-66, wherein the CPP consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-19.

68. The conjugate of any one of claims 64-67, wherein the CPP comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5.

69. The conjugate of any one of claims 64-68, wherein the CPP consists of an amino acid sequence set forth in SEQ ID NO: 4 or 5.

70. The conjugate of any one of claims 64-69, wherein the peptide is conjugated to the CPP via a linker.

71. The conjugate of claim 64, wherein the peptide comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1) and the CPP comprises an amino acid sequence set forth in SEQ ID NO: 4 or 5.

72. The conjugate of claim 71, wherein the conjugate comprises an amino acid sequence with at least 75%, 80%, 85%, 90% or 95% sequence identity YGRKKRRQRRRWGLDVPNLLISVTGGA (SEQ ID NO: 20).

73. The conjugate of claim 72, wherein the conjugate comprises the amino acid sequence of YGRKKRRQRRRWGLDVPNLLISVTGGA (SEQ ID NO: 20).

74. The conjugate of claim 72 or 73, wherein the conjugate consists of the amino acid sequence of YGRKKRRQRRRWGLDVPNLLISVTGGA (SEQ ID NO: 20).

75. A nucleic acid molecule encoding the peptide of any one of claims 50-63 or the conjugate of any one of claims 64-74.

76. An expression vector comprising the nucleic acid molecule of claim 75 operably linked to a control sequence for the expression of the peptide of any one of claims 50- 63 or the conjugate of any one of claims 64-74.

77. A host cell comprising the expression vector of claim 76.Attorney Docket No. 98121.00392 78. A pharmaceutical composition comprising the peptide of any one of claims 50-63 or the conjugate of any one of claims 64-74, and at least one pharmaceutically acceptable excipient.

79. A method for treating or preventing neurological injury or neurological disorder in a subject comprising administering to the subject the peptide of any one of claims 50- 63, the conjugate of any one of claims 64-74, or the pharmaceutical composition of claim 78.

80. The method of claim 79, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits the interaction between TRPM2 and PKCγ.

81. The method of claim 79 or 80, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits the interaction between TRPM2 and esNMDAR.

82. The method of any one of claims 79-81, wherein administering to the subject the agent, the peptide or the pharmaceutical composition decreases surface expression of N-methyl-D-aspartate receptor (NMDAR) in a neuronal cell.

83. The method of any one of claims 79-82, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition decreases the amplitude of NMDAR current in a neuronal cell.

84. The method of any one of claims 79-83, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits an increase of intracellular Ca2+concentration in a neuronal cell.

85. The method of any one of claims 79-84, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition reduces mitochondrial membrane depolarization induced by ischemic injury in neurons.Attorney Docket No. 98121.00392 86. The method of any one of claims 79-85, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition decreases neuronal death.

87. The method of any one of claims 79-86, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition reduces infarct volume and / or improves neurological behavior score.

88. The method of any one of claims 79-87, wherein the neurological injury results from a brain injury.

89. The method of claim 88, wherein the brain injury comprises stroke, traumatic brain injury, cerebral palsy, acquired brain injury, anoxic brain injury, diffuse axonal brain injury, focal brain injury, subdural hematoma, brain aneurysm, coma, or any combination thereof.

90. The method of claim 88, wherein the brain injury is ischemic stroke, hemorrhagic stroke, or transient ischemic attack.

91. The method of claim 88, wherein the brain injury is ischemic stroke.

92. The method of any one of claims 79-91, wherein the conjugate, the peptide, or the pharmaceutical composition is administered to the subject at a dose of about 10 nmol / kg to about 1000 nmol / kg.

93. The method of claim 92, wherein the conjugate, the peptide, or the pharmaceutical composition is administered to the subject at a dose of about 100 nmol / kg.

94. The method of any one of claims 79-93, wherein the conjugate, the peptide, or the pharmaceutical composition is administered to the subject every 2 to 24 hours.

95. The method of claim 94, wherein the conjugate, the peptide, or the pharmaceutical composition is administered to the subject every 10 to 12 hours.Attorney Docket No. 98121.00392 96. The method of any one of claims 79-95, wherein the conjugate, the peptide, or the pharmaceutical composition is administered to the subject less than 6 hours after one or more symptoms of ischemic stroke start.

97. The method of claim 96, wherein the conjugate, the peptide, or the pharmaceutical composition is administered to the subject less than 4 hours after one or more symptoms of ischemic stroke start.

98. The method of any one of claims 79-97, further comprising administering an additional therapeutic to the subject.

99. The method of claim 98, wherein the additional therapeutic comprises an anticoagulant or clot-dissolving medicine (e.g., aspirin), clopidogrel, a tissue plasminogen activator (tPA) (e.g., alteplase), thrombectomy, carotid endarterectomy, a medication for antihypertension (e.g., nicardipine, labetalol, or lisinopril), an antipyretic therapy, warfarin, heparin, apixaban, atorvastatin, rosuvastatin, irbesartan, alteplase, or any combination thereof.

100. A method of treating or preventing a vascular disorder in a subject in need thereof, the method comprising administering to the subject the peptide of any one of claims 50-63, the conjugate of any one of claims 64-74, or the pharmaceutical composition of claim 78.

101. The method of claim 100, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits the interaction between TRPM2 and PKCγ.

102. The method of claim 100 or 101, wherein the vascular disorder is a cardiovascular disease.

103. The method of claim 102, wherein the cardiovascular disease comprises atherosclerosis, in-stent restenosis, aneurysms, vascular calcification, hypertension, heart failure, myocardial infarction, diabetic angiopathy, or any combination thereof.Attorney Docket No. 98121.00392 104. The method of any one of claims 100-103, wherein administering to the subject the peptide, the conjugate, or the pharmaceutical composition inhibits loss of one or more contractile proteins in a vascular smooth muscle cell.

105. The method of claim 104, wherein the contractile protein is selected from the group consisting of α-SMA and SM22α.

106. A method for identifying a compound useful for treating or preventing neurological injury or neurological disorder in a subject in need thereof, comprising a) providing a test compound; b) determining the effect of the test compound on the interaction between transient receptor potential melastatin 2 (TRPM2) and protein kinase C γ (PKCγ); and c) selecting a compound that inhibits the interaction between TRPM2 and PKCγ, thereby identifying a compound useful for treating or preventing neurological injury or neurological disorder in the subject.

107. The method of claim 106, wherein the compound binds to a PKCγ-binding site on TRPM2.

108. The method of claim 106, wherein the PKCγ-binding site comprises residues 151-200 or residues 162-178 of TRPM2.

109. The method of claim 108, wherein the PKCγ-binding site comprises the amino acid sequence of WGLDVPNLLISVTGGA (SEQ ID NO: 1).

110. The method of claim 106, wherein the compound binds to a TRPM2-binding site on PKCγ.

111. The method of claim 110, wherein the TRPM2-binding site on PKCγ is the C2- domain of PKCγ.Attorney Docket No. 98121.00392 112. A kit for treating or preventing neurological injury, neurological disorder, or vascular disorder in a subject, wherein the kit comprises the peptide of any one of claims 50- 63, the conjugate of any one of claims 64-74, or the pharmaceutical composition of claim 78.

113. The kit of claim 112, wherein the kit further comprises: instructions for use; one or more therapeutic agents; one or more reagents; one or more materials for preparing composition for administration to the subject; one or more pharmaceutically acceptable carriers; one or more devices; and / or one or more materials for administration to the subject.

114. The kit of claim 113, wherein the one or more therapeutic agents are for combination therapy and / or emergency therapy of stroke.

115. The kit of claim 113, wherein the one or more reagents comprise one or more diluents.

116. The kit of claim 113, wherein the instructions for use comprise dosages and / or modes of administration to the subject.

117. A kit for diagnosing neurological injury, neurological disorder, or vascular disorder, wherein the kit comprises the peptide of any one of claims 50-63, the conjugate of any one of claims 64-74, or the pharmaceutical composition of claim 78.

118. A kit for use in an imaging assay, wherein the kit comprises the peptide of any one of claims 50-63, the conjugate of any one of claims 64-74, or the pharmaceutical composition of claim 78.Attorney Docket No. 98121.00392 119. A kit for use in an in vitro assay, wherein the kit comprises the peptide of any one of claims 50-63, the conjugate of any one of claims 64-74, or the pharmaceutical composition of claim 78.

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