WO2021097407A2

WO2021097407A2 - Novel peptide compositions and methods of treating neurological injury

Info

Publication number: WO2021097407A2
Application number: PCT/US2020/060681
Authority: WO
Inventors: Paco S. HERSON
Original assignee: Regents Of The University Of Colorado, A Body Corporate
Priority date: 2019-11-15
Filing date: 2020-11-16
Publication date: 2021-05-20
Also published as: EP4058470A4; EP4058470A2; WO2021097407A3

Abstract

TRPM2 is a calcium permeable channel activated by ADPR metabolites and oxidative stress. TRPM2 contributes to neuronal injury in the brain caused by stroke and cardiac arrest among other diseases including pain, inflammation, and cancer. However, the lack of specific inhibitors hinders the study of TRPM2 in brain pathophysiology. Presented is the design of a novel TRPM2 antagonist, tatM2NX and truncated variants thereof, which prevents ligand binding and TRPM2 activation. Mutagenesis of tatM2NX was used to determine the structure- activity relationship and antagonistic mechanism on TRPM2 using whole-cell patch clamp and Ca²⁺ imaging in HEK293 cells with stable human TRPM2 expression, showing that tatM2NX inhibits over 90% of TRPM2 channel currents at concentrations as low as 2μΜ. Moreover, tatM2NX is a potent antagonist with an IC₅₀ of 396nM. These results from tatM2NX mutagenesis demonstrate that specific residues within the tatM2NX C-terminus are required to confer antagonism on TRPM2. Therefore, truncated residues of the C-terminus of the peptide tatM2NX represent a new and potent therapeutic for a number of conditions, including neuronal injury and traumatic brain injury.

Description

NOVEL PEPTIDE COMPOSITIONS AND METHODS OF TREATING

NEUROLOGICAL INJURY

BACKGROUND

The Transient Receptor Potential Melastatin 2 (TRPM2) is a non-selective cation channel from the TRP family. At physiological membrane potentials, activation of TRPM2 results in influx of sodium and calcium into the cell. TRPM2 forms tetramers composed of six transmembrane domains with the N- and C-termini facing the intracellular milieu. The C- terminus contains a unique NUDT9-homology domain (NUDT9-H) required for adenosine diphosphate ribose (ADPR) binding and activation, absent in other TRP family members (Tong et al. 2006; Hantute-Ghesquier et al. 2018). TRPM2 was initially described as a channel/enzyme (chanzyme) due to the presence of the NUDT9-H domain. However, subsequent work has demonstrated that the C-terminal NUDT9-H domain of TRPM2 lacks enzymatic activity (Perraud et al. 2003; lordanov et al. 2016). The NUDT9-H domain of TRPM2 is essential for intra- and interface interactions that regulate TRPM2 channel activation by ADPR (Wang et al. 2018; Huang et al. 2018). Intracellular calcium (Ca2+) serves as a co agonist, modulating channel activity in the presence of ADPR (Herson, Dulock, and Ashford 1997; Herson et al. 1999; Perraud et al. 2001 ; Inamura et al. 2003; McHugh et al. 2003; Kuhn and Luckhoff 2004; Heiner et al. 2006; Olah et al. 2009; Toth, lordanov, and Csanady 2015; Yu et al. 2017; Fliegert, Watt, et al. 2017; Fliegert, Bauche, et al. 2017). Consistent with these physiological observations, the recently solved human TRPM2 channel structure indicates an ADPR binding site in the C-terminus that appears to interact with the N-terminus upon opening and leads to structural changes in a calcium-dependent ‘primed’ state (Huang et al. 2018; Wang et al. 2018).

TRPM2 channels have been implicated in several physiological and pathophysiological conditions in multiple organs (Verma et al. 2012; Shimizu et al. 2013; Gelderblom et al. 2014; Alim et al. 2013; Li and Jiang 2019; Park et al. 2016; Andoh et al. 2019; Haraguchi et al. 2012; Jang et al. 2015; Inamura et al. 2003; Smith et al. 2003; Fonfria et al. 2004; Kraft et al. 2004; Fonfria et al. 2006; Lange et al. 2009; Hoffman et al. 2015; Tan and McNaughton 2016).

TRPM2 channels are highly expressed in the brain, found in neurons and microglia in the cortex, hippocampus, striatum, brainstem and others (Fonfria et al. 2006; Olah et al. 2009; Chung, Freestone, and Lipski 2011). TRPM2 channels are activated following oxidative stress, and the most well-characterized role for these channels is a cell death mediator following oxidative stress, due to excessive Ca2+ influx and consequent cell death (Fonfria et al. 2004; Perraud et al. 2005; Bai and Lipski 2010). However, the lack of specific TRPM2 channel antagonists has hindered the research regarding the role of TRPM2 channels in brain function, with most data coming from cell culture experiments or global TRPM2 channel genetic ablation in mice. Thus far, most described TRPM2 pharmacological inhibitors are not specific to TRPM2 channels, including antifungals, flufenamic acid, fenamate non-steroidal anti-inflammatories, 2- aminoethoxydiphenyl borate and natural compounds with moderate to high potency (Hill, McNulty, and Randall 2004; Chen et al. 2012; Starkus et al. 2017; Zhang et al. 2018). The TRPM2 inhibitor, JNJ-28583113, is a recently described inhibitor that appears promising, with nanomolar potency when applied to the extracellular surface of TRPM2 channels. However, is limited by stability difficulties in the brain (Fourgeaud et al. 2019). The inventors have discovered that the inhibitor, tatM2NX, reduces ischemic injury when administered following focal cerebral ischemia (Shimizu et al. 2016) and global cerebral ischemia (Dietz et al. 2019) in vivo, providing evidence for clinical benefit. Therefore, the inventors characterized the TRPM2 channel inhibitor tatM2NX, a peptide designed to interact with the ADPR binding site on the NUT9-H domain.

SUMMARY

TRPM2 channels have been implicated in ischemic neuronal damage for over a decade (for review, see Aarts MM, Tymianski M. Trpms and neuronal cell death. Pflugers Arch - Eur J Physiol. 2005;451 :243-249; see also US Patent Publication No. 2010/0298394, November 25, 2010), yet the field has been plagued by lack of an inhibitor specific for the channel. Non specific TRPM2 inhibitors, such as clotrimazole (CTZ), have been shown to reduce neuronal death in in vitro cortical and hippocampal neurons to reduce injury in male animals following focal and global cerebral ischemia. The present disclosure identities novel and specific peptide inhibitors of TRPM2 channels.

A distinguishing property of TRPM2 channels is that they are gated by ADPribose (ADPr), via binding to an ADPr hydrolase homology domain (termed “NUDT9-H”) in the C- terminus. The catalytic domain of NUDT9-H is the Nudix domain, which, in coordination with several distant amino acids within the same domain, form the ADPr binding pocket. The ADPr binding pocket was targeted as a strategy to inhibit TRPM2 channel activation. Expression of channels lacking the C-terminal Nudix homology domain are inactive. Therefore, peptides were generated, including GSREPGEMLPRKLKRVLRQEFWV (SEQ ID NO:1 ; “M2NX”), fused to the cell permeable TAT sequence, YGRKKRRQRRR (SEQ ID NO:2; “tat 47-57”) to form the 34- mer, YGRKKRRQRRRGSREPGEMLPRKLKRVLRQEFWV (SEQ ID NO:3; “tat-M2NX”), which specifically inhibits TRPM2 channel activity via interaction with the ADPr binding pocket of the NUDT9-H domain of the channel.

Because the modulation of TRPM2 ion channels has been shown to be significantly associated with cause and/or control of certain disorders and diseases, it is necessary to find agents which are safe and efficacious in inhibiting TRPM2 ion channels. Through a structure- activity relationship (SAR) study, it was found that tatM2NX is an antagonist of human TRPM2 channels using whole-cell patch clamp and calcium imaging in HEK293 cells. Mutagenesis of tatM2NX reveals that the mechanism of action results from tatM2NX C-terminal interactions with TRPM2. Therefore, the inventors generated truncated peptides of the C-terminus and N- terminus of NUDT9-H, fused to the cell permeable TAT sequence (SEQ ID NO:2) to form YGRKKRRQRRRKLKRVLRQEFWV (SEQ ID NO:4, “tat-Cterm”) and

YGRKKRRQRRRGSREPGEMLPR (SEQ ID NO:5, “tat-Nterm”). The interactions of tat-Cterm with the ADPR binding site were stable and the peptide itself moved further into the ADPR binding site over the simulation period. These results demonstrate that the C-terminus hydrophobic interactions with the ADPR binding site are sufficient to maintain the TRPM2-tat Cterm interactions.

Tat-M2NX is a cell-permeable inhibitor with high potency, which makes extensive non- covalent interactions and favorable contacts with a number of residues of the ADPR binding site within the NUDT9-H domain. Many of these interactions were predicted and maintained in the tat Cterm peptide. In contrast, there were no predicted interactions between tatWV-AA (YGRKKRRQRRRGSREPGEMLPR KLKRVLRQEFAA, SEQ ID NO: 16) and the ADPR binding site. Furthermore, both tatM2NX and tat Cterm exhibited competitive inhibition in cells, providing validation of the in silico predictions.

Thus, this disclosure provides methods of treating or preventing neurological damage or injury, or neurodegenerative diseases, or enhancing the restoration of neurological function, in a subject, by administering a pharmaceutical composition comprising a TRPM2-inhibitory peptide of this disclosure, to the subject.

This disclosure also provides methods of treating or preventing neurological damage or injury, or neurodegenerative diseases, or enhancing the restoration of neurological function, in a subject, by administering a pharmaceutical composition comprising a TRPM2-inhibitory peptide of this disclosure, or variants thereof, and at least one additional therapeutic agent to the subject. The additional therapeutic agent(s) may be one or more neuroprotective, neurorestorative or blood clot preventing or dissolving agents. In these methods, the administration may be by parenteral administration. In these methods, the peptide may be administered at a dosage of about 0.05 to about 25 mg/kg. In these methods, the subject may be a human.

One aspect is a pharmaceutical composition comprising a peptide of this disclosure, or a multimer, derivative, or variant thereof, and a pharmaceutically acceptable carrier for the treatment or prevention of neurological damage or injury, or neurodegenerative diseases, or enhancing the restoration of neurological function. These compositions may include at least one additional therapeutic agent, and therefore another aspect is a pharmaceutical composition comprising a peptide of this disclosure, or a multimer, derivative, or variant thereof, at least one additional therapeutic agent, and a pharmaceutically acceptable carrier for the treatment or prevention of neurological damage or injury, including stroke. A related aspect is the use of a peptide of this disclosure or a multimer, derivative, or variant thereof, for the treatment or prevention of neurological damage or injury, including stroke.

This Summary is neither intended nor should it be construed as being representative of the full extent and scope of the present disclosure. Moreover, references made herein to "the present disclosure," or aspects thereof, should be understood to mean certain embodiments of the present disclosure and should not necessarily be construed as limiting all embodiments to a particular description. The present disclosure is set forth in various levels of detail in this Summary as well as in the attached drawings and the Description of Embodiments and no limitation as to the scope of the present disclosure is intended by either the inclusion or non inclusion of elements, components, etc. in this Summary. Additional aspects of the present disclosure will become more readily apparent from the Description of Embodiments, particularly when taken together with the drawings.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows molecular modeling of tatM2NX with the human TRPM2 channel NUDT9- H domain. FIG. 1 A shows the predicted secondary structure of tat-M2NX after MD-based refinement. The peptide is oriented N-term (left) to C-term (right). FIG. 1 B shows the top three predicted TPRM2-tatM2NX complexes, arrowhead indicates the human ADPR binding site within the NUDT9-H domain. FIG. 1C shows the top scoring initial TPRM2-tatM2NX complex (left) and the same complex after 5ns of MD simulation (right).

FIG. 2 shows the peptide tatM2NX inhibits TRPM2 currents in a concentration- dependent manner. FIG. 2A shows the representative TRPM2 initial currents (ADPR,, 3-4nA) activated by 100mM ADPR (red), with 2mM tatM2NX (black), or 20mM CTZ (gray). FIG. 2B shows the TRPM2 current density at 0.15-10mM tatM2NX compared to TRPM2 current at 0.05mM tatM2NX (ineffective concentration control). FIG. 2C shows the dose-response curve showing normalized response (current density) vs. tatM2NX concentration. Potency was determined normalizing each concentration to TRPM2 current density with 0.05mM tatM2NX. All data represented as mean ±SD and significance established at p <0.05 for n>4-7 (at least 3 experimental days/condition) using One-way ANOVA.

FIG. 3 shows that TatM2NX inhibits TRPM2-mediated q8K3b signaling and competes with ADPR to antagonize TRPM2. FIG. 3A shows the co-immunoprecipitation of biotin-tagged tatM2NX with FLAG-tagged TRPM2 in doxycycline-inducible HEK293 cells (+Dox). No biotin- tatM2NX observed in un-induced cells (-Dox). At least 3 independent experimental days (n=3). FIG. 3B shows the Western blot of HEK293 cells expressing TRPM2 treated with 250mM H2O2 (+Con) compared to untreated cells (-Con) and cells pre-incubated with 2mM tatM2NX for 30min-4h followed by H2O2 stimulation (10min). FIG. 3C shows the ADPR_f (control) currents with 500mM ADPR in the presence of 0 or 2mM tatM2NX. No significant differences in ADPR, for control and 2mM tatM2NX. All data is represented as mean ±SD and significance established at p <0.05 for n>4 (at least 3 experimental days/condition) using Student’s t- test, ADPR_f= final ADPR current, ADPR,= initial ADPR current.

FIG. 4 shows the C-terminus of tatM2NX is sufficient to antagonize TRPM2. FIG. 4A shows the TRPM2 current density in the presence of 2mM tat Cterm (blue), tatWV-AA (sky blue), or tat Nterm (green). All peptides were individually compared to control group (ADPR_f). FIG. 4B shows the Representative images for Ca²⁺ imaging experiments at Time Omin and 20min for 250mM H₂0 (control) or H₂0 + 2mM tatM2NX. Fluo5F, AM (5mM, green) is the fluorescent Ca²⁺ indicator. FIG. 4C shows the Quantification of changes in fluorescence/baseline fluorescence (F/Fo) for each peptide (2mM). TatM2NX and tat Cterm significantly inhibit TRPM2 activity in HEK293 cells. F/F₀, corresponding to TRPM2 activity, is inhibited by 20mM CTZ (positive control). FIG. 4D shows the Area under the curve (AUC) analysis for each peptide: H2O2 control (black), tatM2NX (red), tat Cterm (blue), tatWV-AA (sky blue), tat Nterm (green). tatM2NX, tat Cterm, and CTZ significantly decreased AUC for TRPM2 activity, while tatWV-AA significantly increased AUC. All data is represented as mean±SD and significance established as p<0.05 using One-way ANOVA with Dunnett’s post-hoc for multiple group comparison, n>4-10 for electrophysiology and n>25-53 cells for Ca²⁺ imaging (4-6 independent experiments).

FIG. 5 shows delayed inhibition of TRPM2 using the truncated peptide C-term (SEQ ID NO:6) reverses synaptic plasticity deficits. FIG. 5A shows a time course of fEPSP slope from MALE mice 7 days after CA/CPR (blue) and in mice administered 10 mg/kg tat C-term 6 days after CA/CPR and recordings obtained 7 days after CA/CPR (red). FIG. 5B shows quantification of change in fEPSP slope 60 minutes after TBS stimulation normalized to 20 minutes of baseline recording.

FIG. 6 shows that TRPM2 protein is expressed and functional in doxycycline-inducible HEK293 cells. FIG. 6A shows that TRPM2 expression is detected in HEK293 cells treated with doxycycline (1 pg/mL) (Dox). No expression in un-treated HEK293 cells (No Dox). FIG. 6B shows that TRPM2 is expressed in HEK293 cells 16-18h after doxycyline treatment (n=3). Biotin-tatM2NX is found in cells within 1 h incubation in the media (yellow arrows). ICC: DAPI (blue), N-terminal FLAG-TRPM2 (green), N-terminal biotin-tagged tatM2NX (red). Scale bar is 25 pm. FIG. 6C shows that rundown (ADPR_f) current density is significantly decreased from ADPRi current, while CTZ completely inhibits ADPR_f. FIG. 6D shows that no TRPM2 activity is observed in HEK293 cells in the absence of doxycyline (No Dox). FIG. 6E shows that ADPR_f compared to 0.05mM tatM2NX (ineffective concentration, same group as Figure 2C). All data represented as mean±SD and significance established as p<0.05 for n>5-6 for electrophysiology.

FIG. 7 shows molecular modeling of tat Cterm and tatWV-AA with human TRPM2 channel NUDT9-H domain. FIG. 7A shows the top three scoring clusters of tat-M2NX (cyan), tat-Cterm (green), and tatWV-AA (red) in complex with a single monomer of TPRM2 (orange). FIG. 7B shows the Predicted secondary structure of tat-M2NX (cyan) compared to tat-Cterm (green) and tatWV-AA (red) after MD-based refinement. Peptides are oriented N-term (left) to C- term (right). FIG. 7C shows the top scoring initial TPRM2-tat-Cterm complex (left) and the same complex after 5ns of MD simulation (right). FIG. 7A shows the top scoring initial TPRM2-tatWV- AA complex (left) and the same complex after 5ns of MD simulation (right).

FIG. 8 shows there are no changes in ADPR, current density or frequency distribution in Ca²⁺ fluorescence in the presence of peptides. FIG. 8A shows the ADPR, current density was significant (p=0.0489, n=6) for the peptide tat-Cterm. FIG. 8B is a histogram of number of events vs. frames (1 frame=10sec, total 120 frames) in HEK293 cells expressing TRPM2 in response to H₂0₂ in the presence of peptides. All electrophysiology data represented as mean ±SD, statistical significance at p<0.05 for n>4-10 (at least 3 experimental days/condition). For Ca²⁺ imaging, the histogram was generated from n>25-53 (4-6 experimental days).

FIG. 9 Comparison between the tertiary structures of human TRPM2 (orange), human TRPM4 (blue), mouse TRPM7 (green), and TRPM8 (magenta) (PDB IDs: 6MIX, 6BQR, 5ZX5, and 6NR4, respectively). Arrow indicates the proposed binding site for tatMXN2 within the human ADPR binding pocket, which is not present in the other TRPM family members. This suggests a low potential for tatMXN2 to inhibit other TRPM family members. DETAILED DESCRIPTION

This disclosure provides TRPM2 inhibitors that act to disrupt the ligand (ADPribose)- binding pocket of the TRPM2 channel, thereby preventing activation. In one aspect, this disclosure relates to peptides and peptide constructs that are neuroprotective and neurorestorative, and the use of these peptides, and methods of administering such peptides to a subject suffering a neurological injury, neurological disorder, or neurodegenerative disease, or at risk of sustaining a neurological injury, or developing a neurological disorder or neurodegenerative disease.

Definitions

As used herein, the singular forms "a", "and", and "the" include plural referents, unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this technology belongs unless clearly indicated otherwise.

The terms “peptide” and “polypeptide” are used synonymously herein to refer to polymers constructed from amino acid residues.

"Substantially pure", as used herein (for example, in the context of a pharmaceutical composition), means that the peptide makes up greater than about 50% of the total content of the composition (e.g., total protein of the composition), or greater than about 80% of the total protein content. For example, a "substantially pure" peptide refers to compositions in which at least 80%, at least 85%, at least 90% or more of the total composition is the peptide (e.g. 95%, 98%, 99%, greater than 99% of the total protein). The peptide may make up greater than about 90%, greater than about 95%, greater than 98%, or greater than 99%, of the total protein in the composition. In some embodiments, a peptide is substantially pure when the peptide is at least 60% or at least 75% by weight free from organic molecules with which it is associated during production. In some embodiments, the peptide is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure. For example, in some embodiments, an immunomodulatory peptide is substantially pure when the immunomodulatory peptide is at least 60% or at least 75% by weight free from organic molecules with which the peptide(s) is associated during production, in some embodiments, the immunomodulatory peptide is at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, by weight, pure.

The terms "subject", "patient", and "individual", are used herein interchangeably, and refer to a multicellular animal (including mammals (e.g., humans, non-Human primates, murines, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc.), avians (e.g., chicken), amphibians (e.g. Xenopus), reptiles, and insects (e.g. Drosophila). "Animal" includes guinea pig, hamster, ferret, chinchilla, mouse, and rat. These terms specifically include humans, and specifically include male mammals, and therefore, human males.

The term “neuroprotective” as used herein, refers to any property of a peptide that may be evaluated, and/or, that reduces or inhibits, or would be expected to reduce or inhibit, death, apoptosis, destruction or injury to a neuron and/or reduces or inhibits neurodegeneration in a subject.

The term “neurorestorative” as used herein, refers to any property of a peptide that may improve brain function, synaptic function, neuron firing, brain network function, independent of changes in cell death. Neurorestorative applies to agents administered near the time of injury or at chronic timepoints to reverse stable deficits in brain function.

An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A "therapeutically effective amount" of a peptide or pharmaceutical composition comprising a peptide of this disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the peptide or composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the peptide or composition comprising the peptide are outweighed by the therapeutically beneficial effects.

Reference herein to any numerical range (for example, a dosage range) expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range. For example, but without limitation, reference herein to a range of 0.5 mg/kg to 100 mg/kg explicitly includes all whole numbers of and fractional numbers between the two.

An individual referred to as "suffering from" neurological injury, including stroke, TBI, cardiac arrest, as described herein, has been diagnosed with and/or displays one or more symptoms of neurological injury, including stroke.

As used herein, the term "at risk" for a neurological injury, including stroke, refers to a subject (e.g., a human) that is predisposed to developing stroke and/or expressing one or more symptoms of the disease. This predisposition may be genetic or due to other factors. It is not intended that the present disclosure be limited to any particular signs or symptoms. Thus, it is intended that the present disclosure encompasses subjects that are experiencing any range of neurological injury, including stroke, from sub-clinical to full-blown, wherein the subject exhibits at least one of the indicia (e.g., signs and symptoms) associated with neurological injury, including stroke.

The terms "treat," "treatment," or "treating", as used herein, refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., neurological injury, neurodegenerative disease). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

The terms "comprises" and "comprising", have the broad meaning ascribed to them in Patent Law and may mean "includes", "including" and the like.

This disclosure may be understood more fully by reference to the following detailed description and illustrative examples, which are intended to exemplify non-limiting embodiments of this disclosure.

Polypeptides

Peptides of this disclosure include fragments of the Nudix domain of the NUDT9-H region of the C-terminus of TRPM2 channels, or variants thereof, which specifically inhibit the TRPM2 channel activity via interaction with the ADPr binding pocket of the NUDT9-H domain of the channel. Thus, a subject peptide of this disclosure is GSREPGEMLPRKLKRVLRQEFWV (SEQ ID NO:1). Additional peptides are KLKRVLRQEFWV (C-terminus truncation, SEQ ID NO:6) and GSREPGEMLPR (N-terminus truncation, SEQ ID NO:7). Any of the peptides of this disclosure may be linked, preferably at the N-terminus, to an internalization peptide that facilitates translocation through the plasma membrane of a cell. Examples of these peptides include TAT derived from HIV (Vives et al., 1997, J. Biol. Chem. 272:16010; Nagahara et al., 1998, Nat. Med. 4:1449), antennapedia from Drosophila (Derossi et al., 1994, J. Biol. Chem. 261 :10444), VP22 from herpes simplex virus (Elliot and D'Hare, 1997, Cell 88:223-233), complementarity-determining regions (CDR) 2 and 3 of anti-DNA antibodies (Avrameas et al., 1998, Proc. Natl. Acad. Sci. U.S.A., 95:5601-5606), 70 KDa heat shock protein (Fujihara, 1999, EMBO J. 18:411 -419) and transportan (Pooga et al., 1998, FASEB J. 12:67-77). For example, the HIV TAT internalization peptide YGRKKRRQRRR (SEQ ID NO:2) may be used. One exemplary peptide of this disclosure, which includes the HIV Tat internalization peptide and an active peptide inhibitor of TRPM2 channel activity is:

YGRKKRRQRRR-GSREPGEMLPRKLKRVLRQEFWV (SEQ ID NO:3, “Tat-M2NX”).

Variants of the standard TAT sequence YGRKKRRQRRR (SEQ ID NO:2) may also be used. Although practice of this disclosure is not dependent on an understanding of mechanism, it is believed that both the capacity to cross membranes and binding to N-type calcium channels of TAT are conferred by the unusually high occurrence of positively charged residues Y, R and K in the peptide. Variant peptides for use in this disclosure should retain ability to facilitate uptake into cells but have reduced capacity to bind N-type calcium channels. Some suitable internalization peptides comprise or consist of an amino acid sequence XGRKKRRQRRR (SEQ ID NO:8), in which X is an amino acid other than Y. A preferred TAT variant has the N-terminal Y residue substituted with F. Thus, a TAT variant comprising or consisting of FGRKKRRQRRR (SEQ ID NO:9) may be used. Another preferred variant of the TAT internalization peptide consists of GRKKRRQRRR (SEQ ID NO:10). If additional residues flanking XGRKKRRQRRR (SEQ ID NO:10) are present (beside the active peptide), the residues may be for example, natural amino acids flanking this segment from a TAT protein, spacer or linker amino acids of a kind typically used to join two peptide domains, e.g., Gly(Ser)4 (SEQ ID NO:11), TGEKP (SEQ ID NO:12), GGRRGGGS (SEQ ID NO:13), or LRQRDGERP (SEQ ID NO:14) (see, e.g., Tang et al. (1996), J. Biol. Chem. 271 , 15682-15686; Hennecke et al. (1998), Protein Eng. 11 , 405- 410)), GSRVQIRCRFRNSTR (SEQ ID NO:15) (see U.S. Patent Publication No. 2014/0235553; August 21 , 2014), or may be any other amino acids that do not detectably reduce capacity to confer uptake of the variant without the flanking residues and do not significantly increase inhibition of N-type calcium channels relative to the variant without the flanking residues. Preferably, the number of flanking amino acids, other than an active peptide, does not exceed ten residues on either side. Preferably, no flanking amino acids are present and the internalization peptide is linked at its C-terminus directly to an active TRPM2 inhibitor peptide of this disclosure.

A “variant” of a peptide described herein is a polypeptide that is substantially similar to a polypeptide disclosed herein and retains at least one TRPM2 inhibitor property or neuroprotective activity of the peptides of this disclosure. Variants may include deletions (i.e., truncations) of one or more amino acid residues at the N-terminus or the C-terminus of a polypeptide disclosed herein; deletion and/or addition of one or more amino acid residues at one or more internal sites in the polypeptide disclosed herein; and/or substitution of one or more amino acid residues at one or more positions in the polypeptide disclosed herein. For polypeptides that are 12 amino acid residues in length or shorter, variant polypeptides preferably include three or fewer (e.g., two, one, or none) deleted amino acid residues, whether located internally, at the N-terminal end, and/or at the C-terminal end.

Accordingly, the inventive methods and compositions are likewise contemplated for neuroprotective polypeptides that are at least 50% identical (e.g., have at least 60%, 70%, 80%, 90%, 95% or more sequence identity) to the TRPM2 inhibitory polypeptides disclosed herein and that retain at least one neuroprotective property of SEQ ID NO:4. Ordinarily, a protein 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 a full-length native sequence protein sequence as disclosed herein, or any other specifically defined fragment of a full-length protein sequence as disclosed herein. Optionally, the variant polypeptides will have no more than one conservative amino acid substitution as compared to the native protein sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to the native protein sequence.

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.

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

Modified polypeptides Also contemplated in the context of the inventive methods and compositions is the modification of any neuroprotective polypeptides 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 polypeptides 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 polypeptides may be modified by glycosylation and/or phosphorylation.

In addition, the polypeptides 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 polypeptides of this disclosure may be modified by PEG polymers and the like. PEG or PEG polymers means a residue containing polyethylene 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; 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 side-chains 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, C, 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, F. M., et al., Journal of Bioactive and Compatible Polymers 12 (1997) 196-207. Suitable processes and preferred reagents for the production of 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) 405-417, 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.

Additionally or alternatively, 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, 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.

The polypeptides 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.

This disclosure also provides nucleic acid molecules which encode a neuroprotective peptide of this disclosure, preferably an active inhibitor of the TRPM2 ion channel, as defined herein and which has at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length inhibitor peptide sequence of this disclosure. Ordinarily, a variant polynucleotide of this disclosure will have at least about 80% nucleic 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% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length TRPM2 inhibitor protein sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.

Ordinarily, variant polynucleotides are at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length. Variant polypeptides of this disclosure may be those that are encoded by a variant polynucleotide of this disclosure.

These polynucleotides may include control sequences, which are DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

This disclosure also provides isolated peptide inhibitors of the TRPM2 ion channel. "Isolated," when used to describe the various peptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the protein natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

The peptides of this disclosure may include "epitope tagged" peptides, which refers to a chimeric polypeptide comprising a TRPM2 inhibitor peptide of this disclosure fused to a "tag polypeptide." The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the inhibitory polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

The peptides of this disclosure may be linked to or associated with a "solid phase" or “solid support” which is a non-aqueous matrix to which a peptide of this disclosure can adhere or attach. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.

"Active" or "activity" for the purposes herein refers to peptides that inhibit the activity of a TRPM2 ion channel, such as reducing the flux of calcium ions across the TRPM2 ion channel. This disclosure also provides “antagonists” of TRPM2 ion channels, including any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native TRPM2 protein disclosed herein. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TRPM2 proteins, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying antagonists of a TRPM2 protein may comprise contacting a TRPM2 protein with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TRPM2 ion channel protein.

T reatment/Therapy

In certain embodiments, 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 stroke, TBI or cardiac arrest or one or more symptoms associated with stroke, brain injury or neurological damage following stroke, brain injury in a subject. The methods and compositions are also useful to treat and/or prevent neurological damage resulting from cerebral ischemia, for example global cerebral ischemia following cardiac arrest. The methods and compositions are also useful to treat traumatic brain injury (TBI). Additionally, the methods and compositions may also be useful to aide in a patient’s recovery from these neurological injuries, 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. Indeed, data indicates that delayed administration of the active peptides of this disclosure improves memory in both males and females following stroke, cardiac arrest, and TBI.

Additionally or alternatively, the methods and compositions may be useful to treat and/or prevent a neurodegenerative disorder, peripheral neuropathy, or neuropathic pain, wherein the neurodegenerative disorder is selected from Alzheimer's Disease, Multiple Sclerosis, HIV- associated dementia, Huntington's Disease, Parkinson's Disease, and Amyotrophic Lateral Sclerosis. Data indicates that TRPM2 channels play a role in the development of neurodegenerative diseases, as TRPM2 channels are activated under conditions of oxidative stress and consequently contribute to injury and dysfunction. For example, Parkinson’s Disease and Alzheimer’s Disease are both neurodegenerative disorders in which oxidative stress has been strongly implicated, making a role for TRPM2 in the etiology of these disorders logical. Thus, this disclosure also provides methods and compositions that are useful in treating and/or preventing neurodegenerative disorders including Parkinson’s Disease and Alzheimer’s Disease.

Additionally or alternatively, the methods and compositions may be useful to enhance cognitive function in a subject. For example, the methods and compositions may be administered to a subject to enhance synaptic function and/or enhance memory. These effects may reduce or slow the progress of a neurodegenerative disorder, or enhance recovery from a neurological injury.

Additionally or alternatively, the methods and compositions may be useful to treat and/or prevent inflammation, ischemia, atherosclerosis, asthma, autoimmune disease, diabetes, arthritis, allergies, transplant rejection, infection, pain from diabetic neuropathy, gastric pain, postherpetic neuralgia, fibromyalgia, surgery, or chronic back pain.

In these methods, the subject may be human. The subject may be male or female. In specific embodiments, the subject may be a human male.

These treatment methods comprise administering to a subject a pharmaceutical composition comprising a peptide of this disclosure. In certain embodiments, the treatment methods further comprise inhibiting the activity of TRPM2 in the subject (by at least 10% (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more).

These methods may include the administration of a peptide of this disclosure to a subject having a neurological injury, including a stroke, or suspected of having a neurological injury, after the injury has been sustained by the subject. The peptide may be first administered to the subject within a month of the time the neurological injury occurred. Preferably, the peptide is first administered to the subject within 96 hours, or 8 days, of the time the neurological injury occurred. More preferably, the peptide is first administered within a time period of 1 hour to 96 hours of the time neurological injury occurred. More preferably, the peptide is first administered within a time period of 1 minute to 5 hours of the time the neurological injury occurred.

Combination therapy

Additionally, disclosed herein are methods of treatment (and compositions) in which the neuroprotective peptides of this disclosure (or pharmaceutical compositions comprising such peptides) may be administered in combination with at least one other drug or therapy currently known or later discovered to be effective in the prevention and/or treatment of stroke, or neurological damage following stroke. The drug may be an anticoagulant or clot-dissolving medicine, such as aspirin, clopidogrel or tissue plasminogen activator (tPA). The drug may be an ACE Inhibitor, such as Lisinopril, or a blood thinner, such as warfarin, or heparin, or apixaban, or a statin, such as atorvastatin or rosuvastatin, or irbesartan, or reteplase, or alteplase.

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.

The neuroprotective/neurorestorative peptides of this disclosure may be administered prior to, concurrently with, or after the administration of the additional drug 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.

Thus, by "in combination with," it is not intended to imply that the peptides 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.

In general, each agent (in this context, one of the "agents" is a peptide of this disclosure) 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.

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.

Diagnosis

In one embodiment, the inventive treatment method additionally comprises diagnosing a subject with a neurological injury, disorder or neurodegenerative disease, or, during treatment, diagnosing, or evaluating or monitoring the efficacy of the treatment method. Stroke may be diagnosed by medical history and physical exam, brain computed temography, magnetic resonance imaging, computed tomography arteriogram and magnetic resonance arteriogram, carotid ultrasound, carotid angiography, EKG (Electrocardiogram), Echocardiography, and/or blood tests.

Compositions for treating and administration

Compositions for treating neurological injuries, diseases, and disorders, and enhancing cognitive functions of this disclosure may be formulated according to any of the conventional methods known in the art and widely described in the literature. Thus, the active ingredient (e.g., a peptide of this disclosure) may be incorporated, optionally together with other active substances, with one or more conventional pharmaceutically acceptable carriers, diluents and/or excipients, etc., appropriate for the particular use of the composition, to produce conventional preparations that are suitable or may be made suitable for administration. Carriers may include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. They may be formulated as liquids, as semi solids or solids, liquid solutions, dispersions, suspensions, and the like, depending on the intended mode of administration and therapeutic application. In some embodiments, the inventive composition is prepared in a form of an injectable or infusible solution. Peptides of this disclosure may be formulated in a "liposome" which is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as an inhibitory peptide of this disclosure) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

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. By mixing the neuroprotective polypeptide(s) in the pharmaceutical composition with serum album, the neuroprotective polypeptides may be effectively “loaded” onto the serum albumin, allowing a greater amount of neuroprotective polypeptide to be successfully delivered to a site of neurological injury.

Methods of treating neurological injuries, diseases, or neurodegenerative diseases of this disclosure may include administration of a peptide of this disclosure via any one of a variety of routes, including intravenous (IV), intramuscular (IM), intra-arterial, 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 and 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.

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%.

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.

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 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.

Thus, suitable doses of the peptide 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 a prophylactically effective amount, depending on the nature of the treatment involved. In related embodiments, the dosages constitute a neuro-restorative- or rehabilitation-enhancing amount. The ability of the peptide to elicit a desired response in the individual will also be a factor. Exemplary daily doses are: 0.1 to 250 mg/kg, or 0.1 to 200 or 100 mg/kg, or 0.5 to 100 mg/kg, or 1 to 50 or 1 to 10 mg/kg, of the active ingredient. 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.

Kits for treating neurological injuries, neurological disease, or neurodeaenerative diseases

In one aspect, this disclosure further provides kits for the treatment of neurological injury, neurological diseases, or neurodegenerative diseases comprising 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.

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.

EXAMPLES

The following methods were used to conduct the experiments described in Examples 1- 4, below:

Protein Structure Prediction and Molecular Modeling: All molecular modeling studies were conducted using Biovia Discovery Studio 2018 (Biovia, Inc, Sand Diego, CA; www.3dsbiovia.com) and YASARA Structure 18.4 (YASARA Biosciences GmbH, Vienna, Austria; www.yasara.org). Structural coordinates for the human TRPM2 ion channel (Wang et al. 2018) were downloaded from the Protein Data Bank (www.rcsb.org, PDB accession: 6MIX). Ab initio prediction of the secondary and tertiary structures for the wild-type, truncated, and mutant peptides was performed using the online QUARK server (https://zhanglab.ccmb.med.umich.edu/QUARK/) (Xu and Zhang 2012, 2013). The top 5 predicted peptide structures for each designed peptide were subjected to 1 nanosecond (ns) of explicit solvent-based molecular dynamic (MD) simulation utilizing the YASARA2 force field (Krieger et al. 2006; Krieger et al. 2009; Krieger et al. 2012; Krieger and Vriend 2015), which combines the AMBER (ff 14SB) force field (Maier et al. 2015) with self-parameterizing knowledge-based potentials (Krieger, Koraimann, and Vriend 2002), to refine each of the predicted peptide structures. The snapshots from the resulting trajectories were assessed using the WHATJF and WHAT CHECK (Hooft et al. 1996; Vriend 1990) structure validation tools to quantitatively evaluate the overall quality of each predicted structure, with the highest scoring structure for each peptide selected for further analysis. The ZDOCK (Chen, Li, and Weng 2003), ZRANK (Pierce and Weng 2007), and RDOCK (Li, Chen, and Weng 2003) algorithms were employed within Discovery Studio to predict the most likely protein-peptide complexes and refine their respective intermolecular interactions, as we have described previously (Ryan et al. 2012; Smith et al. 2018). To test the stability of the predicted interactions, we first removed the transmembrane domain of the TRPM2 subunit (residues 697-1165) and the top scoring complex for each peptide was placed in a simulation cell under periodic boundary conditions, filled with water, 0.9% NaCI and counter ions, pH7.4, at a temperature of 298K (Krieger et al. 2004). The main MD simulation was run for 5 ns using the AMBER (ff 14SB) force field (Maier et al. 2015) with GAFF (Wang et al. 2004) / AM1 BCC (Jakalian, Jack, and Bayly 2002) parameters, particle mesh Ewald (PME) summation, an 8.0 A cutoff for non-bonded forces, a 5 fs time-step, LINCS- constrained hydrogen atoms (Hess et al. 1997), and at constant pressure and temperature (the NPT ensemble), as described previously (Krieger and Vriend 2015). Figures were generated using Lightwave 2019 (NewTek Inc, Burbank, CA; www.lightwave3d.com) and Marmoset Toolbag 3.07 (Marmoset, LLC, Portland, OR; www.marmoset.co).

Cell Culture: Doxycycline-inducible N-terminal FLAG-TRPM2-expressing human embryonic kidney (HEK293) cells, provided by Anne L. Perraud (University of Colorado Anschutz Medical Campus, CO, USA), were cultured as previously described (Perraud et al. 2001 ; Shimizu et al. 2016). Briefly, cells were grown in Advanced DMEM medium containing 10% fetal bovine serum, 2mM Glutamatax (Life technologies, Carlsbad, CA, USA), and MycoZap-Plus (Lonza, Switzerland). Cell line authentication was confirmed as female human embryonic kidney cells and mycoplasma contamination was negative (BioResources Core, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA). During growth, selection markers Zeocin (1pg/ml) (Invitrogen, Carlsbad, CA, USA) and Blastocidin-S (0.4pg/ml) (Gibco, Carlsbad, CA, USA) were utilized for the selective expression of Tet repressor and human TRPM2 in HEK293 cells until 80-90% confluence. Cells were grown for up to 20 passages (P3- P20). For Western blot experiments, HEK293-derived cells were maintained in Zeocin and Blastocidin-S (un-induced cells) or in doxycycline (1pg/mL) (induced cells) for 16-18h prior to protein lysate collection. For electrophysiology experiments, HEK293 cells were seeded on 12mm glass coverslips at a density of 12,000 cells/ml for 16-24h for doxycycline-inducible human TRPM2 expression prior to experiments. For Ca2+ imaging experiments, HEK293 cells were seeded on Mattek glass bottom dishes (Mattek Corporation, Ashland, MA, USA) at a density of 25,000 cells/ml for 16-24h prior to experiments.

Western blot: HEK293 cells were collected 16-18h after induction via centrifugation at 3,000rpm for 3min, washed in phosphate buffered saline (1xPBS, pH 7.4), and lysed for 10min using neuronal protein extraction reagent (Thermo Scientific, Rockford, IL, USA). For Q5K3b expression and phosphorylation, HEK293 cells were pre-incubated with tatM2NX for 30min-4h followed by 250mM H202 stimulation (10min) and protein lysates collected immediately. Lysates were centrifuged at 12,000rpm for 15min and supernatant collected for protein quantification. Protein samples (15-20pg) were resolved using SDS-PAGE, and transferred in PVDF membranes (b-actin, 50min; FLAG-TRPM2, 90min; ΰ5K3b, 50min). PVDF membranes were blocked in 5% bovine serum albumin for 1h and incubated overnight at 4°C in primary antibody. Human TRPM2 expression was assessed with mouse anti-FLAG (1 :1000, F1804, Sigma, St. Louis MO, USA) (Brizzard, Chubet, and Vizard 1994), and normalized to mouse anti^-actin peroxidase (1 :10000, A3854, Sigma, St. Louis, MO, USA). ΰ5K3b phosphorylation and expression was assessed with rabbit anti-pGSK3 or ΰ5K3b (1 :1000, 9323S; and 1 :1000, 12456S, Cell Signaling Technology, Danvers, MA, USA). All membranes were washed 3 times, incubated in secondary horseradish peroxidase-conjugated goat-anti mouse or goat-anti rabbit antibody (1 :10000, 115-035-174 or 115-035-003, ImmunoResearch Laboratories, USA; or 1 :10000, 31460, ThermoFisher Scientific, USA) for 1h at room temperature. Western blot bands were detected using the SuperSignal™ West Femto Maximum Sensitivity Substrate (34096, ThermoFisher Scientific, USA) and imaged with BioRad ChemiDoc MP Imaging System (Hercules, CA, USA).

Immunocytochemistry: Doxycycline-inducible human TRPM2-expressing HEK293 cells (16-18h) were fixed with 4% paraformaldehyde for 10min on ice, washed in 1xPBS, permeabilized using 0.3% TritonX-100 dissolved in 1xPBS for 10min at room temperature, and blocked in 4% bovine serum albumin overnight at 4°C. For biotin-tatM2NX detection, HEK293 cells were incubated with the peptide for 1 h prior to fixation. Primary antibody (mouse-anti- FLAG, 1 :1000, F1804, Sigma, St. Louis MO, USA) was incubated for 2h at room temperature in 2.5% BSA/PBS and secondary antibody (594-conjugated streptavidin, 1 :1000, 016-540-084; or 488-donkey-anti-mouse, 1 :1000, 715-545-150; ImmunoResearch Laboratories, West Grove,

PA, USA) for 1 h at room temperature in 2.5% BSA/PBS. Then, coverslips were mounted using Prolong Gold Antifade Agent (Thermo Fisher Scientific, USA). Images of HEK293 cells were taken at room temperature on an Olympus IX83 (Olympus Fluoview FV1200 Laser Scanning Confocal Microscope; Olympus Life Science, MA, USA) using a 20x objective. Two individual observers analyzed all images.

Co-immunoprecipitation: Protein lysates (500pg) from doxycycline-inducible human TRPM2-expressing HEK293 cells (16-18h) containing a N-terminal FLAG tag were incubated for 1h with N-terminal biotin-tagged tatM2NX (20pg) following addition of prewashed streptavidin- conjugated agarose (60mI, 16-126, Sigma Aldrich, St. Louis, MO, USA) at 4°C on a rocker. Samples were washed three times with phosphate buffer (100mM NaCI, pH 7.4). Purified biotin- tatM2NX complexes with FLAG-TRPM2 were boiled at 95°C for 5min in Laemmli dye (40-50mI, BioRad, USA). Bands were resolved using Precast Miniprotean gradient gels (4-20% acrylamide, BioRad, USA) cut in half and transferred 30min for biotin-tatM2NX (5kDa) and 90min for FLAG-TRPM2 (170kDa) in PVDF membranes (0.22 or 45pm). FLAG-TRPM2 was probed using primary mouse anti-FLAG M2 antibody (F1804, Sigma Aldrich, USA), and secondary horseradish peroxidase-conjugated goat anti-mouse (1 :10000, 115-035-003 or 111- 035-003 ImmunoResearch Laboratories, USA). See Western blot for membrane development.

Peptide synthesis: Tat-M2NX and derivatives were synthetized commercially with >95% purity (Chi Scientific, Maynard, MA, USA). All peptides contained a cell-permeant N- terminus tat-HIV sequence (YGRKKRRQRRR) fused to the M2NX sequence variations. For co- immunoprecipitation, biotin was conjugated to tatM2NX.

Electrophysiology: All recordings were performed in HEK293 cells and currents amplified with Axopatch 200B (Axon Instruments, USA), digitized with DigiData1550B, and controlled using pClamp10.7 software (Molecular Devices, CA, USA). Signals were filtered at 5kHz and digitized at 1 kHz. Glass borosilicate electrodes (3.5-5MW) were used to record human TRPM2 currents activated by +40mV voltage-step from OmV in whole-cell voltage clamp configuration. The protocol was chosen to produce an outward current and therefore reduce TRPM2-mediated calcium influx and subsequent cell death. All experiments were performed with HEPES-buffered saline containing in mM: 140 NaCI, 2.5 KCI, 10 HEPES, 5 glucose, 1 MgCI2, 1 CaCI, pH 7.4. The internal solution contained in mM: 145 K-gluconate, 0.05 EGTA (for physiological buffering of Ca²⁺ at 100-200nM), 1 MgCI2, 10 HEPES, pH 7.3. For antagonism, the internal solution contained 100mM ADPR (A0752, Sigma Aldrich, St. Louis, MO, USA) and 0, 0.05, 0.15, 0.3, 0.5, 2, 5 or 10mM tatM2NX. For competitive antagonism, the internal solution contained 500mM ADPR and 0 or 2mM tat-M2NX. For structure-activity relationship (SAR) experiments, the internal solution contained 100mM ADPR and 2mM peptide (tatWV-AA, tat- Cterm, tat-Nterm). We added clotrimazole (CTZ, 20mM, C6019-G, Sigma, St. Louis, MO, USA) after steady-state inhibition as a positive control. Exclusion criteria was based on access resistance Ra<15MQ and remaining leak current <350pA after CTZ inhibition.

Ca2+ Imaging: Live cell imaging of HEK293 cells was performed at room temperature on an Olympus IX83 (Olympus Fluoview FV1200 Laser Scanning Confocal Microscope; Olympus Life Science, MA, USA) using a 10x objective. For all imaging experiments in this study, controls and peptides were tested each experimental day to account for passage differences. All images were processed using FIJI Software (Rueden et al. 2017). Each plate was washed 3 times with HEPES-buffered saline (see electrophysiology above). A total volume of 2ml was added to each plate with 5mM of the Ca²⁺ indicator Fluo5F, AM (Invitrogen, Eugene, OR, USA), with or without 2mM peptide (tatWV-AA, tat-Cterm, tat-Nterm), or 20mM CTZ and incubated at 37°C for 40-50min prior to experiments. Then, plates were washed and fresh solution added with or without drugs. Excitation illumination was delivered every 10 sec for 20min. After 1min baseline, TRPM2 activity was stimulated using 250mM H2O2 and fluorescence recorded for 20min.

Statistical Analysis: For analysis of steady-state inhibition of TRPM2 currents, a paired or independent samples t-test was performed with statistical significance of p<0.05 for n>4-10. Potency (IC50) was determined using a non-linear regression analysis (Log of inhibitor concentration versus normalized response) equation: Y=100/(1 +10^A((X-LoglC50))); x= log of concentration, y=normalized response (current density pA/pF), using Graph Pad Prism version 8.0.2 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com. For Ca²⁺ imaging, changes in F/F0 (n=6-12 individual cells/plate) was used to compare groups after normalizing to background fluorescence. Individual ROI were drawn for each cell and analyzed using Time Series Analyzer plugin in FIJI Software (Rueden et al. 2017). Area under the curve for each group was determined and groups were compared to control H202 using One-Way ANOVA with Dunnett’s post-hoc test for multiple group comparison. Statistical significance was determined as p <0.05 and all groups represented as mean ±SD.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of this disclosure.

Example 1

Modeling for tat-M2NX antagonism on TRPM2 To characterize the mechanism of TRPM2 inhibition by tat-M2NX, molecular modeling (MD) was performed to allow prediction of tatM2NX interaction/inhibition of activity and potential key residues responsible for efficacy. Ab initio prediction of the secondary and tertiary structure of the parent tat-M2NX peptide was predicted to adopt a single, alpha helical structure that remained stable during MD-based refinement and analysis (FIG. 1 A).

Protein-peptide complex prediction for tatM2NX indicated that the top scoring cluster for the peptide involved direct interaction with the human ADPR binding site (FIG. 1B), which suggests occlusion of this site and the resulting inhibition of ADPR binding as a potential mechanism for the observed functional inhibition of TRPM2 in vitro. To test the stability of the predicted complex, we performed a 5ns MD-based simulation in the presence of an explicit solvent. The TRPM2-tat-M2NX complex remained stable throughout the simulation and appeared to settle further into the ADPR binding site and enhance the number of favorable interactions between tat-M2NX and TRPM2 compared to the initial predicted complexes (FIG. 1C). At the end of the MD simulation period, tat-M2NX made several favorable intermolecular interactions, including salt-bridges with Arg1280 and Arg1433 as well as an extensive network of intermolecular hydrogen bonds and hydrophobic interactions. Tat-M2NX also appeared to form a wedge-like conformation, with the C-terminus portion interacting with the ADPR binding site and the Tat-HIV tag (N-terminus) interacting with the opposite side the TRPM2 monomer (FIG 1C).

Example 2

Human TRPM2 channels are expressed and functional in HEK293 cells

TRPM2 protein expression and function was measured in HEK293 cells. Human TRPM2 (N-terminal FLAG tag) is expressed in HEK293 cells after doxycycline treatment (16-18h) (FIGs. 6A, 6B). To validate human TRPM2 channel function, we performed whole cell patch-clamp experiments in HEK293 cells with ADPR present in the pipette solution to stimulate TPRM2- mediated currents. We found that human TRPM2 exhibited large initial current density (ADPR,, 184.4 ± 63.73 pA/pF, n=5) exclusively under doxycycline treatment (FIGs. 2A, 6C). A significant rundown of TRPM2 initial current density (ADPR_f, 86.45 ± 46.68 pA/pF, n=5; p<0.05) was observed, which reached steady-state levels after 2-3min, as previously described (Toth and Csanady 2012) (FIG. 6C). Therefore, subsequent experimental analysis was performed using the steady state current density at 3min as the active TRPM2 current (ADPR_f). Multiple control experiments were performed to confirm the ADPR-induced current in HEK293 cells were carried by TRPM2 channels. Importantly, the TRPM2 channel pore-blocker Clotrimazole (20mM CTZ) abolished ADPR currents (6.359 ± 3.666 pA/pF, n=5; p<0.05) (FIG. 6C). Also, un-induced (no- doxycycline treatment) cells lacked TRPM2 expression or activity (0.58 ± 0.765 pA/pF vs 0.09± 1 .67 pA/pF, p>0.05) (FIGs. 6A, 6D). These results validate TRPM2 expression and function in doxycycline-treated HEK293 cells.

Example 3

TatM2NX is a potent TRPM2 antagonist

To characterize tat-M2NX as an antagonist, a dose-response study was performed. To achieve a known concentration of ligand and antagonist within each HEK293 cell, whole-cell patch clamp was used with 100mM ADPR and tat-M2NX added to the internal solution (pipette) allowing them to freely dialyze into the cell once whole cell access was obtained. These results show a concentration-dependent decrease in TRPM2 current density. Approximately 75% inhibition of initial TRPM2 current at 0.5mM tatM2NX (37.82 ± 53.19 pA/pF, n=5; p<0.05) was observed, and approximately 90% inhibition at 2mM (10.27 ± 9.52 pA/pF, n=7; p<0.05), 5mM (7.76 ± 6.387 pA/pF, n=5; p<0.05), and 10mM tatM2NX (13.17 ± 13.36, n=4; p<0.05) (FIGs. 2A, B). This inhibition was reached within 6-8min. No significant differences were observed for 0.15mM (121.6 ± 62.65 pA/pF, p>0.05), 0.3mM (118.0 ± 50.68 pA/pF, p>0.05) tatM2NX compared to 0.05 mM (142.2 ± 90.81 pA/pF, n=7) (FIG. 2B), which was similar to ADPR alone (ADPR_f, 86.45 ± 46.68 pA/pF, n=5, p>0.05) (FIG. 6E). A non-linear regression method (log inhibitor concentration versus normalized response) of the tatM2NX dose-response estimated a potency (IC₅o) of 3.96e-007 ± 0.05868 (396nM) (FIG. 2C). These results demonstrate that tat- M2NX is a potent antagonist of TRPM2 currents in the presence of ADPR.

To validate tatM2NX binding to TRPM2 as predicted in the molecular modeling, co- immunoprecipitation was performed using the N-terminal biotin-tag on tat-M2NX. To do this, whole-cell protein lysates were extracted from HEK293 cells and incubated protein lysates (500pg) with biotinylated-tat-M2NX (20pg). The peptide biotin-tat-M2NX formed a complex with FLAG-TRPM2 when co-immunoprecipitated using streptavidin-conjugated agarose (FIG. 3A). This binding interaction is specific to HEK293 cells expressing TRPM2 (lane 3) and absent in un-induced cells (lane 4) or beads without biotin-tatM2NX (lane 1). These results demonstrate that tat-M2NX directly interacts with TRPM2.

The ability of tat-M2NX to inhibit TRPM2-mediated dephosphorylation of q8K-3b (activation) was next assessed, as this signaling pathway has recently been shown in cell culture (Fourgeaud et al. 2019) and brain slices (Dietz et al. 2019). TRPM2 stimulation with H₂q₂(250mM) activates qdK3b (dephosphorylation) in HEK293 cells (+control) compared to untreated cells (-control) (FIG. 3B). Pre-incubation with tatM2NX (2mM) for 2-4h prevents qdK3b activation indicating that tatM2NX inhibits TRPM2-mediated qdK3b signaling (FIG. 3B).

To determine whether tatM2NX acts as a competitive antagonist for human TRPM2 channels, the antagonistic capacity of 2mM tatM2NX in the presence of 500mM ADPR was tested. In these ligand saturating conditions, ADPR, were similar in control (173.1 ± 45.22 pA/pF, n=4) and in the presence of 2mM tatM2NX (193.4 ± 78.00 pA/pF, n=4) (FIG. 3C). Interestingly, 2mM tatM2NX failed to inhibit ADPR_f current density (89.22 ± 42.22 pA/pF, n=4; p>0.05) compared to control (117.0 ± 10.53, n=4) indicating that higher concentrations of ADPR can prevent tatM2NX antagonism (FIG. 3C). These results demonstrate that tat-M2NX is a competitive antagonist of TRPM2 and serves to validate the results of the molecular modeling simulations.

Example 4

TatM2NX C-terminus is sufficient to antagonize TRPM2 in cells

Based on the initial molecular modeling of tatM2NX in complex with TRPM2, two bulky hydrophobic residues were identified, tryptophan in position 33 and valine in position 34 (W33, V34) within the C-terminus that remained buried within the ADPR binding site throughout the molecular modeling simulation. It was hypothesized that mutating these two residues would potentially abrogate the binding of tat-M2NX to the ADPR binding site. Molecular modeling of tat-M2NX with W33A and V34A mutations to W33V34 (tat-WVAA) indicated a loss of interaction with the ADPR binding site in any of the top 3 scoring interaction clusters (FIG. 7A). No substantive interactions were observed with this site in any of the lower scoring results. Additional computational simulations were performed to assess the capacity of a truncated form of tat-M2NX (tat-Cterm; containing the last 23 amino acids, SEQ. ID No:4) to preserve these interactions with the ADPR binding site. The peptide tat-Cterm interacts with the ADPR binding site similarly to the parent peptide tatM2NX (FIGs. 7A, 7C). This suggested that the C-terminus of tat-M2NX was sufficient to interact with the ADPR binding site and would likely recapitulate the inhibitory effects of the parent peptide tat-M2NX on TRPM2. Similar to tat-M2NX, the tat- Cterm peptide was predicted to maintain and enhance interactions with the ADPR binding site over the simulation period (FIGs. 7B, 7C). In contrast, the interactions of the tat-WVAA peptide did not appear stable and had substantial dissociation from TRPM2 (FIGs. 7B, 7D) suggesting that tatWV-AA lacks physiologically relevant interactions with TRPM2. The overall structure of both tat-Cterm and tatWV-AA peptides were highly similar in terms of secondary/tertiary structure (FIG. 7B). FIG. 9 sets for a comparison of TRP proteins from different species.

Our molecular modeling predicted that stable interactions of tat-Cterm and that mutating W33V34 to AA would result in a loss of interaction with the ADPR binding site. To validate the molecular modeling results, we performed electrophysiology and cell-based functional assays using Ca²⁺ imaging. In whole-cell patch clamp, the peptide tat-WVAA contained within the C- terminus lacks antagonism on TRPM2 (116.3 ± 66.90 pA/pF, n=6; p>0.05) compared to ADPR_f (89.39 ± 32.38 pA/pF, n=5), indicating that either/both residues are required to confer antagonism (FIG. 4A). Because the tat-Cterm peptide clustered by the ADPR binding site, we tested N- and C-termini truncated peptides (23 amino acids each) to determine the extent of antagonism by these truncated forms of tatM2NX (Table 1). In whole-cell patch clamp experiments, tat-Cterm displayed significant antagonism (15.56 ± 7.52 pA/pF, n=6; p< 0.05) compared to ADPR_f (FIG. 4A). The N-terminus truncation (tat Nterm) did not inhibit TRPM2 (141 .2 ± 31 .47 pA/pF, n=4; p<0.05) compared to ADPR_f (FIG. 4A). These results suggest that tatM2NX C-terminus is sufficient to inhibit TRPM2 while the N-terminus alone lacks antagonistic effect. Also, these confirms that the tat-HIV tag, and not the N-terminus sequence, is responsible for direct interactions with TRPM2 and requires the C-terminus residues W33V34 in proximity to the ADPR binding site. Lastly, all ADPR, currents were similar in the presence of peptides compared to control (FIG. 8A).

To verify that tatM2NX was cell permeable, we incubated biotin-tatM2NX with HEK293 cells for 1 h, followed by immunocytochemistry for N-terminal tagged biotin-tatM2NX and N- terminal tagged FLAG-TRPM2 within HEK293 cells (FIG. 6B). To test whether these peptides show antagonistic effects when applied extracellularly, we measured Ca²⁺ in HEK293 cells in response to H₂0₂ stimulation. H₂0₂ stimulated Ca²⁺ influx through TRPM2 in control group (77,441 ± 34,790 F/F₀x min; n=53) as previously described (Herson et al. 1999; Shimizu et al. 2016; Kraft et al. 2004; Olah et al. 2009) (FIGs. 4B, 4C). TatM2NX, tat-Cterm and CTZ significantly decreased Ca²⁺ fluorescence (F/F₀) after 20min (47,904 ± 31 ,245 F/F₀x min; n=48, p<0.05; 50,600 ± 26,993 F/F₀x min; n=49, p<0.05; 31 ,910 ± 10,880 F/F₀x min; n=25, p<0.05, respectively) (FIGs. 4B, 4C, 4D). However, tat-Nterm (67,851 ± 33,830 F/F₀x min; n=48, p>0.05) had no effect on Ca²⁺ fluorescence activated by H₂0₂ (FIGs. 4C, 4D). On the other hand, Ca²⁺ fluorescence with tat-WVAA is higher than H₂0₂ (99,192 ± 39,653 F/F₀x min; n=52, p<0.05) (FIG. 4D). All cellular responses in the presence of peptides had similar fluorescent kinetics as H₂0₂ stimulation alone (H₂0₂, 105.27 ± 16.81 frames; tatM2NX, 106.48 ± 18.73 frames; tat-Cterm, 107 ± 19.06 frames; tat-Nterm, 110.19 ± 14.07 frames; tat-WVAA, 102.81 ± 22.10 frames) (FIG. 8B). These data suggest that tat-Cterm inhibits TRPM2 activity in cells at similar levels as tat-M2NX and residues W33V34 are essential for antagonism.

The various features and processes described above may be used independently of one another, or may be combined in various ways. All possible combinations and subcombinations are intended to fall within the scope of this disclosure. In addition, certain method or process blocks may be omitted in some implementations. The methods and processes described herein are also not limited to any particular sequence, and the blocks or states relating thereto can be performed in other sequences that are appropriate. For example, described blocks or states may be performed in an order other than that specifically disclosed, or multiple blocks or states may be combined in a single block or state. The example blocks or states may be performed in serial, in parallel, or in some other manner. Blocks or states may be added to or removed from the disclosed example embodiments. The example systems and components described herein may be configured differently than described. For example, elements may be added to, removed from, or rearranged compared to the disclosed example embodiments.

Conditional language used herein, such as, among others, “can,” “could,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements, and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment. The terms “comprising,” “including,” “having,” and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list.

Whereas certain example embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions disclosed herein. Thus, nothing in the foregoing description is intended to imply that any particular feature, characteristic, step, module, or block is necessary or indispensable. Indeed, the novel methods and systems described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods and systems described herein may be made without departing from the spirit of the inventions disclosed herein. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of certain of the inventions disclosed herein.

Table 1. Description of peptides

Claims

CLAIMS What is claimed is:

1. An isolated peptide comprising the amino acid sequence XGRKKRRQRRRKLKRVLRQEFWV or a multimer, derivative, or variant thereof, wherein X=Y, F, or any amino acid other than Y, or no amino acid.

2. The peptide of claim 1 , wherein the peptide is linked to the internalization peptide through a linker selected from Gly(Ser)4, TGEKP, GGRRGGGS, and LRQRDGERP.

3. The peptide of claim 1 , wherein the peptide comprises as at least one of a dimer, trimer, or a tetramer of the sequence.

4. An isolated peptide comprising an amino acid sequence with at least 80% amino acid sequence identity to amino acid sequence YGRKKRRQRRRKLKRVLRQEFWV (SEQ ID NO. 4), and the ability to inhibit the flux of calcium ions through a TRPM2 protein ion channel.

5. The peptide of claim 4, comprising an amino acid sequence with at least 95% amino acid sequence identity to SEQ ID NO. 4.

6. The peptide of claim 4, comprising between 1 and 3 conservative amino acid substitutions compared to SEQ ID NO. 4, and the ability to inhibit the flux of calcium ions through a TRPM2 protein ion channel.

7. An isolated peptide comprising an amino acid sequence with at least 80% amino acid sequence identity to KLKRVLRQEFWV (SEQ ID NO. 6), wherein C-terminal amino acid residues W V are not substituted.

8. The isolated peptide of claim 7, further comprising amino acid sequence XGRKKRRQRRR (SEQ ID NO. 8), at the N-terminus of SEQ ID NO. 2, wherein X=Y, F, or any amino acid other than Y, or no amino acid.

9. The isolated peptide of claims 7 or 8, comprised of the following amino acid sequence: YGRKKRRQRRRKLKRVLRQEFWV (SEQ ID NO. 4).

10. A composition comprising an isolated peptide of any of claims 1-9, and a pharmaceutically acceptable carrier.

11 . A method of inhibiting activity of an TRPM2 protein ion channel in a subject, the method comprising administering to the subject an inhibiting amount of the composition according to claim 10.

12. A method of treating or preventing a neurological injury or neurological disorder in a subject in need, the method comprising administering a therapeutically effective amount of the composition according to claim 10.

13. The method of claims 11 or 12, wherein the subject is a mammal.

14. The method of claim 13, wherein the mammal is a human.

15. The method of claims 11-14, further comprising administering an additional therapeutic agent in combination with the composition.

16. The composition of claim 10, further comprising an additional therapeutic agent.