Phlpp1 activator and uses thereof

Abstract

Disclosed are activators of the phosphatase PHLPP1 (PH domain and Leucine rich repeat protein phosphatase 1), a widely conserved enzyme involved in the regulation of two signaling networks, mTOR and AMPK, the balance and coordination of which controls the cellular response to either ample or restricted fuel supply to cells. These activators are referred to as SNAP (Snail-derived Activators of PHLPP) compounds as they are based on the structure of a Dormancy Inducing factor (DIF) discovered in hibernating snails. SNAP compounds are demonstrated to increase the activity of both human recombinant PHLPP, and mouse PHLPP1. One such example of a SNAP compound is 3-(2-methyl-8- (2-((oxidanidylsulfonyl)oxy) ethyl)-3,4-dihydro-2H-chromen-2-yl) propanoate-pyridine.

Description

PHLPP1 ACTIVATOR AND USES THEREOFInventors: Evangelos D. Michelakis; Gopinath Sutendra; Yuan Zhang; John Ussher; Seyed Amirhossein Tabatabaei DakhiliAssignee: The Governors of the University of AlbertaField of the Invention

[0001] The present invention relates to compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof and combinations thereof, and methods of using the same as activators of phosphatase PHLPP1.Background and prior art

[0002] During hibernation (under low temperatures) or aestivation (under higher temperatures) many species, from snails to mammals, enter a state of dormancy similar to cellular quiescence, with profound metabolic and signaling changes [1-9], the molecular trigger of which remains unknown since the identity of circulating hibernation / estivation-inducing factor(s), presumably secreted from the brain in response to environmental sensors, remains unknown. Such factors may activate the many signaling pathways contributing to the stressresistance that underlies these dormancy states, including metabolic / mitochondrial remodeling, autophagy, apoptosis-resistance, cell-cycle exit and proteostasis [10-12], Intriguingly, these features of quiescence under stress also characterize stem cells [13-15] or resistant bacteria [16,17], but, once again, the quiescence trigger remains unknown. Because animals enter and exit hibernation quickly, it has been suggested that these dormancy -inducing factors (DIFs) may be kinases or phosphatases [18-20], Attractive putative targets of DIFs may be the mTOR [10-12] and AMPK networks [18-21], which through phosphorylation cascades, regulate the cellular response and transition from nutrient restriction (ischemia), which activates AMPK, to nutrient abundance (like in normal or reperfusion states), which activates mTOR. AMPK promotes metabolic remodeling toward ATP conservation, glycolysis and fatty acid oxidation (FAO, as opposed to glucose oxidation, GO), cell-cycle exit, activation of autophagy and suppression of 1WSLEGAL\055326\00505\42539390v6protein synthesis rates and apoptosis. In contrast, mT0R / S6Kl promotes growth with cell-cycle entry, increased proteinosynthesis, inhibition of autophagy and a metabolic shift toward nucleotide synthesis over GO or FAO. The two networks are regulated by multiple feedback loops, including inhibiting each other, an important being through activation of AKT via phosphorylation at S473. While mTOR activates AKT during fuel abundance, metabolic stress (low O2 and glucose in ischemia, or ROS in reperfusion) can also acutely activate AKT [22-26], which can in turn activate mTOR [22,27-29] even during fuel restriction. This important feedback may reset the balance of mTOR and AMPK, particularly during the acute transition from dormancy under fuel restriction to exit from it at reperfusion. Both the widely conserved mTOR and AMPK networks are involved in hibernation [10,30-34] and cellular quiescence (the cellular equivalent of hibernation). We speculated that these networks are attractive putative effectors of DIFs and may be common among different dormancy states. DIF secretion triggered by decrease in fuel supply or humidity (as in hibemation / aestivation) may promote dormancy, with its absence allowing exit from it (i.e., reperfusion).

[0003] Hibernating mammals survive in perfect health during hibernation, when their respiration and heart rates drop to extremely low levels (e.g. ~2 / min

[0035] ), otherwise incompatible with life, exhibiting remarkable tolerance to starvation [36,37] and ischemia [7,9,31,33], Hibernating animals do not suffer from ischemia or reperfusion injury when they enter or exit hibernation. Echocardiography in hibernating bears shows preserved systolic and diastolic myocardial function

[0038] , In contrast, non-hibernating species like mice and humans are very vulnerable to ischemic and IR injury. IR injury in normal organs is a major limitation in transplant medicine since most of the offered organs are not suitable for transplantation (unless within a very short window of time) by the time they reach the recipient because of damage due to both ischemia during transport and reperfusion during placement in the recipient [39-43], IR injury is a major clinical problem in transplant medicine [41-43], but also in coronary interventions and coronary artery bypass surgery

[0044] ,Summary of the Invention

[0004] Generally, disclosed are activators of the phosphatase PHLPP1 (PH domain and Leucine rich repeat protein phosphatase 1), a widely conserved enzyme involved in the 2WSLEGAL\055326\00505\42539390v6regulation of two signaling networks, mTOR and AMPK, the balance and coordination of which controls the cellular response to either ample or restricted (respectively for mTOR and AMPK) fuel supply to cells. These activators are referred to herein as SNAP (Snail-derived Activators of PHLPP) compounds as they are based on the structure of a Dormancy Inducing factor (DIF) discovered in hibernating snails. SNAP compounds are demonstrated to increase the activity of both human recombinant PHLPP, and mouse PHLPP 1.

[0005] A SNAP compound can induce quiescence (the cellular equivalent of hibernation) in mice and human cells (fibroblasts) deprived of fuel (O2 and / or glucose). Specifically, upon fuel deprivation, a SNAP compound can limit death (apoptosis), causes cell cycle exit reversibly i.e. when a SNAP compound is withdrawn and fuel is reintroduced, cells start proliferating again without evidence of ischemic or reperfusion damage, suppressed proteinosynthesis rates, rewired metabolism (increased Spare Respiratory Capacity (SRC)) and inhibited oxidative phosphorylation in mitochondria) and significantly activated autophagy, all features of quiescence.

[0006] A SNAP compound can significantly limit ischemia / reperfusion (IR) injury in mouse isolated / perfused hearts and cardiomyocytes, preserving heart function upon ischemia (severe decrease in supplied O2 and glucose) and reperfusion, by rewiring metabolism and activating autophagy in heart cells (fibroblasts and cardiomyocytes).

[0007] A SNAP compound can significantly decrease growth in human melanoma cancer cells and human prostate cancer organoids.

[0008] Therefore, in one aspect, disclosed are compounds which activate PHLPP, comprising at least one compound of Formula (I) of the following structure:A;(I)wherein:3WSLEGAL\055326\00505\42539390v6R1 is cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; branched or unbranched acyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;R2 is hydrogen, hydroxy, methoxy, halogen (such as F, Cl, or Br), or CX-O-S(=O)2-OH, where x is zero or 1, 2, 3, 4 or 5;R3, R4 and R5 are each, independently of each occurrence, hydrogen; hydroxy, methoxy, or halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl.

[0009] In some embodiments, R1 is preferably ethanoic, propanoic or butanoic acid, more preferably propanoic acid.

[0010] In some embodiments, R3, R4 and R5 are each H, F or CX-O-S(=O)2-OH, where x is zero or 1, 2, 3 or 4.

[0011] In some embodiments, the PHLLP activating compound is one of SNAP001 to SNAP020 below.4WSLEGAL\055326\00505\42539390v6<WSLEGAL\055326\00505\42539390v6

[0012] In one preferred embodiment, the compound comprises SNAP001 (IUPAC name 3-(2-methyl-8-(2-((oxidanidylsulfonyl)oxy)ethyl)-3,4-dihydro-2H-chromen-2-yl) propanoic acid) or a pharmaceutically acceptable salt thereof, such as a potassium, sodium, calcium, magnesium, or a pyridinium salt, as shown om Formula l.A:I ■Q - ■ ' OH

[0013] In another aspect, disclosed are compounds which activate PHLPP, comprising at least one compound of Formula (II) of the following structure:wherein R1 is hydrogen; hydroxy; methoxy; halogen (such as F, Cl, Br); oxygen; nitrogen; sulphur; sulfonyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranchedWSLEGAL\055326\00505\42539390v6aliphatic; or cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic.

[0014] In some embodiments, the compound is one of SNAP021 to SNAP024, where R1 is selected from the group consisting of:

[0015] In another aspect, disclosed is a pharmaceutical composition comprising at least one SNAP compound and pharmaceutically acceptable carrier.

[0016] In another aspect, disclosed is a method of using at least one compound disclosed herein, in a therapeutically effective amount, to protect an organ which is at risk of ischemia, whether in vivo or ex vivo.

[0017] In some embodiments, the method comprises the use of at least one compound disclosed herein to treat a disease or a condition in a patient in need thereof, wherein the disease or condition is marked by a risk of human organ ischemia.

[0018] In some embodiments, a SNAP compound can be used to: a) protect solid organs from the IR injury that occurs in transplant donor organs that suffer damage during their transport to a transplant recipient; (b) treat human hearts that suffer IR injury upon thrombolysis or coronary interventions during a myocardial infarction; (c) treat hearts suffering IR injury during coronary artery bypass surgery (CABG); and (d) treat hearts and / or other internal organs suffering injury during septic or hemorrhagic shock.

[0019] In another aspect, disclosed are methods of using at least one compound disclosed herein to induce a hibernation-like state in a person, which may be desirable in circumstances 7WSLEGAL\055326\00505\42539390v6such as travelling long distances, such as astronauts during ultra-long space travel, such that the person will not suffer the well-known consequences of fuel restriction and immobility.

[0020] In another aspect, disclosed are methods of using at least one compound disclosed herein in a therapeutically effective amount to treat patients suffering from cancer, in that at least one compound disclosed herein activates PHLPP and thus inhibits AKT, which is known to promote cancer growth.

[0021] In another aspect, disclosed are methods of using at least one compound disclosed herein to promote the longevity of a mammal, or slow the rate of aging in a mammal, which may include a human. This use follows from the showing that during dormancy in hibernating animals, ageing rates decrease.

[0022] In another aspect, disclosed are methods of using at least one compound disclosed herein to induce quiescence of human cells, which is a critical feature of stem cells. In a preferred embodiment, at least one compound disclosed herein can be used in the methodology of inducing pluripotent or hemopoietic stem cells or inducing quiescence of human ovarian oocytes (eggs) to promote their preservation without freezing them.Brief Description of the Drawings

[0023] Figure 1. Discovery of SNAP in aestivating snails.(A) A representative image of a non-aestivating and an aestivating snail with seal formation. (B) A representative snail activity (left) and group data (right) measured by the distance covered under the cameras in non-aestivating snails (kept in normal humidity) injected with hemolymph from either control (non-aestivating) or aestivating (humidity -deprived) snails. (C) O2 consumption rates of normal, aestivating and normal with aestivation hemolymph-injected snail tissues, measured with a Seahorse XF24 Extracellular Flux Analyzer. (D) Representative live images and quantification of fluorescence intensity of TMRM staining to measure mitochondrial membrane potential (DYm) of normal and aestivating snail tissues (Scale bar: 20 pm). (E) Extracted chromatogram of the ion at m / z 343.0859 (negative electrospray ionization mode) using an unbiased HPLC coupled with an Orbitrap Elite high-resolution mass spectrometer. A8WSLEGAL\055326\00505\42539390v6peak at a retention time of 5.1 minutes was observed in the aestivating but not the control snail tissues. (F) The chemical structure of the ion at m / z 343.0859, identified and synthesized (as described in methods), that we named SNAP when it was realized that it specifically binds to and activates PHLPP1. (G) The 3D structure of PHLPP1 and its binding domain for the SNAP shown in situ (left); a zoom of the predicted binding sites of SNAP is shown with the key amino acids that interact with SNAP according to computer modelling (right), as described in the text. (H) Mean distance (and variability) covered by 20 snails in a quadratic best-fit line, showing that snails injected with lOpM SNAP mimicked exactly the snails injected with aestivating (but not control) hemolymph. The dormancy in these snails (inactivity and formation of a seal), was indistinguishable from natural hibernation due to humidity deprivation. Data in all bar plots are shown as mean ±S.D. and represent ten (C and H), or five (D) biological replicates in each group, p values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (C) or two-sided unpaired Student’s t-tests (D). * / ><0.05, ***p< 0.001.

[0024] Figure 2. SNAP synthesis and identification.(A)1H-NMR spectra of the intermediate SNAP (upper) and the final product SNAP pyridinium salt (lower) as described in the SNAP synthesis section in the methods section. (B) Fragment mass spectrum of the ion at m / z 343.0859 under negative electrospray ionization mode: synthesized SNAP (Top left), original snail sample (bottom left), and interpreted fragment pattern with m / z values derived from its chemical structure (right). Note that the positively charged pyridine does not appear under negative electrospray ionization mode and thus the synthesized and snail molecules are identical (other than the pyridine part that was added during the SNAP synthesis to form a stable salt).

[0025] Figure 3. SNAP is a specific PHLPP1 activator.(A) A phosphatase activity assay measuring phosphate release was performed in mouse fibroblast lysates from scramble or siPHLPPl -transfected cells after 72 h of ischemic stress, using a phospho-peptide derived from p-AKT, a PHLPP1 target, as the substrate. (B) Immunoprecipitation (IP) of PHLPP1 from mouse fibroblasts shown using immunoblots. (C) PHLPP1 activity assay was conducted with IP-PHLPP1 from 72h-stress-mouse fibroblast and 9WSLEGAL\055326\00505\42539390v6phospho-peptide derived from p-AKT as a substrate. (D) PHLPP1 activity assay performed with human recombinant PHLPPland phospho-peptide derived from p-AKT as substrate in the presence of the required cofactor manganese. (E) Co-IP of PHLPP1 and total AKT (t-AKT) and t-S6Kl from mouse fibroblasts. (F) p-AKT, t-AKT, p-S6Kl and t-S6Kl protein levels between DMSO and SNAP -treated cells were detected by immunoblots analysis, quantified using Image J. (G) p-AKT, t-AKT, LC3B and GAPDH protein levels between different doses of SNAP-treated cells were detected by immunoblots and quantified using Image J. (H) PHLPP1, p-AKT, t-AKT, p-S6Kl and t-S6Kl protein levels between scramble and siPHLPPl -transfected cells detected with immunoblots and quantified using Image J. (I) PHLPP1 activity assay was performed with IP-PHLPP1 from 72h-stress-mouse fibroblasts transfected with mutant PHLPP1 cDNAs, where the different amino acids at the predicted SNAP binding sites were mutated to non-polar amino acids: in the presence of the substrate phospho-peptide derived from p-AKT there was no phosphate release suggesting that PHLPP1 was not activated because SNAP failed to bind with it. (J) PHLPP1, p-AKT, t-AKT, p-S6Kl and t-S6Kl protein levels among EV (empty vector), WT (wild type PHLPP1 cDNA) and MUT (mutant PHLPP1 cDNA where all the amino acids binding SNAP were simultaneously mutated)-transfected cells were detected by immunoblots, and quantified with Image J: the absence of effects of SNAP in the mutant is in keeping with the lack of activity when the mutant sites were individually mutated as shown in I. Data in all bar plots are shown as mean ± S.D., and all data are representative of three independent experiments, p values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (A, C, H, I and J) or two-sided unpaired Student’s t-tests (D and F). * / ><0.05, ***p< 0.001; ns, no statistical significance.

[0026] Figure 4. SNAP treatment induces quiescence in mouse lung fibroblasts under long-term fuel deprivation.(A) The protocol for stressed mouse fibroblasts studies: after seeding, the cells were cultured in stress conditions for 24 or 72 hours with DMSO or SNAP, then returned to normal conditions without DMSO or SNAP for another 2 days. (B) Representative live images of mitochondria and lysosomes stained with MitoTracker and LysoTracker under stress conditions for 24 hours (Scale bar: 50 pm) shows early activation of lysosomes suggesting autophagy. (C) The protein levels of 10WSLEGAL\055326\00505\42539390v6the autophagy markers LC-3B II and LAMP-1 between DMSO-treated and SNAP -treated cells under stress conditions for 24 hours were detected by immunoblots and quantified with Image J. (D) Cleaved caspase-3 protein levels were detected after a 72-hours of stress using immunoblots and quantified with Image J. (E) Cell numbers were detected and calculated using a Holomonitor microscope, which allows imaging of live cells based on optical density, avoiding the additional stress of staining: survival rate was calculated after stress for 3 days and population doubling time was calculated after returning to normal conditions for 2 days. (F) Ki-67, p-CDK2, t-CDK2, p-Rb and t-Rb protein levels were detected at day 0, 3 and 5 by immunoblots, and their expression levels were quantified using Image J. (G) Schematic shows the SNAP protects mouse fibroblasts during stress by putting them into reversible quiescence and allow them to re-enter the cell cycle after stress (unlike the irreversible state of senescence. (H) After seeding fibroblasts, cells were cultured in stress for 72 hours with DMSO or SNAP and then immediately studied as shown in subsequent panels. (I) OCR (oxygen consumption rate) of cells after normal vs stress conditions for 72 hours was assessed using a Seahorse XF24 Extracellular Flux Analyzer and SRC (the difference between maximal OCR triggered by FCCP and minimum OCR triggered by Oligomycin) were calculated. (J) Representative live images of TMRM and MitoSOX staining with cells after culturing in stress conditions for 72 hours (Scale bar: 20 pm) with TMRM and MitoSOX fluorescence intensity quantification. (K) p-PDHElaSer232, p-PDHElaSer300, p-PDHElaSer293, (all potential sites of inhibiting phosphorylation) and t-PDH protein levels between DMSO and SNAP -treated cells were detected by immunoblots and quantified using Image J. All data are shown as mean ± S.D. and represent three (B, C, E, F, I and L) and eight (J) independent experiments. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (F, J and I) or two-sided unpaired Student’s t-tests (B, C, E, andK). * / ?<0.05, **p< 0.01, ***p< 0.001; ns, no statistical significance.

[0027] Figure 5. SNAP inhibits protein synthesis early after stress.(A) After seeding mouse fibroblasts were cultured in stress conditions for 5 hours with DMSO or SNAP. (B) p-eIF2(Z and t-eIF2(Z protein levels were detected at early time points Oh, 3h and 5h post-stress condition induction with DMSO or SNAP by immunoblots and quantified using Image J. (C) Representative images of OPP staining with cells under stress conditions for 5 hours 11WSLEGAL\055326\00505\42539390v6(Scale bar: 50 m) and OPP fluorescence intensity quantification. OPP is a methionine analogue that incorporates into the translation machinery, allowing the detection of translated proteins after OPP addition to the culture media. The decreased signal induced by SNAP suggests supressed translation rates, a feature of cellular quiescence. Data in all bar plots are shown as mean ± S.D. and represent three (B and C) independent experiments. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (C). **p< 0.01.

[0028] Figure 6. SNAP effects on autophagy and PDH compared to the mTOR / S6K inhibitor rapamycin.(A) After seeding mouse fibroblasts were cultured in stress conditions for 5 hours with DMSO or SNAP or rapamycin, and p-mTOR, t-mTOR, p-S6Kl, t-S6Kl, p-AKT, t-AKT, p-PDHEla, t-PDHEla and LC-3B II protein levels were detected by immunoblots and quantified using Image J. (B) After seeding mouse lung fibroblasts, cells were cultured in normal conditions for 5 hours with DMSO or SNAP or rapamycin, and p-mTOR, t-mTOR, p-S6Kl, t-S6Kl, p-AKT, t-AKT, p-PDHEla, t-PDHEla and LC-3B protein levels were detected by immunoblots and quantified using Image J. Data in all bar plots are shown as mean ±S.D. and represent three (A and B) independent experiments, p values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (A and B). * / ><0.05, ** <0.01, *** <0.001; ns, no statistical significance.

[0029] Figure 7. SNAP protects mouse cardiomyocytes from acute IR injury.(A) In vitro IR model: after seeding, mouse cardiomyocytes were cultured in stress conditions for 3 hours with DMSO or SNAP and returned to normal environment without DMSO or SNAP for another 0.5 hours. (B) p-AMPK, t-AMPK, p-AKT, t-AKT, p-PDHEla, t-PDHEla, LC3B and cleaved caspase-3 protein levels were detected by immunoblots and quantified with Image J. (C) Representative live images of TMRM and MitoSOX staining of cardiomyocytes after reoxygenation for 0.5 hours (Scale bar: 20 pm) and quantification of fluorescence intensity. (D) Representative immunofluorescence images of citric synthase and p-Akt in cardiomyocytes after 1 hr of ischemic stress (Scale bar: 20 pm), with an increased mitochondrial p-Akt signal with DMSO, diminished by SNAP. (E) Cytosol and mitochondrial fractionation in cardiomyocytes 12WSLEGAL\055326\00505\42539390v6after 1 hour stress, and PHLPP1, NHERF-1, p-AKT, t-AKT, p-PDHElaSer300, t-PDHEla, PDK1 and LDHA protein levels detected by immunoblots (low and high exposure) and quantified with Image J. The mitochondrial enzyme PDH also served as a marker for mitochondria and the cytosolic enzyme LDH as a cytoplasm marker. Note that the PHLPP1 p-AKT levels increased in the mitochondrial fraction with stress and the mitochondrial, but not cytoplasmic p-AKT, was decreased by SNAP. (F) Protocol for the experiments with cardiomyocytes lacking PDH: after separately seeding aMHC-MerCreMer and PclhaPM'<i'x~ ~ mouse cardiomyocytes, cells were cultured in stress conditions for 3 hours with DMSO or SNAP and returned to a normal environment without DMSO or SNAP for another 0.5 hours. (G) Measured OCR (Seahorse XF24 Extracellular Flux Analyzer) and calculated SRC (max OCR by FCCP minus min OCR by oligomycin). (H) Cardiomyocyte contractility (cell shortening) after reoxygenation. (I to J), After reoxygenation, ATP (I) and acetyl CoA (J) levels in cardiomyocytes were measured by HPLC / MS / MS. (K) p-AKT, t-AKT, p-PDHEla, t-PDHEla, p-S6Kl, t-S6Kl and LC-3B protein levels were detected by immunoblots and quantified with Image J. Data in all bar plots are shown as mean ± S.D. and represent at least three (C, D, G, H, I and J) biological replicates per group and three (B, E and K) independent experiments, p values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (B, C, E, G, H, I, J and K). * / ?<0.05, ** / ?< 0.01, *** <0.001; ns, no statistical significance.

[0030] Figure 8. SNAP reduced ER stress while maintaining higher 'I' in and lower mROS levels in the cardiomyocytes after a 5-hour-long IR protocol.(A) After seeding, mouse cardiomyocytes were cultured in stress conditions for 3 hours with DMSO or SNAP and returned to normal environment without DMSO or SNAP for another 2 hours. (B) p-PERK, t-PERK, p-JNK, t-JNK and cleaved ATF6 protein levels were detected by immunoblots and quantified with Image J. (C) Representative live images of TMRM staining in cardiomyocytes (Scale bar: 20 pm) and TMRM fluorescence intensity quantification. (D) Representative live images of MitoSOX staining in cardiomyocytes (Scale bar: 20 pm) and MitoSOX fluorescence intensity quantification. Data in all bar plots are shown as mean ± S.D. and represent three (B to D) independent experiments. P values were calculated by one-way13WSLEGAL\055326\00505\42539390v6ANOVA with Tukey’s multiple comparisons post hoc tests (C to F) or two-sided unpaired Student’s t-tests (B). *p<Q.Q5, *** <0.001; ns, no statistical significance.

[0031] Figure 9. SNAP dephosphorylates AKT and S6K1 in fibroblasts under stress conditions for 1 hour.(A) After seeding, mouse fibroblasts were cultured under stress conditions for 1 hour with DMSO or SNAP. (B) p-AKT, t-AKT, p-S6Kl and t-S6Kl protein levels were detected by immunoblots.

[0032] Figure 10. NHERF1 and PHLPP1 co-immunoprecipitate and under 1-hour stress conditions, their total expression levels remained unchanged.(A) After seeding, mouse cardiomyocytes were cultured under stress conditions for 1 hour with DMSO or SNAP. (B) Co-IP of NHERF1 and PHLPP1 from mouse cardiomyocytes as shown by immunoblot. (C) PHLPP1 and NHERF1 protein levels were detected by immunoblots and remained unchanged. This suggests that the increased cytoplasmic and mitochondrial PHLPP1 in stress shown in Fig. 3E is due to PHLPP1 translocation from the plasma membrane where it is typically found in normal conditions.

[0033] Figure 11. Under stress conditions, cytoplasmic PHLPP1 levels remain unchanged, while PHLPPla specifically accumulates in mitochondria within 1 hour, with no significant changes in nuclear PHLPP1.After seeding, mouse cardiomyocytes were cultured under stress conditions for 1 hour in the presence of DMSO or SNAP, followed by cell fractionation. (B) Cytosolic, mitochondrial, and nuclear fractions were prepared after 1 hour of stress, and PHLPP1, Histone H3, citrate synthase, and LDHA protein levels were analyzed by immunoblotting and quantified using ImageJ. To confirm equal protein loading, Ponceau S staining is shown. Histone H3 served as a nuclear marker, citrate synthase as a mitochondrial marker, and LDHA as a cytosolic marker. Each fraction was normalized to its respective marker. PHLPPip (which, in contrast to PHLPPla has an NLS and can translocate from the cytoplasm to the nucleus) levels were quantified relative to the cytoplasmic control group, and PHLPPla levels were quantified relative to the mitochondrial14WSLEGAL\055326\00505\42539390v6control group. Data are presented as mean ±S.D. from three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons post hoc test. **p<0.01.

[0034] Figure 12. SNAP increased levels of acetyl-CoA and decreased lactate levels in aMHC-MerCreMer cardiomyocytes after the 2-hour-long IR injury, in keeping with preserved PDH activity and glucose oxidation, compared to vehicle.After separately seeding aMHC-MerCreMer and Pdhal^^'1' mouse cardiomyocytes, cells were cultured in stress conditions for 3 hours with DMSO or SNAP and returned to a normal environment without DMSO or SNAP for another 2 hours. (B and C) After reoxygenation, acetyl CoA (B) and lactate (C) levels in cardiomyocytes were analyzed by HPLC / MS / MS. Data in all bar plots are shown as mean ± S.D. and represent three (B and C) independent experiments. P values were calculated by two-sided unpaired Student’ s t-tests (B and C). *** / ?<0.001.

[0035] Figure 13. SNAP protects from IR injury in pe vivo perfused hearts.(A) Protocol 2 for Langendorff ex vivo heart IR model. (B) Representative traces of left ventricular pressure (LVP) and at different time points. (C to G) Mean data for-time-to-stable-arrhythmia-free contraction after reperfusion (C), heart rate (D), LVP (E), max dp / dt (F), and min dp / dt (G). The different stages of the protocol are depicted by Latin numerals and correspond to those shown in the protocol (A). SNAP improved all the parameters measured compared to vehicle. (H) Recovery rates for LVP, max dp / dt, and Min dp / dt were calculated based on the pacing at 300bpm data compared to the initial baseline for each mouse to address variability. (I) Representative live images of TMRM and MitoSOX-stanned heart tissue after reperfusion (Scale bar: 20 pm) and quantified TMRM and MitoSOX fluorescence intensities. (J and K). Concentration of lactate (J) and acetyl CoA (K) in the repercussed heart tissues (Langendorff model) measured by HPLC / MS / MS. (L) GO and FAO rates measured in the mouse working heart model (see text) by using radiolabeled glucose and palmitate and comparing the input perfusate to the coronary sinus output, to calculate the % changes from baseline in the vehicle vs SNAP perfusions. Data in all bar plots are shown as mean ± S.D. and represent seven (C to H) three (J and K) or four (L) biological replicates per group. P values were calculated by 15WSLEGAL\055326\00505\42539390v6one-way ANOVA with Tukey’s multiple comparisons post hoc tests (D to I) or two-sided unpaired Student’s t-tests (C, J, K and L). *p<0.05, ***p<0.01, *** <0.001; ns, no statistical significance.

[0036] Figure 14. SNAP under normoxia conditions has no significant effects, other than min dp / dt in an ex vivo perfused mouse heart IR model.(A) Protocol 1 for Langendorff IR ex vivo heart IR model. (B) Representative LVP traces of mouse ex vivo perfused hearts at different time points. (C to G) Time to stable arrhythmia-free contraction after reperfusion (C), heart rate (D), LVP (E), max dp / dt (F), and min dp / dt (G) were measured at different stages of the protocol, depicted by Latin numerals corresponding to the protocol schematic (A). Data in all bar plots are shown as mean ± S.D. and represent six (C to F) biological replicates per group, p values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (D to G) or two-sided unpaired Student’s t-tests (C). *p<0.05.

[0037] Figure 15. SNAP under normoxia conditions has some protective effects even after 40-minute-long occlusion.(A) Experimental scheme for Langendorff IR ex vivo heart model. (B to F) Time for stable contraction after reperfusion initiation (B), heart rate (C), LVP (D), Max dp / dt (E), and Min dp / dt (F) were measured. Data in all bar plots are shown as mean ± S.D. and represent six (B to F) biological replicates per group. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (C to F) or two-sided unpaired Student’s t-tests (B). *p<0.05.

[0038] Figure 16. During 30 min ischemia, SNAP preserves PDH activity and increases mitochondrial membrane potential (increasing the apoptosis threshold) without increasing mROS levels.(A) Experimental scheme for Langendorff IR ex vivo heart model. (B) Representative images of PDH activity assay and PDH activity signal intensity from the images were quantified. (C) p-PDHElaSer232, p-PDHElaSer300, p-AKTser473and t-PDHEla protein levels between DMSO and16WSLEGAL\055326\00505\42539390v6SNAP -perfused heart homogenates were detected by Western blot analysis. Their expression levels were quantified using the Image J program, and the p-PDHA / t-PDHA calculated levels between DMSO and SNAP -perfused heart were detected by Western blot analysis. Their expression levels were quantified using the Image J program, and the p-PDHEla / t-PDHEla was calculated. (D) Representative live images of TMRM and MitoSOX staining in heart tissue (Scale bar: 20 pm) and TMRM and MitoSOX fluorescence intensity were analyzed. Data in all bar plots are shown as mean ± S.D. and represent four (B to D) biological replicates per group. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests (D) or two-sided unpaired Student’s t-tests (B and C). ** <0.01, *** <0.001; ns, no statistical significance.

[0039] Figure 17. SNAP’s cardioprotective effects in a mouse perfused heart IR model are absent in PdhalCardiac / ~ mice hearts.(A) Protocol for the Langendorff IR model. (B) Representative traces for Left ventricular pressure (LVP) at various time points. (C to F) Mean data for the time to stable arrhythmia-free contraction after reperfusion (C), LVP (D), max dp / dt) (E) and min dp / dt) (F). SNAP’s cardioprotective effects (also documented in wild type mice in Fig. 4) are present in the control hearts but absent in the hears with a myocardial cell-specific KO of PDH. (G) Recovery rates for LVP, max dp / dt, and Min dp / dt were calculated based on the pacing at 300bpm data compared to the initial baseline for each mouse to address variability. Data in all bar charts are presented as mean ± S.D. and represent five or six (Cre n=5, PDH KO n=6) (C to G) biological replicates, p values were determined using one-way ANOVA with Tukey’s multiple comparisons post hoc tests (D to G) or one-sided unpaired Student’s t-tests (C). * / ><0.05, ** <0.01, ***p< 0.001; ns indicates no statistical significance).

[0040] Figure 18. Effect of SNAP on metastatic melanoma cell proliferation, death and migration.A. 24 hours after seeding, A2058 cells were treated with DMSO or SNAP. Cell proliferation was monitored for 72 hours using a Holomonitor microscope. Scale bar indicates 100 pm. (A) Representative holographic images of cells tracked over 72 hours are shown. The quantification 17WSLEGAL\055326\00505\42539390v6data shown represent means ± SEM of one representative experiment from three independent replicates, each with 15 technical replicates (*p = 0.0195, ***p < 0.0002, and ****p < 0.0001, Mann-Whitney U test). B. Proteins were collected at 72 hours post-treatment, and the effectiveness of SNAP on the expression of p-AKT and the proliferation marker Ki67 were analyzed via western blot. Data shown represent means ± SEM of four independent experiments (***p < 0.001, Two-way ANOVA). (C) A2058 cells were exposed to DMSO or SNAP for 72 hours and then stained with Alexa Fluor 488 annexin V (AV) and propidium iodide (PI) to analyze apoptosis. The nuclei are stained with Hoechst (blue). Scale bar indicates 20 pm. Histograms show the quantification of AV / PI signal in early and late apoptotic cells, defined as AV-positive / PI-negative and AV-and Pl-positive, respectively. Image J software was used to analyze the Z-stack images. Data shown represent means ± SEM of two independent experiments (****p < 0.0001, Two-way ANOVA). Immunoblot analysis of apoptosis marker C-caspase3 after 72h of exposure to SNAP compared with DMSO is also shown. Data shown represent means ± SEM of four independent experiments (**p < 0.003, Two-way ANOVA). P-actin was used as a loading control in all Western blot analysis. All treatments were performed under metabolic stress condition (2.5mM glucose, 1% serum, 1% Oxygen). D. In addition to pAKT, SNAP also inhibits another known PHLPP1 target, pERK (1-2) which is critical for cancer cell survival. When gigen in addition to doxorubicin, SNAP has additive effects in terms of induced cancer cell death. E. DA2058 cells were treated with DMSO or SNAP under metabolic stress (2.5mM glucose, 1% FBS, 1% Oxygen) for 48 hours before migration and adhesion analysis. After 48 hours, 15 000 cells were resuspended in DMEM (2.5mM glucose, No FBS) and loaded in the upper chamber of transwell. Stress media containing 2.5mM glucose and 1% FBS is added as chemoattractant in the bottom chambers. Cells were then left under hypoxia to migrate to the lower surface of the transwell membrane for 15 hours before fixing, staining and counting, SNSP inhibited migration, decreased Focal adhesions (FA) as well as the key FA protein Paxillin. The quantification bar graph represents mean ± SEM from three independent experiments. Immunofluorescence analysis of paxillin-enriched focal adhesion (FA) was performed 48 hours post-treatment. Representative confocal microscopy images of paxillin staining, with nuclei counterstained with DAPI, are shown. Scale bar indicates 10 pm. FA complexes were divided into three types according to their size: Nascent (Early), < 0.5 pm2; Intermediate, [0.5-1] pm2; Elongated / mature, >1 pm2. ImageJ 18WSLEGAL\055326\00505\42539390v6software was used to analyze the Z-stack images and determine the number of paxillin-enriched FA and it size distribution per cell. Charts show means ± SEM from three independent experiments. All statistical significance between the groups was evaluated using a two-tailed Mann-Whitney U test (**p < 0.002, and ****p < 0.0001).

[0041] Figure 19. SNAP inhibits prostate tumor organoid growth.Prostate cancer-derived tissues from two patients were embedded in Matrigel to generate 3D organoids. The resulting organoid were treated in regulated conditions with DMSO or SNAP (100 pM) for 10 days, with media and treatment renewed every two days. Organoids were imaged on day 0 and day 10. The raw organoids sizes (pm2) were quantified (A), as well as the relative growth of the organoids at day 10, normalized to day 1 (B). For each patient, charts show mean ± SEM of more than 100 organoids from 5 replicates. Statistical significance between the groups was evaluated using a two-tailed Mann-Whitney U test (*p = 0.02, **p = 0.008, ****p < 0.0001). (C-D) After 14 days, live-dead cell viability was assessed with Cyto3D kit to evaluate the effect of SNAP on cell death. Representative confocal microscopy images of live cells (Cyan, acridine orange), dead cells (Red, propidium iodide), are shown with nuclei counterstained with Hoechst. Scale bars indicate 20 pm. For each patient, ImageJ software was used to analyze the Z-stack images, quantify the dead cell signal and represent as the mean ± SEM of 43 organoids from 5 replicates. Statistical significance between the groups was evaluated using a two-tailed Mann-Whitney U test (***p = 0.0001, ****p < 0.0001). (E) After 14 days, Proteins were extracted from each patient and the target of PHLPP1, p-Akt / p-ERKl / 2, and apoptosis marker c-casp3 were analyzed by western blot.Detailed Description

[0042] Chemical Definitions: Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999,19WSLEGAL\055326\00505\42539390v6Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989, and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0043] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0044] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer, geometric isomer, or a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ of Notre Dame Press, Notre Dame, IN 1972) The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0045] In the compositions provided herein, an enantiomerically pure compound can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising enantiomerically pure R-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R-compound.

[0046] The compounds described herein can be provided as amorphous solids or crystalline solids. Lyophilization can be employed to provide the compounds as amorphous solids.20WSLEGAL\055326\00505\42539390v6

[0047] It should further be understood that solvates (e.g., hydrates) of the compounds described herein are also within the scope of the present invention. The term “solvate” means a physical association of a compound of Formula (I) with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solutionphase and isolable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, isopropanolates, acetonitrile solvates, and ethyl acetate solvates. Methods of solvation are known in the art.

[0048] The compounds of the present invention are intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium (D) and tritium (T). Isotopes of carbon include 13C and 14C. Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. For example, methyl ( — CH3) also includes deuterated methyl groups such as — CD3.

[0049] Any reference to a compound or a SNAP compound includes pharmaceutically acceptable salts thereof. The term “pharmaceutically acceptable salts” is meant to include salts of active compounds that are prepared with relatively nontoxic acids or bases, depending on the substituents found on the compounds described herein. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium salt, or a similar salt. Pharmaceutically acceptable salts are well known in the art and are the subject of numerous reviews and monographs such as P. H. Stahl and C. G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich:Wiley-VCH / VHCA, 2002.21WSLEGAL\055326\00505\42539390v6

[0050] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0051] We hypothesized that DIFs across many species perhaps evolved in parallel with the mTOR and AMPK networks, and that some species may have lost the need to produce DIFs when they gained control of their dependence on the environment for food / water and thus the need to hibernate, while maintaining the mTOR / AMPK networks, because of their overall importance in survival. We also speculated that DIFs are diffusible circulating small molecules / metabolites that may be transferable across hibernating or aestivating species, but also able to induce hibernation in non-hibernating species, since their putative effectors, i.e., the mTOR / AMPK networks, are widely conserved.

[0052] Using a model of aestivating snails and unbiased metabolomics

[0045] , we discovered a putative DIF small molecule that was produced in the snail brain and diffused to the body through the snail blood (hemolymph), with its levels dropping immediately prior to exit from dormancy. We characterized its structure with high-performance liquid chromatography (HPLC) and mass spectrometry (MS), and with a structural Similarity Ensemble Approach, we found that it has a very high affinity for a target in the mouse / human proteome, namely PHLPP1

[0046] (PH Domain and Leucine-rich repeat Protein Phosphatase 1), a widely conserved phosphatase that sits in the heart of the mTOR / AMPK networks as its targets include p-AKT and p-S6Kl. Intriguingly, PHLPP1 was originally discovered in the hypothalamus as a product of a circadian-regulated gene

[0047] . We synthesized the metabolite chemically and named it SNail Activator of PHLPP1 (SNAP) because we found it directly activates PHLPP1 phosphatase activity. SNAP can induce dormancy in snails, indistinguishable from physiologic aestivation / hibernation, quiescence in ischemic mouse fibroblasts and a cardioprotective hibernation-like state in the mouse heart (a species that does not normally hibernate), with significant protection from ischemic and IR injury.22WSLEGAL\055326\00505\42539390v6

[0053] SNAP001 was synthesized based on the structure of the Dormancy Inducing Factor (DIF) discovered in snails (which is absent from the non-hibernating mouse and human metabolome and activates PHLPP1) and had not been previously described in terms of its structure and role. We believe that SNAP001 and other SNAP compounds described herein can offer non-hibernating species like humans, the advantages and protection from ischemia and IR injury of hibernating species. This is because PHLPP1 and its targets are widely conserved from snails to mammals.

[0054] Without restriction to a theory, it is believed that by inhibiting the PHLPP1 targets AKT and S6K1, SNAP can reset the balance of the competing AMPK and mTOR networks, to optimize fuel utilization in fuel-deprived states, promoting quiescence at the cellular level and hibernation at the organ or animal level.Compounds

[0055] Here, we provide evidence that novel compounds synthesized based on a putative Dormancy Inducing Factor (DIF) discovered in a snail hibernation model, can induce quiescence in mice or human fibroblasts, hibernation in the hearts of mice, species that do not normally hibernate; and decrease the growth of human cancer. In one preferred embodiment, the molecule comprises SNAP.

[0056] Following our initial molecular modeling analysis of SNAP binding to and activating PHLPP, two distinct series of derivatives using bioisosteric modifications were conceived in a manner that mimick SNAP’s binding pattern and interaction with PHLPP 1 amino acids, resulting in the activation of the enzyme: Series A, consisting of 19 compounds, retains the conserved core scaffold of SNAP, incorporating systematic variations at substituents R1-R5 that retain chemical functionalities and optimize binding interactions. The parent compound, SNAP001, is the reference structure for comparative analysis of Series A derivatives, where all are in the form of pyridinium salt (or other salt formulations).

[0057] A reference to “SNAP” can be a reference to at least one compound of Formula (I) or Formula (II) described below.23WSLEGAL\055326\00505\42539390v6

[0058] Disclosed herein are compounds which activate PHLPP, comprising at least one compound of Series A (Formula (I) in the form of a pyridinium salt, of the following structure:Rfwherein, independently of each occurrence, Rl, R2, R3, R4 and R5 are selected from the groups identified below:24WSLEGAL\055326\00505\42539390v6WSLEGAL\055326\00505\42539390v6

[0059] Also disclosed are compounds which activate PHLPP designated herein as Series B (also in the form of pyridinium or other salt formulations) (Formula (II)), which compounds comprise 4 structurally distinct compounds that diverge from the SNAP core structure, but maintain the same properties of interaction with the amino acids in the PHLPP pocket as in Series A. Here, the aromatic benzene ring is removed from the core scaffold, introducing a new structural framework designed to probe alternative binding conformations within the PHLPP binding site. This modification allows for increased conformational flexibility, enabling additional binding interactions and potentially enhancing target engagement.

[0060] Formula II has the following structure:WSLEGAL\055326\00505\42539390v6wherein R1 is selected from the group consisting of

[0061] Despite being a small molecule, SNAP compounds are specific in terms of PHLPP activation, as predicted by computer modeling. Experimentally, SNAP increases phosphatase activity of PHLPP 1 in mice cells and human recombinant PHLPP even outside of the cellular environment. Within cells, SNAP’s ability to dephosphorylate the known PHLPP 1 targets pAKT and pS6K, is absent when PHLPP 1 expression is inhibited by siRNA or when the amino acids that SNAP was predicted to interact with to increase enzymatic activity are mutated, supporting its specificity. The fact that SNAP, under these conditions, does not dephosphorylate the known PHLPP1 targets pAKT and pSKl and does not cause a release of phosphates in the enzymatic assays, means that SNAP does not activate any phosphatases other than PHLPP 1 (e.g., see Figure 3).

[0062] Our claim that SNAP can induce cell quiescence is based on its ability to induce all features of cellular quiescence in mouse and human fibroblasts (resistance to stress-induced death, reversible cell-cycle exit, metabolic remodeling, suppressed proteinosynthesis rates and autophagy) cellular quiescence in mouse and human fibroblasts.

[0063] In one aspect, disclosed herein is the use of a SNAP compound in the preparation of stem cells or the non-freezing preservation of ovarian cells (eggs), both of which are characterized by cellular quiescence.

[0064] Our claim that SNAP can induce heart hibernation is based on the fact that SNAP can acutely preserve cardiomyocyte and whole heart systolic and diastolic heart function under 27WSLEGAL\055326\00505\42539390v6both ischemia and reperfusion. In one example, an application can be the use of SNAP in solid organ transplantation, where the majority of donated organs are not usable because of the cumulative effects of ischemia and reperfusion injury during organ transport. If SNAP can preserve function in an organ with a high fuel demand like the heart, it is likely that it may also be beneficial in organs will less fuel demand like the lungs, liver or kidneys.

[0065] In another example, SNAP can protect human hearts from the IR injury they undergo during thrombolysis or by-pass surgery.

[0066] In another example, SNAP may protect internal organs (like heart, brain, lungs, kidneys or liver) from the ischemic damage they undergo during the hypotension that develops in septic or hemorrhagic shock (e.g., sepsis, trauma). In such cases, SNAP can also be added to coma-inducing medications used in ventilated patients, to offer the patients that hibernation-related organ stress protection that induced coma by itself does not offer.

[0067] Our claim that SNAP can cause hibernation in whole animals, like humans, is supported by our finding that SNAP induces hibernation in a whole animal model (snails) that is indistinguishable from naturally occurring hibernation. One application may be the potential of SNAP inducing hibernation in astronauts during ultralong space travel where research is active but currently restricted to cryopreservation strategies. One of the attractive features of SNAP is that it came from a model of hibernation that is not temperature dependent (estivating snails), making its application to humans easier since currently this field is dominated by the more challenging cryo-preservation approaches.

[0068] In another example, SNAP may decelerate ageing in humans or animals that do not hibernate normally (e.g. humans, dogs). This because SNAP can induce hibernation in a whole animal (snail) and it was recently discovered that hibernating animals exhibit decreased ageing rates during their hibernation phases.

[0069] Our claim that SNAP can treat cancer is supported by our data showing that SNAP, by inhibiting the cancer-promoting AKT, decreases the growth of human cancer cells, like melanoma or prostate cancer.28WSLEGAL\055326\00505\42539390v6Synthesis

[0070] Synthesis: An exemplary synthesis of 3-(2-methyl-8-(2- ((oxidanidylsulfonyl)oxy)ethyl)-3,4-dihydro-2H-chromen-2-yl) propanoic acid (SNAP001) and 3-(2-methyl-8-(2-((oxidanidylsulfonyl)oxy)ethyl)-3,4-dihydro-2H-chromen-2-yl) propanoate-pyridine (SNAP001 -Pyridine) is provided below in the Examples. Derivatives and variants, such as those described above as Series A and Series B, may be synthesized in a straight-forward manner using analgous techniques well-known to those skilled in the art.

[0071] One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, may utilize a variety of protecting groups. By the term “protecting group,” as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In certain embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. Additionally, a variety of protecting groups are described in Protective Groups in Organic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.Compositions

[0072] Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology and chemistry. In general, such preparatory methods include the steps of bringing a SNAP compound into association with a carrier and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit. Pharmaceutical compositions can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As 29WSLEGAL\055326\00505\42539390v6used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0073] For oral administration, a SNAP compound can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. As used herein, a “pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

[0074] Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, i.e. EDTA for neutralizing internal acid conditions or may be administered without any carriers.

[0075] Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of 30WSLEGAL\055326\00505\42539390v6the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981, “Soluble Polymer-Enzyme Adducts” In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark, et al., 1982, J. Appl. Biochem. 4:185-189. Other polymers that could be used are poly- 1,3 -di oxolane and poly-1, 3, 6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are polyethylene glycol moieties.

[0076] For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the active agent (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.

[0077] To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

[0078] A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used. The shell material of31WSLEGAL\055326\00505\42539390v6cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

[0079] Various additives or agents may be added, such as a colorant or a flavoring agent, an inert material or diluent, a disintegrant, a binder, an anti-frictional agent, a glidant, a surfactant.

[0080] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

[0081] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0082] For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant. Also contemplated herein is pulmonary delivery of the active agents (or derivatives thereof). The active agent (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise active agent (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active active agent per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for active agent stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the active agent caused by atomization of the solution in forming the aerosol.32WSLEGAL\055326\00505\42539390v6

[0083] A SNAP compound may be delivered systemically, such as by parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0084] The active agents and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

[0085] Suitable buffering agents include: acetic acid and a salt (1-2% w / v); citric acid and a salt (1-3% w / v); boric acid and a salt (0.5-2.5% w / v); and phosphoric acid and a salt (0.8-2% w / v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w / v); chlorobutanol (0.3-0.9% w / v); parabens (0.01-0.25% w / v) and thimerosal (0.004-0.02% w / v).Methods of Use33WSLEGAL\055326\00505\42539390v6

[0086] The present disclosure features compounds, compositions, and methods comprising a SNAP compound disclosed herein. In some embodiments, the compounds, compositions, and methods disclosed herein are used in the prevention, treatment or amelioration of a disease, disorder, or condition, or the prevention or amelioration of injury or degradation to an organ, whether in vivo or ex vivo.

[0087] The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0088] For use in therapy, an effective amount of the active agent can be administered to a subject by any mode that delivers the active agent to the desired surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal.

[0089] “Therapeutically effective amount” or an "effective amount" is intended to include an amount of a SNAP compound alone or an amount of the combination including at least one SNAP compound, such as a combination with at least one other active ingredient effective to act as an activator or effective to treat or ameliorate the disease or condition which it intended to treat or ameliorate.

[0090] As used herein, “treating” or “treatment” cover the treatment of a disease-state or condition in a mammal, particularly in a human, and include: (a) preventing the disease-state or condition from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state or condition, i.e., arresting its development; (c) relieving the disease-state or condition, i.e., causing regression of the disease state and / or relieving or mitigating at least one symptom of the diseasestate or condition.34WSLEGAL\055326\00505\42539390v6

[0091] In another aspect, disclosed are methods of using at least one SNAP compound, in a therapeutically effective amount, to protect an organ which is at risk of ischemia, whether in vivo or ex vivo. In some embodiments, the method comprises the use of at least one SNAP compound to treat a disease or a condition in a patient in need thereof, wherein the disease or condition is marked by a risk of human organ ischemia. The organ most vulnerable to ischemia is the heart. Our data support that SNAP compounds can be beneficial in protecting hearts or other solid organs from the IR injury that occurs a) in transplant donor hearts other organs that suffer damage during their transport to a transplant recipient (most donated organs cannot be transplanted because of this damage); b) human hearts that suffer IR injury upon thrombolysis or coronary interventions during a myocardial infarction; c) hearts suffering IR injury during coronary artery bypass surgery (CABG); and (d) hearts and other internal organs suffering injury during septic or hemorrhagic shock.

[0092] In another aspect, because of its ability in inducing quiescence and hibernation in human cells and organs, disclosed are methods of using at least one compound disclosed herein to treat astronauts during ultra-long space travel, where like in hibernating animals, astronauts will not suffer the well-known consequences of fuel restriction and immobility.

[0093] In another aspect, because mTOR and its complex interactions with AMPK, are critical in promoting longevity in multiple species, and because during hibernation the ageing rates of animals decrease, disclosed are methods of using at least one compound disclosed herein to promote longevity of different species, including humans.

[0094] In another aspect, because PHLPP inhibition is known to promote cancer, soon after its discovery, and specifically its ability to inhibit the cancer-promoting AKT, it is considered an “anti-oncogene”, disclosed are methods of using at least one compound disclosed herein in a therapeutically effective amount to treat patients suffering from cancer, in that at least one compound disclosed herein activates PHLPP and thus inhibits AKT, which will promote cancer regression.

[0095] The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references 35WSLEGAL\055326\00505\42539390v6(including literature references, issued patents, published patent applications, and co-pending patent applications, if any) cited throughout this application are hereby expressly incorporated by reference.

[0096] EXAMPLES: The present invention may be described with reference to the following Examples. These Examples are provided for the purpose of illustration only.

[0097] Discovery of a snail circulating Dormancy Inducing Factor (DIF), SNAP’s synthesis, its ability to induce hibernation in snails and proof of its specificity as a PHLPP1 activator. We developed a snail model in which to study aestivation using Oreohelix subrudis snails

[0048] , Snails offer an attractive model because they enter dormancy quickly under conditions of restricted water (humidity) supply either in room temperature (aestivation) or in cold temperatures (hibernation). Their dormancy is easy to detect because it is marked by the formation of a characteristic membrane or "seal", in addition to inactivity (Figure 1A). To measure the activity of individual control and hibernating snails within a mixed population, we “tagged” individual snails and tracked their activity (distance covered) with continuous monitoring, using a customized camera detection system. To determine whether a presumably circulating DIF in dormant snails (deprived of humidity) can be transferred and induce dormancy in control snails (with normal access to food / humidity), we injected hemolymph (i.e., plasma) from dormant snails into control snails. We found that the injection caused the formation of the dormancy seal and inactivity in all snails within a few hours, i.e., a state otherwise indistinguishable from aestivation (Figure IB). This state was maintained upon repeated daily injections despite keeping the snails in normal humidity. However, upon discontinuing the injections and making food / water available, the snails exited dormancy and returned to normal activity, identical to snails that never aestivated, measured by the distance covered under the cameras. We measured the O2 consumption of homogenized control and dormant snail tissues (whether naturally aestivating or due to injection of aestivating blood), using the SeaHorse platform. We found a significant and similar decrease in mitochondrial respiration in both, compared to control snails, in keeping with what is known in many hibernating animals (Figure 1C). We also stained snail tissues with TMRM (a positively charged dye that is commonly used to measure mitochondrial membrane potential (ATm), since the positively charged TMRM is 36WSLEGAL\055326\00505\42539390v6uptaken preferentially by the most negatively charged organelles in the cell, i.e., mitochondria (Figure ID). Dormant snail tissues had more hyperpolarized mitochondria, compatible with a hypometabolic state of suppressed respiration and resistance to mitochondria-dep endent apoptosis (which is triggered by mitochondrial depolarization). These data suggested that a circulating putative DIF is transferable across different snails.

[0098] To discover the putative snail DIF(s), we proceeded with a methanol / water extraction of metabolites from homogenized snail heads versus bodies, followed by HPLC coupled with Orbitrap high-resolution MS under positive and negative electrospray ionization modes. We focused on the detection of metabolites that were only present in aestivating but not in control tissues. A metabolite with a negative charge at m / z 343.0859 at retention time of 5.1 min from HPLC was observed in all aestivating snail heads, hemolymph and body but was absent in all controls (Figure IE). The metabolite appeared first in the head and then peaked in the body but disappeared just before snails exited hibernation upon provision of humidity. We found that the chemical formula of the ion at m / z 343.0859, based on its HPLC / mass spec profile and by using Xcalibur software, was C15H19O7S. Using the PubChem database, we identified a structural template, which we modified to fit the fragmentation pattern observed in the snail metabolite. This process resulted in the structure shown, where specific parts of the molecule aligned with the fragmentation pattern displayed in the mass spectrum (Figure IF). We then applied the Similarity Ensemble Approach to identify its possible protein targets / ligands in the human proteome from the ChEMBL database. Despite being a small molecule, it exhibited a high predicted affinity (maximum Takimoto coefficient = 0.31, p = 8.8 / I055) for PHLPP1. Computational modelling indicated that the compound binds to an allosteric pocket near the catalytic domain of PHLPP

[0049] , (Figure 1G). This binding allows the molecule to interact with the residues Q1407, Cl 109, El 132, and H1412, resulting in biophysical interactions predicted to enhance PHLPP1 enzymatic activity (Figure 1G) through stabilizing the active site loop through interaction with H1412 and inducing conformational changes that optimize the distance between Cysl411 and Aspl413 at the catalytic site, effectively reducing the enzyme's activation threshold. We named it SNail Activator of PHLPP 1 (SNAP) and proceeded to confirm its predicted ability to activate PHLPP 1.37WSLEGAL\055326\00505\42539390v6We then chemically synthesized SNAP001 in the form of a stable pyridinium salt (as described in the methods) and gave it to snails by injection. We found that it caused reversible hibernation, indistinguishable to natural hibernation caused by humidity restriction. Furthermore, in another group of control snails we gave plasma (hemolymph) from hibernating snails. We found that the hibernation induced by SNAP001 versus hibernating hemolymph was identical (Figure I H). We then confirmed its purity and identical structure with the snail metabolite, using NMR spectroscopy (Figure 2A). Subsequent analysis with HPLC / MS / MS (Figure 2B) confirmed a perfect match between the synthesized compound and the snail metabolite.

[0099] SNAP001 is a specific PHLPP1 activator. Given that phosphorylated AKT (p-AKT) at Ser473 is a well-established target of PHLPP1, we employed a validated PHLPP1 activity assay using a synthetic peptide (HFPQFPSYSAS) corresponding to the p-AKT Ser473 sequence to examine the effect of SNAP001 on PHLPP1 -mediated dephosphorylation. We utilized three enzyme sources that represent increasing levels of biochemical specificity and purity and measured phosphate release: (i) stressed mouse fibroblast lysates under fuel deprivation (O2, glucose, FBS) from scramble versus siPHLPPl -transfected cells (Figure 3A), (ii) immunoprecipitated PHLPP1 from stressed fibroblast lysates (Figures 3B and 3C), and (iii) recombinant human PHLPP1 incubated directly with the peptide substrate and Mn2+, a required cofactor (Figure 3D). Across all three conditions, SNAP001 enhanced phosphate release, indicating increased PHLPP1 activity; except in the presence of PHLPP1 siRNA, suggesting that snap does not activate other phosphatases in the cell. In fuel -restricted fibroblasts, SNAP coimmunoprecipitated with PHLPP1 and promoted dephosphorylation of known PHLPP1 targets, p-AKT and p-S6Kl (Figures 3E and 3F). With a dose-response experiment, we found that the lOpM SNAP001 was sufficient to inhibit p-AKT, as well as p-S6Kl to induce autophagy (LC3B-II) (Figure 3G), and the lOpM dose was used throughout the paper. In keeping with Figure 3A, these SNAP001 effects were abolished when cells were transfected with siRNA for PHLPP1 (Figure 3H). To further confirm its specificity for PHLPP1, we also utilized site-directed mutagenesis of the 4 amino acids predicted to be essential for SNAP’s binding to PHLPP1 (Q1407, Cl 109, El 132, and H1412). Mutating them individually or simultaneously had the same effect, preventing enzymatic activation by SNAP001 (Figures 31 and 3 J).38WSLEGAL\055326\00505\42539390v6

[0100] SNAP001 induces quiescence in fuel-deprived fibroblasts. Because PHLPP1 and the putative DIF effectors AKT and S6K are widely conserved, we speculated that SNAP001 may also induce quiescence in fuel-stressed fibroblasts from mice, a species that normally does not hibernate. To explore its effects on mouse cells, we gave SNAP001 to fibroblasts exposed to normal (P02~100mmHg, pH 7.35, glucose 25mM) versus moderately ischemic conditions (PC>2~50 mmHg, pH7.35, glucose 2.5 mM, 50% reduced FBS) in order to determine whether SNAP001 can induce dormancy (i.e. quiescence) and thus decrease damage from ischemia. We studied several critical features of cellular quiescence, i.e., autophagy, mitochondrial metabolism, proteinosynthesis and cell-cycle exit50. To assess whether these effects are reversible, we also studied ischemic fibroblasts after returning them to normal conditions and removing SNAP001 from the media (Figure 4A). Overall, SNAP001 did not have any effects under normal conditions. Under ischemia however, compared to vehicle, SNAP001 induced autophagy as it increased markers of autophagy (lysosome activation, LC3B and LAMP) (Figures 4B and 4C), while suppressing apoptosis and increasing confluency (Figures 4D and 4E). This was associated with increased markers of cell-cycle exit (p-CDK2, p-Rb) and decreased markers of proliferation like Ki67 (Figure 4E-G). Upon return to normal conditions with no SNAP001, cells entered the cell cycle again and started proliferating again with doubling times similar to normal cells (Figure 4E), suggesting resistance to injury during ischemia and reversible entry to quiescence and not senescence.

[0101] Because ischemic effects on mitochondria are drivers of injury through mitochondrial depolarization (i.e., decreased A'Pm, which decreases the threshold of mitochondria-induced apoptosis) and increase in mitochondrial reactive O2 species (mROS) (which induce ER stress) we studied SNAP’s effects on mitochondrial respiration, A'Pm and mROS (Figures 4H-4J). Under ischemia, SNAP001 caused an increase in spare respiratory capacity (SRC), which was also present when we studied cells after coming out of ischemia (Figure 41). SRC is defined as the difference between max O2 consumption rate (OCR) due to FCCP and the minimum OCR due to oligomycin

[0051] and reflects the flexibility of mitochondria to adapt to varying fuel supply / demand states, maintaining ATP production and avoiding injury

[0051] , SNAP prevented a large decrease in A'Pm and suppressed mROS compared to vehicle 39WSLEGAL\055326\00505\42539390v6(Figure 4J). One of the reasons that increased SRC can be achieved is by activating Pyruvate Dehydrogenase (PDH)

[0051] , the gatekeeper of GO in mitochondria. Indeed, SNAP prevented a decrease in PDH activity reflected by a decrease in all 3 inhibiting phosphorylation sites of PDH compared to vehicle (Figure 4K). PDH is suppressed in ischemia either through the HIF-la induced increase in Pyruvate Dehydrogenase Kinase (PDK) expression, which phosphorylates and inhibits PDH; or acutely through phosphorylation and activation of PDK by mitochondrial p-AKT, as we discuss below. While fibroblasts do not depend on PDH as much as more energydependent cells like cardiomyocytes (which we studied next), the preservation of PDH activity (and thus GO and ATP) is critical because it explains the increase in SRC and furthermore supports the robust increase in autophagy, an energy-dependent process. Another feature of quiescence is proteostasis. We found that SNAP suppresses proteostasis by inhibiting the S6K1 target eIF2a and inhibiting new protein synthesis (i.e., OPP incorporation) (Fig 5).

[0102] We next studied the effects of SNAP001 in autophagy if human and mouse fibroblasts, and compared them to a known activator of autophagy, i.e. the mTOR / S6Kl inhibitor rapamycin

[0052] (Fig 6). We found that while the SNAP induction of autophagy was not as large as with rapamycin, SNAP did not cause the significant inhibition of PDH seen with rapamycin. This suggests that the induction of the ATP-dependent autophagy may be more sustained due to the preservation of PDH function and may not lead to death and toxicity, as can happen with excessive autophagy without energetic support, as seen with rapamycin. The SNAP effects on autophagy and PDH can be explained by the activation of PHLPP1 and the inhibition of the mTOR / S6Kl axis and the acute inhibition of AKT, respectively (mitochondrial AKT is known to phosphorylate and activate PDK, which in turn phosphorylates and inhibits PDH

[0053] ).

[0103] SNAP001 protects mouse cardiomyocytes from ischemia and acute IR injury. We studied freshly isolated cardiomyocytes subjected to physiologic fuel deprivation (PC>2~50 mmHg, pH 7.35, glucose 5 mM) with SNAP vs vehicle for only 3hrs (Figure 7A), rather than longer-term culture used for fibroblasts, because cardiomyocytes are especially susceptible to fuel deprivation, and measured mitochondrial function, apoptosis, autophagy, and ER stress. We also studied cardiomyocytes under re-exposure to normal conditions (PCh-HO mmHg, pH 7.35, glucose 10 mM) without SNAP for an additional 0.5 or 2hs. SNAP caused dephosphorylation of 40WSLEGAL\055326\00505\42539390v6p-S6Kl and p-AKT, two established targets of PHLPP1 [46,54], as well as PDH, and decreased apoptosis under IR stress (Figure 7B). Compared to vehicle, SNAP -treated cells had less drop in A'Pm and less increase in mROS within 30 minutes after reoxygenation (Figure 7C). PHLPP1, its membrane scaffold protein NHERF-1 and pAKT were translocated from the plasma membrane to the cytosol and mitochondria, where SNAP inhibited the mitochondrial fraction of pAKT, explain the decreased levels of pPDK (which is phosphorylated by AKT and in turn in phosphorylates and inhibits PDH) and the preserved levels of pPDH (as opposed to the increased levels caused by vehicle) under IR stress (Fig 7C, D, E). The total mitochondrial PDK1 levels did not increase during the 1 hour of ischemia, suggesting that its function was activated by AKT rather than its levels through induction by HIFla. Indeed, while hypoxia activates HIFla, 1 hour is not enough time to increase its transcription and translation in our model, although this may happen at longer time intervals. SNAP treated cells also had higher SRC, (Figure 7G) along with improved contractility (Figure 7H), increased ATP and acetyl-CoA levels compared to vehicle (Figure 7I-J). All of the above benefits of SNAP in the above figures were absent in cardiomyocytes isolated from a myocardial cell specific inducible PDH knock our model that lacks myocardial PDH, confirming the central role of PDH on SNAP’s benefits under IR stress.

[0104] When the reperfusion phase was extended to 2 hours (Figure 8A), SNAP-pretreated cells had significantly reduced ER stress (Figure 8B) while still maintaining higher A m and lower mROS levels compared to vehicle (Figures 8C and D), suggesting that SNAP protects the energy-demanding mouse cardiomyocytes against IR injury in a sustained manner, similarly to the less energy-dependent fibroblasts. In fibroblasts, a significant increase in pAKT and pS6K occurred within only 1 hour of moderate ischemia and this was inhibited by SNAP (Figure 9), prompting us to investigate how PDH phosphorylation and inhibition were prevented during this short period of stress. Under stress conditions, p-AKT is known to translocate to mitochondria via a chaperone

[0053] , In normal conditions, PHLPP1 is known to bind the scaffold protein NHERF-1 [55,56] in the plasma membrane, but under stress, PHLPP la translocates to mitochondria

[0057] , These reports are in keeping with our data in Fig 7 D and E. With additional data we confirmed the binding of PHLPP1 to NHERF-1 with co-immunoprecipitation (Figure 10). While we had found more PHLPP la and NHERF-1 in the cytoplasm and mitochondria 41WSLEGAL\055326\00505\42539390v6under 1 hour of ischemia (Fig 7 D-E), we also found that the total PHLPPl and 0 as well as NHERF-1 levels in whole cell lysates remained unchanged (Figure 11), suggesting translocation of PHLPPla and NHERF-1 to cytosol and mitochondria. Importantly, we found no PHLPP1 levels in the nuclear fraction. This means that potential known targets of PHLPP1 in the nucleus (e.g. STAT 1 and other transcription factors and histone) are not involved in the mechanism of protection from acute stress by SNAP unlike its cytoplasmic and mitochondrial targets pAKT and S6K1. Lastly, even after 3 hours of stress, the energetic benefits of SNAP that we had shown after 1 hour of stress, (increased ATP levels, decreased lactate levels) persisted (Figure 12), in keeping with the preserved PDH activity under stress.

[0105] Collectively, our findings illustrate that SNAP acutely minimize injury and orchestrates stress recovery by increasing autophagy through the inhibition of cytoplasmic S6K1 and by preserving PDH activity through the inhibition of mitochondrial AKT.

[0106] SNAP001 induces a hibernation-like state with acute protection from IR injury in mice hearts. We studied isolated perfused mouse hearts using the Langendorff and the working heart models. In the former, the afterload is low (i.e., gravity), and for this reason, at the end of the protocol we paced the hearts at 300 beats / min in order to assess performance at higher workloads and also standardize the rate, which showed some variation among different mice heart preparations; while, in the later model, the afterload is higher, i.e., the hearts are pumping against an aortic pressure of 50 mmHg. We used two protocols: protocol 7, where following an adjustment period, the hearts were exposed to normal conditions prior to disruption of perfusion with occlusion (PO2 110 mmHg, pH7.35, glucose 10 mM) (Figures 13A and 14A); and protocol 2 where following adjustment, hearts were exposed to global ischemia (PO2 50 mmHg, pH7.35, glucose 5 mM) . Protocol 2 simulated the conditions that an offered transplant heart undergoes from the donor (normal conditions, stage I) to the ischemia during transfer (stage IL ischemia with low workload), to anoxia during surgery (stage III: occlusion), to placement in the donor (pacing-induced increased workload and reperfusion, stage IV). We measured the time to return to normal contractions without arrhythmias, and left ventricular pressures (LVP) with Millar catheters and calculated max and min dp / dt. In protocol 7, SNAP had small effects compared to the vehicle, mostly an improvement in min dp / dt (which is most sensitive to ischemia),42WSLEGAL\055326\00505\42539390v6indicating preservation of diastolic function (Figure 14). In protocol 2 SNAP improved all the parameters we followed significantly (Figures 13). SNAP was cardioprotective even when the occlusion phase was prolonged from 30 min (standard in this model) to 40 min (Figure 15). In stage II, SNAP preserved PDH function compared to vehicle, as p-PDHEl levels decreased markedly, along with lower p-AKT levels (Figure 16A-C). This was associated with higher A'Pm and lower mROS in the SNAP -treated vs vehicle-treated hearts (Figure 16D). The preserved PDH function suggested higher GO rates. Increased GO is associated with decreased FAO rates, in part through the Randle feedback

[0060] , Increased GO / FAO rates are a feature of many cardioprotective agents like the PDK inhibitor dichloroacetate [61-63], malonate

[0064] and GLP-l(28-36)

[0065] , To measure GO / FAO ratio we used the working heart model (that in contrast to the Langendorff perfusate where we only used glucose, we used [5-3H] palmitate and U-14C glucose. We compared the input perfusate to the output from the coronary sinus, revealing a 50% increase in the GO / FAO ratio in SNAP vs vehicle-treated hearts (Figure 13 L).

[0107] To assess the critical role of PDH, like we did with cardiomyocytes, we perfused heats from wild-type versus / k / / 7a / Cardiac' "mice [58,59], under protocol 2 (Figure 17). We found that SNAP significantly improved the recovery of contractility (determined by examining the data from the last 40 minutes of reperfusion against the initial baseline for each mouse) in WT mice (. SNAP better restored LVP, maximum dp / dt, and minimum dp / dt in aMHC-MerCreMer hearts (i.e., WT hearts: because Cre may have detrimental effects we used these mice as a control simulating WT mice) compared to Pdhal^^'1' hearts. These ex-vivo experiments corroborate the in vitro results, supporting the beneficial role of SNAP in IR injury and the critical role of PDH in SNAP’s cardioprotection.

[0108] SNAP001 decreases growth and induces death (apoptosis) in aggressive (metastatic) cancer cells: 24 hours after seeding, A2058 cells (a metastatic melanoma cell line) were treated with vehicle (DMSO) or SNAP (lOpM). Cell proliferation was monitored for 72 hours using a Holomonitor microscope. Proteins were collected at 72 hours post-treatment, and the effectiveness of SNAP on the expression of p-AKT and the proliferation marker Ki67 were analyzed via western blot. Cells were also stained with Alexa Fluor 488 annexin V (AV) and43WSLEGAL\055326\00505\42539390v6propidium iodide (PI) to analyze apoptosis. We found that SNAP decreases cell growth and induces cell death by dephosphorylating and inhibiting its target pAKT (Fig 18A-C).

[0109] In addition to AKT, SN AP001 also inhibits the PHLPP1 target pERK (1-2) and sensitizes cells to chemotherapy-induced death. SNAP decreased the levels of pERK (1-20) and potentiated the cell death induced by chemotherapy (doxorubicin), suggesting that it can be used an adjuvant to chemotherapy (Fig 18D). Cancer cells often grow and migrate under hypo-perfused tissues (ischemic) as the vascularization of tumors is incomplete, due to suboptimal and anarchous angiogenesis, particularly at the edges of a tumor. Thus, A2058 cells were treated with DMSO or SNAP under metabolic stress (2.5mM glucose, 1% FBS, 1% Oxygen) for 48 hours before migration and adhesion analysis using standard trans-well methodology. SNAP suppressed the migration of cancer cells, and this was associated by a significant inhibition of paxillin a critical part of focal adhesions required for effective migration (Fig 18E). As AKT is known to increase paxillin levels, these effects are explained by SNAP’s ability to inhibit AKT.

[0110] SN AP001 inhibits prostate tumor organoid growth and survival. Prostate cancer-derived tissues from two patients that underwent radical prostatectomy, were embedded in Matrigel to generate 3D organoids. And treated with DMSO or SNAP (10 pM) for 10 days, with media and treatment renewed every two days. Organoids were imaged on day 0 and day 10. SNAP significantly decreased organoid size from both patients. After 14 days, live-dead cell viability was assessed with Cyto3D kit to evaluate the effect of SNAP on cell death. SNAP increased cell death in the prostate cancer organoids, once again inhibiting pAKT and pERK (1-2) (Fig 19). These data suggest that SNAP can be an effective anti-cancer agent in the setting of a cancer organoid, that reflects the in vivo conditions much better than cancer cells in a 2D culture, that also lacks the important tumor stroma cells.

[0111] MethodsAll animal studies were approved by the University of Alberta Animal Care and Use Committee (ACUC). Investigators performing data collection and analysis were blinded to the groups.Antibodies for immunoblots44WSLEGAL\055326\00505\42539390v6Antibody dilution was 1:1000 for the following immunoblots: p-Rbser807 / 811(Cell Signaling Technology, 8516), Rb (Invitrogen, SY63-03), p-CDK2Thr160(Cell Signaling Technology, 2561), t-CDK2 (Abeam, ab32147), ?-Actin (Cell Signaling Technology, 3700), t-AMPK (Cell Signaling Technology, 5831), p-AMPKThr172(Cell Signaling Technology, 2535), AKT (Cell Signaling Technology, 9272), p-AKTSer473(Cell Signaling Technology, 9271), t-S6Kl (Cell Signaling Technology, 2708), and p-S6KlThr389(Cell Signaling Technology, 9205), p-PDHElaser232(Millipore Sigma, AP1063), p-PDHElaser300(Millipore Sigma, AP1064), t-PERK (Cell Signaling Technology, 3192), p-PERKThr982(Invitrogen, PA5-40294), ATF6 (Abeam, ab37149), LC3B (Cell Signaling Technology, 2775), cleaved caspase-3 (Cell signaling Technology, 9664), t-mTOR (Cell Signaling Technology, 2983), p-mTORser2448(Cell Signaling Technology, 5536), Ki67 (Abeam, abl6667), p-eIF2<zSer51(Cell Signaling Technology, 9721), t-eZF2<z (Cell Signaling Technology, 5324), Custom p-PDKlThr344(generated by Neobiolab), t-PDK1 (Abeam, abl 10025), LDHA (Cell Signaling Technology, 2012), p-JNKTlirl83 Tvrl85(Cell Signaling Technology, 9251), t-JNK (Cell Signaling Technology, 3708), cr-Tubulin (Cell Signaling Technology, 2144), NHERF-1 (Santa Cruz Biotechnology, sc-271552).Antibody dilution was 1:5000 for the following immunoblots: PHLPP (Proteintech, 22789-1-AP), p-PDHEl<zser292(Abeam, ab92696), t-PDHElcr (Abeam, abl68379), GAPDH (Abeam, ab8245).Snail experiment: Maintenance: Oreohelix subrudis snails48were collected from Southern Alberta under Alberta collection permit # 19-528 and maintained in a room -temperature colony in a snail facility at the University of Alberta. This species of terrestrial snail is native to Southern Alberta and frequently undergoes periods of aestivation during dry periods of the summer and enters a period of long-term hibernation over winter. Aestivation was induced by removing snails from the optimal conditions of their housing colony and placing them in equivalent housing conditions in the absence of food and moisture. Under these conditions, visible signs of an aestivation state are visible after ~24 hours, when a thin translucent film covers the shell aperture.Isolation of haemolymph: Snails were induced into a state of aestivation and maintained under these conditions for at least 48 hours prior to bleeding. First, snails were visually examined for 45WSLEGAL\055326\00505\42539390v6the presence of the translucent film over the shell opening to indicate those in an aestivating state. Hemolymph was extracted from snails following the head foot-retraction method. Following collection, haemolymph was then centrifuged for 5 minutes at 500g to remove the cellular component before use in subsequent studies.Haemolymph and SNAP injections: To determine whether the state of aestivation could be transferred to a non-aestivating snail through hemolymph transfusion, ten control snails and 10 experimental snails were each marked with a unique paint color. These snails were monitored using a top-down camera set up that took a picture of the entire population every minute for 1 hour. After the 1-hour calibration, during which movement of each individual snail was measured each minute based on distance travelled between consecutive images, the 10 control snails were injected with haemolymph isolated from a separate group of 10 control snails kept in normal conditions. The experimental snail group was injected with haemolymph isolated from snails that had been in an aestivation state for 7 days. The movement of the 10 control and 10 experimental snails was then monitored each minute for another 5 hours. The snails were kept in the monitoring tank for 24 hours after the 5-hour monitoring period ended, and then movement was tracked for 30 minutes to determine if the aestivation state induced by injection of aestivating snail haemolymph was reversible. After SNAP001 synthesis, a SNAP001 treatment group was included in the movement analysis using the above methodology. SNAP was injected at a concentration of 10 pM. Each movement trial was replicated three times.Detection of snail metabolites by HPLC / MS / MS analysis; Snail samples extraction: 80 / 20 of methanol / water was added to snail samples (hemolymph, head and body) at of 40pl per mg for tissue and 1 / 20 for hemolymph samples (volume ratio). The mixture was vortexed, homogenized (12 pulses at 10) and incubated on an ice bath for 30 mins and vortexed every 5 minutes during the incubation, followed by centrifugation at 10,000 rpm for 15 mins at 4 °C. The supernatant was collected, and the extraction procedure was repeated one more time. The combined supernatants were dried by speed-Vac and then re-dissolved in lOOpl of methanol / acetonitrile (50 / 50, V / V) prior to HPLC / MS / MS analysis.Snail sample extract solutions were analyzed using an ARIA MX HPLC system (Thermo Fisher Scientific, San Jose, CA) coupled to Orbitrap LTQ XL Mass spectrometer (Thermo Scientific,46WSLEGAL\055326\00505\42539390v6San Jose, CA). Data acquisition and analysis were performed using the Thermo Xcalibur software. An Xbridge BEH amide column (150 mm x 2.1 mm Amide column, 2.5 pm particle size, Waters, Milford, MA) was employed for HPLC separations. The column temperature was controlled at 25 °C. The mobile phase A was acetonitrile, and B was 10 mM ammonium acetate in 95:5 water / acetonitrile at pH 9.0, which was adjusted using ammonium hydroxide. The gradient was as follows: 0-2min, 10% B 2-18min, linear gradient to 30% B and then back to 10% B and hold for 15 min. The flow rate of the mobile phase was 150 pl / min and the cycle time was 32 min / inj ection. The Orbitrap mass spectrometer was operated under electrospray (ESI) in negative ion mode. The ionization voltage was set at -2.5 KV. For positive ionization mass spec analysis, mobile phase A was acetonitrile, and B was lOmM ammonium acetate and 0.1% formic acid. The gradient was the same as above. The ionization voltage was set at 3KV. Nitrogen was used as sheath gas, aux gas and sweep gas. They were set at sheath gas 35, aux gas 30 and sweep gas 3 (arbitrary units). The ion source temperature and capillary temperature were at 300°C and 350°C, respectively. Acquisition was carried out in full scan mode with a mass range from 70 to 1000 amu with resolving power set to a nominal value of 60,000 at full-width half-maximum at m / z 400. Within the same analysis under negative ionization mode, tandem mass spectrometry (MS / MS) was performed for ions at m / z 343.08 using high energy collision dissociation (HCD) at 70 eV at a resolution of 30,000. Mass calibration and tuning were done by infusing LTQ Velos ESI negative Ion Calibration Solution (Thermo Fisher Scientific, Rockford, IL) prior to performing HPLC / MS / MS analysis.Computer modelling: The Takimoto coefficient was used to quantify structural similarities by comparing SNAP against known ligands within the ChEMBL database. SNAP001 had a very high affinity (maximum Tanimoto coefficient=0.31, / ?=8.8xl0'55) for a single human protein, i.e., PHLPP1, from the database. The computer modelling predicted that the molecule binds to an allosteric site pocket adjacent to the catalytic domain of PHLPP1, with Q1407, Cl 109, El 132 and H1412 and predicted to enhance enzymatic activity by stabilizing the active site loop and, through conformational changes, optimizing the distance at the catalytic site. ChimeraX was used for molecular visualization74.47WSLEGAL\055326\00505\42539390v6SNAP001 synthesis: 3-[8-(2-hydroxyethyl)-2-methyl-3,4-dihydro-2H-l-benzopyran-2-yl] propanoic acid (intermediate SNAP): 2-(2-hydroxyethyl) phenol (1 equivalent), anhydrous 1,4-di oxane, ZnC12 (0.5 equivalent), and concentrated HC1 (catalytic amount) were added to a round bottom flask. A solution of 5-ethenyldihydro-5-methyl-2(3H)-furanone (1.3 equivalent) phenol in anhydrous 1,4-di oxane was then added dropwise with stirring over 2 hours. Following this, the reaction mixture was heated to 110°C under an argon atmosphere and stirred for 24 hours. After cooling to room temperature, the dioxane was removed under reduced pressure. Water was added, and the product was extracted with ethyl acetate before being purified using flash chromatography (hexane / ethyl acetate / acetic acid 3:1:0.002) (Yield: 23.4%). A full scan high-resolution mass spectrometry was conducted under negative mode showing the most intense ion at m / z 263.1293 with chemical formula C15H20O4. 1 H NMR (600 MHz, DMSO) 8 12.12 (s, 1H), 6.92 (ddd, 2H), 6.70 (t, 1H), 4.50 (s, 1H), 3.57 - 3.48 (m, 2H), 2.77 - 2.60 (m, 4H), 2.44 - 2.31 (m, 2H), 1.90 - 1.77 (m, 2H), 1.79 - 1.70 (m, 2H), 1.20 (s, 3H).3-(2-methyl-8-(2-((oxidanidylsulfonyl)oxy) ethyl)-3,4-dihydro-2H-chromen-2-yl) propanoate-pyridine (SNAPOOl-Pyridinium salt): After the intermediate structure was confirmed by mass spec and NMR, it was used for the second step synthesis for SNAP. The intermediate obtained from the previous step (3-[8-(2-hydroxyethyl)-2-methyl-3,4-dihydro-2H-l-benzopyran-2-yl] propanoic acid) (1 equivalent) was dissolved in dry dichloromethane (DCM) in a round-bottom flask. Sulfur trioxide pyridine complex (2 equivalent) was added, and the mixture was stirred for 24 hours at room temperature. The solution was then cooled to 0°C on ice, and the precipitate was filtered out. The DCM solvent was removed using rotary evaporation to yield the final product, (yield: 97.6%). A full scan of high-resolution mass spectrometry analysis was performed under negative mode, with the most intense ion at m / z 343.0859 and the chemical formula C15H20O7S.XH NMR (600 MHz, DMSO) 6 8.54 (ddd, 2H), 8.53 (m, 1H), 7.98 (dd, 2H), 6.98 (dd, 2H), 6.77 (t, 1H), 4.16 - 4.11 (m, 2H), 2.93 - 2.68 (m, 4H), 2.51- 2.48 (m, 2H), 1.97 - 1.83 (m, 2H), 1.76 - 1.71 (m, 2H), 1.17 (s, 3H) confirmed that the synthesized SNAP matched the isolated snail metabolite.Cardiomyocyte-specific < / / taI-deficient (PdhalCarAtae'l'')mice generation: Cardiomyocyte-specific / W / ia / -deficient mice were generated as previously described58,59. C57BL / 6J wild-type 48WSLEGAL\055326\00505\42539390v6(WT), alpha-myosin heavy chain (aMHC)-MerCreMer (stock no. 005657), and Pdhalflox (stock no. 017443) mice were sourced from the Jackson Laboratory in the USA. To create Pdhalc,xA'^ mice, aMHC-MerCreMer transgenic mice that express tamoxifen-inducible Cre in cardiomyocytes were crossed with Pdhaln"'' mice to create Pdhalc,xA'^ mice. The inactivation of the Pdhal gene through Cre was achieved by administering 6 intraperitoneal injections of tamoxifen (50 mg / kg) over 8 days to male mice aged 6-7 weeks. All mice underwent a five-week washout period following the tamoxifen treatment before experimentation.Isolated hearts perfusion: Langendorff model: Mice were anesthetized by i.p. injection of 0.1 ml sodium pentobarbitone 20% w / v (Pentoject, Animal care Ltd., York, UK). Hearts were rapidly excised and arrested in ice-cold ”s perfusion buffer (118.5 mM NaCl, 25 mM NaHCCL, 4.7 mM KC1, 1.2 mM MgSO4, 1.2mM KH2PO4, 1.4 mM CaCl2, 5.5 mM Glucose). The aorta was cannulated and secured with sutures (Mersilk 3-0; Ethicon, Somerville, NJ, USA). Hearts were perfused with Krebs perfusion buffer at 3-5 ml / min at a constant perfusion pressure of 100cm H2O by gravity. Buffer was bubbled with 21% O2, 5% CO2 at 37°C for normoxic condition (PO2~130mmHg). For hypoxic conditions the buffer was equilibrated with 95% N2 and 5% CO2 (PO2~50mmHg). The pH for both solutions was 7.35. A Millar catheter (microtip, 1.4F, AD Instruments), attached to a pressure transducer, was inserted into the left ventricle. A pacemaker which would be used at the final perfusion stage was connected to the myocardium with a pacing rate at 300 / min. For protocol 1, hearts were isolated, mounted on a perfusion system, and adapted with Krebs perfusion buffer under normoxia for 10 minutes. They were then perfused with SNAP or vehicle (DMSO; same concentration of SNAP) under hypoxia for 30 minutes, followed by a complete flow occlusion. After 30 minutes, the heart was reperfused with normal Krebs perfusion buffer for 40 minutes, and pacing was set at 300 bpm by a pacemaker. For protocol 2, isolated hearts were perfused with Krebs perfusion buffer for 10 minutes in normoxia, followed by 30 minutes with SNAP or vehicle (DMSO; same concentration of SNAP) under normoxia. The flow was completely occluded for 30 minutes before SNAP reperfusion for 40 minutes. The hearts were paced at 300 bpm at the end by a pacemaker. Left ventricular pressure (LVP), rate of contraction (maximal dp / dt) and rate of relaxation (minimal dp / dt) were recorded using a PowerLab recorder and LabChart 8.0 software (ADInstruments Inc, Colorado49WSLEGAL\055326\00505\42539390v6Springs, Colo). Working heart model: Mice were anesthetized with sodium pentobarbital (O.lg / kg), and hearts were quickly excised from fully anesthetized mice. Following this, hearts were perfused in the working mode at an 11.5 mmHg left atrial preload and 50 mmHg aortic afterload, as described previously75. Hearts were perfused with modified Krebs-Hensel eit solution containing 118.5 mM NaCl, 25 mM NaHCCE, 4.7 mM KC1, 1.2 mM MgSC , 1.2 mM KH2PO4, 2.5 mM CaCh, 0.8 mM palmitate pre-bound to 3% albumin, and 5 mM glucose. To measure palmitate oxidation and glucose oxidation, the perfusate was radiolabeled with [U-14C] glucose and [5-3H] palmitate respectively. Glucose oxidation rates were assessed by measuring14CC>2 production. Palmitate oxidation rates were assessed by measuring3H2O production, as described previously. At the end of the aerobic perfusion protocol, hearts were immediately frozen in liquid N2 and stored at - 80 °C.Mouse fibroblast isolation and culture: Mouse lung tissue was rinsed with HBSS (Gibco, 14025-092) and cut into small pieces. These pieces were digested with 0.2 % type I collagenase (Worthington Biochemical Corporation, LS004196) at 37 °C for 30 minutes. Following digestion, the cell suspension was centrifuged and then resuspended in 0.25 % Trypsin-EDTA (Gibco, 25200-072) at 37 °C for 10 minutes before being centrifuged again and washed three times with lx PBS, after which it was resuspended in complete culture medium. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, 11995), augmented with 10 % FBS (Sigma-Aldrich, F1051) and 1% penicillin / streptomycin / amphotericin (PSF) (Gibco; 15240-062) under a 9% CO2 atmosphere (PO2: llOmmHg, pH 7.35). To create fuel-deprivation conditions, cells were allowed to grow to 70-80 % confluence, washed once with PBS, and then cultured in glucose-free DMEM (Gibco, 11966-025), supplemented with 1 % FBS, 1 % P / S, and 2.5 mM of D-(+)-glucose at 1 % O2 and 9 % CO2 (PO2: 50mmHg, pH 7.35).Cardiomyocyte isolation and culture: Adult cardiomyocytes were isolated from C57BL / 6 using type II collagenase digestion (Worthington Biochemical Corporation, LS004177) with a modified Langendorff perfusion apparatus (Harvard Apparatus; 73-4393), following our previously described method76. Briefly, post-digestion, the cardiomyocytes were pelleted at 20g for 3 minutes and then resuspended in a stopping buffer containing 2mM ATP. This was followed by pelleting and subsequent resuspension in stopping buffer with increasing 50WSLEGAL\055326\00505\42539390v6concentrations of CaCh at 100 pM, 400 pM, and 900 pM. Prior to plating, culture plates were coated with laminin (Sigma-Aldrich; 11243217001) for 2 hours at room temperature. The plating medium consisted of MEM with Hanks’ salts and 2 mM glutamine (Gibco, 11575032), supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich; F1051), 10 mM 2,3-butanedione monoxime (BDM) (Sigma-Aldrich; B0753), 2 mM ATP (Sigma-Aldrich; A6419), and 1% PSF (Gibco; 15240-062), and was maintained in an incubator at 37°C with 2% CO2. Cells were plated for 1 hour, after which the medium was replaced with fresh culture medium containing MEM with Hanks’ salts and 2 mM glutamine, 1% PSF, 0.1% bovine serum albumin (Sigma-Aldrich; A7906), 10 mM BDM, 1% insulin transferrin selenium (ITS) (Sigma-Aldrich; 11884) and 10 mM of D-(+)-glucose at 2% CO2 (PO2: llOmmHg). To create fuel-deprivation conditions, the medium was replaced with fresh culture medium containing MEM with Hanks’ salts and 2 mM glutamine, 1% PSF, 0.1% bovine serum albumin, 10 mM BDM, 1% insulin ITS and 5 mM of D-(+)-glucose 1 % O2 and 2 % CO2 (PO2: 50mmHg).SNAP001 treatment: For the in vitro study, mouse fibroblasts were seeded and cultured in DMEM containing 10% FBS and 1% P / S. At 24 h after the media change, SNAP was added to the cell culture in the final dose of 10 pM in glucose-free DMEM, supplemented with 1% FBS, 1% P / S and 2.5 mM of D-(+)-glucose at 1% O2 and 9 % CO2 (PO2: 50 mmHg). The same concentration of SNAP was used in the in vitro study of cardiomyocytes.For the ex vivo study, SNAP was added to Krebs perfusing buffer containing 120 mM NaCl, 25 mM NaHCO3, 10 mM Dextrose, 1.75 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.7 mM KC1, 2 mM Sodium Pyruvate (pH 7.4) in the final dose of 10 pM and bubbled with 95 % O2 and 5 % CO2 at 37°C for normoxic conditions (PO2: HO mmHg) or equilibrated with 95% N2 and 5% CO2 at 37°C to induce hypoxic condition (PO2: 50 mmHg).Pure mitochondria and cytosol isolation: Mitochondria and cytosol were isolated from cardiomyocytes using the Mitochondria Isolation Kit (ThermoFisher Scientific, 89874). According to the kit instructions, 800 pl of Reagent A was added to the cells and incubated on ice for 2 minutes. Following this, lOpl of Reagent B was introduced, and the cells were collected and transferred to a Dounce homogenizer for homogenization for 5 minutes. Subsequently, all cells along with the reagents were collected and mixed with 800 pl of Reagent C before 51WSLEGAL\055326\00505\42539390v6centrifugation at 700*g for 10 minutes at 4°C. The supernatant was collected and centrifuged again at 12000 *g for 15 minutes at 4°C to obtain the cytosolic fraction, while the pellet (mitochondrial fraction) was washed with 500 pl of Reagent C and centrifuged at 12000 / g for an additional 15 minutes at 4 °C for purification.Cell Fractionation: Cytosolic, mitochondrial, and nuclear fractions were isolated using the Cell Fractionation Kit (Abeam, ab 109719) following the manufacturer’s protocol with minor modifications. Cells grown on 100-mm dishes per condition were harvested with scrapers and pelleted at 300 * g for 5 min. Pellets were washed once in lx Buffer A and resuspended. An equal volume of Buffer B (Detergent I diluted 1:1000 in Buffer A) was added, mixed by pipetting, and rotated for 7 min at room temperature. Samples were centrifuged at 5000 / g for i min at 4 °C, and the supernatants were clarified at 10000 x g for 1 min to obtain the cytosolic fraction. Pellets were resuspended to the original volume in Buffer A and extracted with an equal volume of Buffer C (Detergent II diluted 1:25 in Buffer A) for 10 min on a rotator, followed by centrifugation at 5000 x g for 1 min and 10000 x g for 1 min to yield the mitochondrial fraction. The remaining pellets were resuspended in Buffer A and collected as nuclear fractions. And nuclei were lysed by using lx RIPA buffer. All buffers were supplemented with protease and phosphatase inhibitors.Assessment of cardiomyocyte contractility: Cell length shortening was evaluated using a video-based edge-detection system (lonOptix, Milton, MA, USA) to assess cell contractility. Ventricular myocytes were placed on a 25 mm round cover glass coated with laminin. After treatment with SNAP and vehicle (DMSO; same concentration of SNAP) in hypoxia for 3 hours, the cover glass was glued into an experimental chamber containing a pair of electrodes, which were perfused with modified Krebs buffer (140 mM NaCl, 5 mM KC1, 10 mM HEPES buffer, 2 mM CaCh, 1.4 mM MgCh, and 10 mM glucose) bubbled with 21 % O2, 5% CO2 at 37 °C for normoxic condition at a rate of 1 ml / min. The cells were stimulated with 100 volts at a frequency of 2 Hz (1 msec duration) using a field stimulator, capturing the real-time trace of cell length during contraction. The percentage of cell length shortening was determined as (diastolic cell length-systolic cell length) / diastolic cell length) x 100 %.52WSLEGAL\055326\00505\42539390v6Small Interfering RNA (siRNA) transfection: Cells were grown in 35 mm dishes to 50-60 % confluence and then transfected in antibiotic-free medium with 30 pmol siRNA diluted in Opti-MEM reduced serum medium (ThermoFisher Scientific, 31985070) and mixed with LipofectamineTM RNAiMAX reagent (ThermoFisher Scientific; 13778100). The mixture was incubated for 10 min at room temperature and then added to the cells to incubate for 7 hours, changed to fresh culture media, which were used in the next experiment. siRNA used was phlppl (ThermoFisher Scientific, s23365)Plasmid transfection: Cells were grown in 35 mm dishes to 50-60 % confluence and then transfected in antibiotic-free medium with 20 pmol DNA diluted in Opti-MEM reduced serum medium (ThermoFisher Scientific, 31985070) and mixed with Lipofectamine 3000 reagent (ThermoFisher Scientific, L3000150). The mixture was incubated for 10 min at room temperature, then added to the cells to incubate overnight, changed to fresh culture media, and collected after 72 hours. Wildtype and mutagenic human phlppl (GenScript) were designed and used for phlppl overexpression and mutagenesis experiments.Confocal microscopy: All images were obtained on a ZEISS LSM 710 confocal microscope (Carl ZEISS AG, Oberkochen, Germany), equipped with a GaAsp detector and the Airy scan module, allowing us to obtain super-resolution images with a lateral resolution of 140 nm. Images were acquired with a 403 Oil objective at optimal pixel size and interval (for z-stacks) based on the zoom factor and the fluorophores used in each experiment. After the acquisition, the images were processed with the ZEISS ZEN Blue software.Live imaging: Snail tissue was cut into approximately 20 mm sections using a blade and placed in confocal dishes. TMRM (ThermoFisher Scientific, T668) was added to fresh media without FBS, following the datasheet instructions, for a duration of 30 minutes at 9% CO2. Prior to live imaging, the tissue was washed twice with fresh medium.Cultured cells were maintained in confocal dishes and treated as specified for each experiment. MitoTracker (ThermoFisher Scientific, M22426), LysoTracker Yellow (ThermoFisher Scientific, L12491), TMRM, MitoSox Green (ThermoFisher Scientific, M36006), MitoSox Red (ThermoFisher Scientific, M36008), and Hoechst (ThermoFisher Scientific, 33342) were 53WSLEGAL\055326\00505\42539390v6incorporated into fresh media lacking FBS at concentrations detailed in the datasheet for 30 minutes at 9 % CO2. Afterward, the cells underwent two washes with the fresh medium before live imaging.Heart tissue was sliced around 20 mm by the blade and put into confocal dishes and TMRM and / or MitoSox were added to the fresh media without FBS in a concentration according to the datasheet for 30 min at 9% CO2. Tissue was washed with fresh medium 2 times before live imaging.O-propargyl-puromycin (OPP) staining: Cells are plated in glass bottom confocal dishes until they reach 70 % confluency. On the experiment day, they are incubated in fuel-deprived DMEM containing DMSO or SNAP for 5 hours within 1% hypoxia containers. Afterward, they are treated with OPP reagent A from the OPP staining kit (Thermo Fisher Scientific, C10458) at a dilution of 1:1000 for another 30 minutes, followed by fixation with 2 % PFA at 37°C for 10 minutes. The cells are rinsed with PBS and then permeabilized using 0.25% Triton-X at 37°C for 10 minutes, with three more rinses in PBS. Finally, ClickIT reaction and nucleus staining were performed according to kit instructions.PHLPP1 activity assay: The catalytic activity of PHLPP1 was assessed using a modified Malachite Green assay method, as previously shown49. In brief, a serine-phosphorylated peptide (HFPQFpSYSAS), representing the sequence involved in Akt reaction, was used as the substrate for PHLPP -mediated dephosphorylation. The enzymatic reaction was carried out at 37°C for 2 hours in a reaction mixture containing 0.02 mg / ml BSA, 100 mM NaCl, 0.4 mM Mn2+, and 100 mM tricine (pH 7.4). For the reaction, 1.5 pM mouse fibroblast lysates, or 1.5 pM immunoprecipitated PHLPP 1 from stressed mouse fibroblast lysates, or 1.5 pM human recombinant PHLPP1 (OriGene Technologies, TP303930) were utilized, along with 300 pM of the substrate. Phosphate resulting from the PHLPP 1 enzymatic activity was quantified using a commercial Malachite Green assay kit (Cell Signaling Technology, 12776).Immunoblots: Cultured cells were collected and lysed in ice-cold RIPA buffer (ThermoFisher Scientific, 89900) supplemented with protease inhibitor cocktail (Sigma- Aldrich, P2714), sodium orthovanadate (Sigma-Aldrich, 13721-39-6), sodium fluoride (New England Biolabs,54WSLEGAL\055326\00505\42539390v6P0759S) and PMSF (Sigma-Aldrich, 93482) for 30 min with vortexing every 10 min. Samples were then spun down at 12,000 rpm for 20 min in a tabletop centrifuge (Eppendorf AG, Hamburg, Germany). After centrifugation, the supernatant was collected. Protein concentration was quantified with a BCA kit (ThermoFisher Scientific, 23227) and measured on a SpectraMax iD3 plate reader (Molecular Devices, San Jose, CA, US). Samples were then diluted to a final concentration of 0.5 mg / mL in RIPA buffer and 2 Laemmli Sample Buffer (Sigma-Aldrich, S3401). Finally, they were boiled at 96 °C for 5 min. All samples were loaded on homemade SDS-PAGE gels. Proteins were then transferred onto 0.45 pm pore nitrocellulose membranes using a Trans-blot Turbo transfer system (Bio-Rad) according to the manufacturer’s instructions. After transfer, membranes were incubated with Ponceau S (Sigma- Aldrich, P7170) to verify the efficient transfer of the proteins. Blocking of the membrane was performed with 5% skim milk or bovine serum albumin for 1 h. After blocking, the membranes were incubated with the primary antibodies in 5% non-fat dry milk (or BSA) in TBST overnight at 4°C with gentle rotation. The following day, membranes were washed with IxTBST and incubated with the appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, US). Proteins were detected after incubation of the membranes with ECL buffer (Cytiva Amersham, 45000875) or Clarity Max Western ECL Substrate (Biorad, 1705062) and visualized on a ChemiDoc imaging system (Bio-Rad). The expression level was quantified using the Image J program.Heart tissue was transferred to a round-bottomed jar and snap-frozen by immersing it in liquid nitrogen. For 5 mg of tissue, 400 pl of ice-cold RIPA buffer was added for 30 min, with vortexing every 10 min. The other steps were the same as those for cell lysate preparation above.Immunoprecipitation: Dynabeads™ Protein A for Immunoprecipitation (Invitrogen 10008D) and Pierce™ IP Lysis Buffer (ThermoFisher Scientific 87788) were used as per the manufacturer’s recommendations. Cardiomyocytes were lysed with IP buffer. 1 mg of protein was added to Ipg of PHLPP or IgG isotype control (Cell Signaling 5415) antibody incubated rotating overnight at 4°C. Protein conjugated to antibodies were added to Protein A beads and incubated at 4°C for 2 hours. For the PHLPP activity assay, beads were collected and washed 3 times with assay buffer (see PHLPP activity assay part) and the protein concentration was 55WSLEGAL\055326\00505\42539390v6quantified with a BCA kit (see Immunoblot part). For immunoblots, beads were washed and eluted with an equal volume of Pierce™ IP Lysis Buffer and 2* Laemmli Sample buffer (Sigma Aldrich, S3410) and boiled for 10 minutes at 55°C.Co-immunoprecipitation (Co-IP) was performed using the Pierce™ Crosslink Magnetic IP / Co-IP Kit (Thermo Fisher Scientific, 88805) according to the manufacturer’s instructions. Briefly, 25 pl of Pierce Protein A / G Magnetic Beads were washed three times with 1 x Coupling Buffer and incubated with 20 pg of NHERF1 antibody or control IgG at room temperature for 15 minutes. After washing 3 times with lx Coupling Buffer, 50 pl of Binding Solution was added, and the mixture was incubated for 30 minutes at room temperature to crosslink the antibody and beads. The antibody-crosslinked beads were washed 3 times with 100 pl of Elution Buffer, followed by incubation with 3 mg of total cardiomyocyte lysates overnight at 4°C with rotation. Beads were then washed twice with cold IP Lysis / Wash Buffer and once with purified water. To elute the bound proteins, 100 pl of Elution Buffer was added, followed by a 5-minute incubation at room temperature on a rotator. The supernatant containing the target antigens was collected after magnetic separation, neutralized with 20 pl of Neutralization Buffer, and subjected to immunoblotting as described above.Seahorse analyzer: Oxygen consumption (OCR) rate was assessed using a Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, US) according to the manufacturer’s instructions. Briefly, mouse fibroblasts and ventricular myocytes were seeded and cultured overnight in Seahorse XF-24 plates (Agilent Technologies, 102342100) at a density of 6 x 104cells for fibroblasts and 4,000 cells for ventricular myocytes per well respectively for overnight. After treatment, the culture medium of fibroblasts was removed and replaced with bicarbonate-free Seahorse XF Base medium without phenol red (Agilent Technologies, 103575-100) supplemented with 2 mM L-glutamine (Sigma-Aldrich, 607983) and either 25 mM (baseline) or 2.5 mM (low) D-(+)-glucose (Sigma-Aldrich, G5767). The culture media of cardiomyocytes were changed to bicarbonate-free Seahorse XF Base medium without phenol red (Agilent Technologies, 10375-100) supplemented with 2 mM L-glutamine and 5.5 mM D-(+)-glucose. Following incubation of the cells in the CCh-free incubator for 1 h, cells were placed in the Seahorse Analyzer. OCR was measured using 3 -minute mix and 3 -minute measure cycles.56WSLEGAL\055326\00505\42539390v6After three baseline cycles, sequential injections were performed: 1 pM Oligomycin, 1 pM FCCP. After the run, cells were washed with PBS and lysed in 100 ml per well of RIP A buffer (supplemented with protease inhibitors) per well for 30 min at 4°C with constant agitation. Protein concentration was measured with a BCA assay (see Immunoblots part) and used to normalize the OCR value. Spare respiratory capacity (SRC) was defined as the difference between maximum OCR (triggered by FCCP) and basal OCR (triggered by oligomycin). At least 4 replicate wells were used per group.PDH activity assay: PDH activity (mOD / min / mg) was assessed using commercial kits from Abeam (ab 109882). Following the provided protocol, 25 pl of blocking solution was placed into an empty microplate well, followed by the addition of 25 pg of tissue lysates, which were thoroughly mixed using a pipette. A dipstick was gently inserted into the microplate, allowing the sample to absorb into it for 20 minutes. Next, the well was washed with 40 pl of sample buffer for 10 minutes, and 300 pl of activity buffer was added to a separate empty well for each dipstick. The dipstick was then transferred into the activity buffer to react for another 20 minutes. Once signal development was complete, the dipstick was rinsed with deionized water for 5 minutes. Finally, the dipstick was visualized using a ChemiDoc imaging system from BioRad, and the resulting signal was quantified with Image J.Quantification and statistical analysis: All statistical analyses were performed on GraphPad Prism 9 (GraphPad Software, CA, US). Values are expressed as mean ±SEM or SD as shown. Probability values of less than 0.05 were considered statistically significant. An unpaired, two-tailed Student t-test and ANOVA with Tukey’s multiple comparisons post hoc test were used for all data. Statistical significance: *p < 0.05,< 0.01, *** / > < 0.001.

[0112] Definitions and Interpretation: The description of the present invention has been presented for purposes of illustration and description, but it is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Embodiments were chosen and described in order to best explain the principles of the 57WSLEGAL\055326\00505\42539390v6invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention.

[0113] The corresponding structures, materials, acts, and equivalents of all means or steps plus function elements in the claims appended to this specification are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed.

[0114] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to combine, affect or connect such aspect, feature, structure, or characteristic with other embodiments, whether or not such connection or combination is explicitly described. In other words, any element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility between the two, or it is specifically excluded.

[0115] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation. The terms “preferably,” “preferred,” “prefer,” “optionally,” “may,” and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention.58WSLEGAL\055326\00505\42539390v6

[0116] The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0117] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.

[0118] As will also be understood by one skilled in the art, all ranges described herein, and all language such as "between", "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number(s) recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above.

[0119] The following references, referred to in this description, are indicative of the level of ordinary skill in the art, and are incorporated herein by reference, in their entirety.1. Carey, H.V., Andrews, M.T., and Martin, S.L. (2003). Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol Rev 83, 1153-1181.10.1152 / physrev.00008.2003.2. Jastroch, M., Giroud, S., Barrett, P., Geiser, F., Heldmaier, G., and Herwig, A. (2016). Seasonal Control of Mammalian Energy Balance: Recent Advances in the Understanding of Daily Torpor and Hibernation. J Neuroendocrinol 28. 10.1111 / jne.12437.3. Staples, J.F. (2014). Metabolic suppression in mammalian hibernation: the role of mitochondria. J Exp Biol 217, 2032-2036. 10.1242 / jeb.092973.4. Staples, J.F., Mathers, K.E., and Duffy, B.M. (2022). Mitochondrial Metabolism in Hibernation: Regulation and Implications. Physiology (Bethesda) 37, 0. 10.1152 / physiol.00006.2022.5. Storey, K.B. (1997). Metabolic regulation in mammalian hibernation: enzyme and protein adaptations. Comp Biochem Physiol A Physiol 118, 1115-1124.59WSLEGAL\055326\00505\42539390v66. Storey, K.B. (2003). Mammalian hibernation. Transcriptional and translational controls. Adv Exp Med Biol 543, 21-38.7. Storey, K.B. (2010). Out cold: biochemical regulation of mammalian hibernation - a mini -review. Gerontology 56, 220-230. 10.1159 / 000228829.8. Wu, C.W., Biggar, K.K., and Storey, K.B. (2013). Biochemical adaptations of mammalian hibernation: exploring squirrels as a perspective model for naturally induced reversible insulin resistance. Braz J Med Biol Res 46, 1-13. 10.1590 / 1414-431x20122388.9. Storey, K.B., and Storey, J.M. (2010). Metabolic rate depression: the biochemistry of mammalian hibernation. Adv Clin Chem 52, 77-108.10. Wu, C.W., and Storey, K.B. (2021). mTOR Signaling in Metabolic Stress Adaptation. Biomolecules 11. 10.3390 / bioml 1050681.11. Panwar, V., Singh, A., Bhatt, M., Tonk, R.K., Azizov, S., Raza, A.S., Sengupta, S., Kumar, D., and Garg, M. (2023). Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease. Signal Transduct Target Ther 8, 375. 10.1038 / s41392-023-01608-z.12. Liu, G.Y., and Sabatini, D.M. (2020). mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol 21, 183-203. 10.1038 / s41580-019-0199-y.13. Urban, N., and Cheung, T.H. (2021). Stem cell quiescence: the challenging path to activation.Development 148. 10.1242 / dev.165084.14. van Velthoven, C.T.J., and Rando, T.A. (2019). Stem Cell Quiescence: Dynamism, Restraint, and Cellular Idling. Cell Stem Cell 24, 213-225. 10.1016 / j.stem.2019.01.001.15. Dias, I.B., Bouma, H.R., and Henning, R.H. (2021). Unraveling the Big Sleep: Molecular Aspects of Stem Cell Dormancy and Hibernation. Front Physiol 12, 624950. 10.3389 / fphys.2021.624950.16. Peyrusson, F., Nguyen, T.K., Najdovski, T., and Van Bambeke, F. (2022). Host Cell Oxidative Stress Induces Dormant Staphylococcus aureus Persisters. Microbiol Spectr 10, e0231321.10.1128 / spectrum.02313-21.17. Niu, H., Gu, J., and Zhang, Y. (2024). Bacterial persisters: molecular mechanisms and therapeutic development. Signal Transduct Target Ther 9, 174. 10.1038 / s41392-024-01866-5.18. Drake, J.C., Wilson, R.J., Laker, R.C., Guan, Y ., Spaulding, H.R., Nichenko, A.S., Shen, W., Shang, H., Dorn, M.V., Huang, K., et al. (2021). Mitochondria-localized AMPK responds to local energetics and contributes to exercise and energetic stress-induced mitophagy. Proc Natl Acad Sci U S A 118.10.1073 / pnas.2025932118.19. Herzig, S., and Shaw, R.J. (2018). AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol 19, 121-135. 10.1038 / nrm.2017.95.20. Mihaylova, M.M., and Shaw, R.J. (2011). The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol 13, 1016-1023. 10.1038 / ncb2329.21. Steinberg, G.R., and Hardie, D.G. (2023). New insights into activation and function of the AMPK. Nat Rev Mol Cell Biol 24, 255-272. 10.1038 / s41580-022-00547-x.60WSLEGAL\055326\00505\42539390v622. Li, W., Petrimpol, M., Molle, K.D., Hall, M.N., Battegay, E.J., and Humar, R. (2007). Hypoxia-induced endothelial proliferation requires both mTORCl and mT0RC2. Circ Res 100, 79-87.10.1161 / 01.RES.0000253094.03023.3f.23. Kugo, H., Sukketsiri, W., Iwamoto, K., Suihara, S., Moriyama, T., and Zaima, N. (2021). Low glucose and serum levels cause an increased inflammatory factor in 3T3-L1 cell through Akt, MAPKs andNF-small ka, CyrillicB activation. Adipocyte 10, 232-241. 10.1080 / 21623945.2021.1914420.24. O'Neill, B.T., and Abel, E.D. (2005). Aktl in the cardiovascular system: friend or foe? J Clin Invest 115, 2059-2064. 10.1172 / JCI25900.25. Dossumbekova, A., Berdyshev, E.V., Gorshkova, I., Shao, Z., Li, C., Long, P., Joshi, A., Natarajan, V., and Vanden Hoek, T.L. (2008). Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium. Am J Physiol Heart Circ Physiol 295, H2417-2426.10.1152 / ajpheart.00501.2008.26. Zhang, J., Wang, X., Vikash, V., Ye, Q., Wu, D., Liu, Y., and Dong, W. (2016). ROS and ROS-Mediated Cellular Signaling. Oxid Med Cell Longev 2016, 4350965. 10.1155 / 2016 / 4350965.27. Ghafouri-Fard, S., Khanbabapour Sasi, A., Hussen, B.M., Shoorei, H., Siddiq, A., Taheri, M., and Ayatollahi, S.A. (2022). Interplay between PI3K / AKT pathway and heart disorders. Mol Biol Rep 49, 9767-9781. 10.1007 / sl 1033-022-07468-0.28. Li, Y ., Yang, L., Dong, L., Yang, Z.W., Zhang, J., Zhang, S.L., Niu, M.J., Xia, J.W., Gong, Y ., Zhu, N., et al. (2019). Crosstalk between the Akt / mTORCl and NF-kappaB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs. Acta Pharmacol Sin 40, 1322-1333. 10.1038 / s41401-019-0272-2.29. Miniaci, M.C., Dattolo, M.G., Irace, C., Capuozzo, A., Santamaria, R., and Scotto, P. (2015). Glucose deprivation promotes activation of mTOR signaling pathway and protein synthesis in rat skeletal muscle cells. Pflugers Arch 467, 1357-1366. 10.1007 / s00424-014-1583-2.30. Horman, S., Hussain, N., Dilworth, S.M., Storey, K.B., and Rider, M.H. (2005). Evaluation of the role of AMP -activated protein kinase and its downstream targets in mammalian hibernation. Comp Biochem Physiol B Biochem Mol Biol 142, 374-382. 10.1016 / j.cbpb.2005.08.010.31. Logan, S.M., and Storey, K.B. (2017). Avoiding apoptosis during mammalian hibernation.Temperature (Austin) 4, 15-17. 10.1080 / 23328940.2016.1211071.32. Morin, P., Jr., and Storey, K.B. (2009). Mammalian hibernation: differential gene expression and novel application of epigenetic controls. Int J Dev Biol 53, 433-442. 10.1387 / ijdb.082643pm.33. Rouble, A.N., Hefler, J., Mamady, H., Storey, K.B., and Tessier, S.N. (2013). Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation. PeerJ 1, e29. 10.7717 / peerj.29. 34. Rouble, A.N., and Storey, K.B. (2015). Characterization of the SIRT family of NAD+-dependent protein deacetylases in the context of a mammalian model of hibernation, the thirteen-lined ground squirrel. Cryobiology 71, 334-343. 10.1016 / j.cryobiol.2015.08.009.35. Dawe, A.R., and Morrison, P.R. (1955). Characteristics of the hibernating heart. Am Heart J 49, 367-384. 10.1016 / 0002-8703(55)90031-4.61WSLEGAL\055326\00505\42539390v636. McCue, M.D. (2010). Starvation physiology: reviewing the different strategies animals use to survive a common challenge. Comp Biochem Physiol A Mol Integr Physiol 156, 1-18.10.1016 / j.cbpa.2010.01.002.37. Malan, A. (2014). The evolution of mammalian hibernation: lessons from comparative acid-base physiology. Integr Comp Biol 54, 484-496. 10.1093 / icb / icu002.38. Nelson, O.L., McEwen, M.M., Robbins, C.T., Felicetti, L., and Christensen, W.F. (2003). Evaluation of cardiac function in active and hibernating grizzly bears. J Am Vet Med Assoc 223, 1170-1175.10.2460 / javma.2003.223.1170.39. Buchko, M.T., Boroumand, N., Cheng, J.C., Hirj i, A., Halloran, K., Freed, D.H., and Nagendran, J. (2020). Clinical transplantation using negative pressure ventilation ex situ lung perfusion with extended criteria donor lungs. Nat Commun 11, 5765. 10.1038 / s41467-020- 19581-4.40. Klein, A.S., Messersmith, E.E., Ratner, L.E., Kochik, R., Baliga, P.K., and Ojo, A.O. (2010). Organ donation and utilization in the United States, 1999-2008. Am J Transplant 10, 973-986. 10.1111 / j .1600-6143.2009.03008.x.41. Wang, L., Li, J., He, S., Liu, Y., Chen, H., He, S., Yin, M., Zou, D., Chen, S., Luo, T., et al. (2021). Resolving the graft ischemia-reperfusion injury during liver transplantation at the single cell resolution. Cell Death Dis 12, 589. 10.1038 / s41419-021-03878-3.42. Dery, K.J., Yao, S., Cheng, B., and Kupiec-Weglinski, J.W. (2023). New therapeutic concepts against ischemia-reperfusion injury in organ transplantation. Expert Rev Clin Immunol 19, 1205-1224.10.1080 / 1744666X.2023.2240516.43. Fernandez, A.R., Sanchez-Tarjuelo, R., Cravedi, P., Ochando, J., and Lopez -Hoyos, M. (2020). Review: Ischemia Reperfusion Injury-A Translational Perspective in Organ Transplantation. Int J Mol Sci 21. 10.3390 / ijms21228549.44. Hausenloy, D.J., and Yellon, D.M. (2013). Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J Clin Invest 123, 92-100. 10.1172 / JCI62874.45. Bose, U., Centurion, E., Hodson, M.P., Shaw, P.N., Storey, K.B., and Cummins, S.F. (2016). Global metabolite analysis of the land snail Theba pisana hemolymph during active and aestivated states. Comp Biochem Physiol Part D Genomics Proteomics 19, 25-33. 10.1016 / j.cbd.2016.05.004.46. Baffi, T.R., Cohen-Katsenelson, K., and Newton, A.C. (2021). PHLPPing the Script: Emerging Roles of PHLPP Phosphatases in Cell Signaling. Annu Rev Pharmacol Toxicol 61, 723-743. 10.1146 / annurev-pharmtox-031820-122108.47. Masubuchi, S., Gao, T., O'Neill, A., Eckel-Mahan, K., Newton, A.C., and Sassone-Corsi, P. (2010). Protein phosphatase PHLPP 1 controls the light-induced resetting of the circadian clock. Proc Natl Acad Sci U S A 107, 1642-1647. 10.1073 / pnas.0910292107.48. Dempsey, Z.W., Goater, C.P., and Burg, T.M. (2020). Living on the edge: comparative phylogeography and phylogenetics of Oreohelix land snails at their range edge in Western Canada. BMC Evol Biol 20, 3. 10.1186 / sl2862-019-1566-l .49. Sierecki, E., and Newton, A.C. (2014). Biochemical characterization of the phosphatase domain of the tumor suppressor PH domain leucine-rich repeat protein phosphatase. Biochemistry 53, 3971-3981.10.102 l / bi500428j .62WSLEGAL\055326\00505\42539390v650. Mareseal, O., and Cheeseman, I.M. (2020). Cellular Mechanisms and Regulation of Quiescence. Dev Cell 55, 259-271. 10.1016 / j.devcel.2020.09.029.51. Marcheti, P., Fovez, Q., Germain, N., Khamari, R., and Kluza, J. (2020). Mitochondrial spare respiratory capacity: Mechanisms, regulation, and significance in non-transformed and cancer cells. FASEB 34, 13106-13124. 10.1096 / g.202000767R.52. Sun, Y., Wang, H., Qu, T., Luo, J., An, P., Ren, F., Luo, Y., and Li, Y. (2023). mT0RC2: a multifaceted regulator of autophagy. Cell Commun Signal 21, 4. 10.1186 / sl2964-022-00859-7.53. Chae, Y.C., Vaira, V., Caino, M.C., Tang, H.Y., Seo, J.H., Kossenkov, A.V., Otobrini, L., Martelli, C., Lucignani, G., Bertolini, I., et al. (2016). Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming. Cancer Cell 30, 257-272. 10.1016 / j.ccell.2016.07.004.54. Lemoine, K.A., Fassas, J.M., Ohannesian, S.H., and Purcell, N.H. (2021). On the PHLPPside: Emerging roles of PHLPP phosphatases in the heart. Cell Signal 86, 110097.10.1016 / j. cellsig.2021.110097.55. Arias, E., Koga, H., Diaz, A., Mocholi, E., Patel, B., and Cuervo, A.M. (2015). Lysosomal mTORC2 / PHLPPl / Akt Regulate Chaperone-Mediated Autophagy. Mol Cell 59, 270-284.10.1016 / j.molcel.2015.05.030.56. Li, J., Zhu, X., Oberdier, M.T., Lee, C., Lin, S., Fink, S.J., Justice, C.N., Qin, K., Begeman, A.W., Damen, F.C., et al. (2023). A cell-penetrating PHLPP peptide improves cardiac arrest survival in murine and swine models. J Clin Invest 133. 10.1172 / JCI164283.57. Ohwada, W., Tanno, M., Yano, T., Ong, S.B., Abe, K., Sato, T., Kuno, A., Miki, T., Sugawara, H., Igaki, Y., and Miura, T. (2020). Distinct intra-mitochondrial localizations of pro-survival kinases and regulation of their functions by DUSP5 and PHLPP-1. Biochim Biophys Acta Mol Basis Dis 1866, 165851. 10.1016 / j .bbadis.2020.165851.58. Gopal, K., Al Batran, R., Altamimi, T.R., Greenwell, A.A., Saed, C.T., Tabatabaei Dakhili, S.A., Dimaano, M.T.E., Zhang, Y ., Eaton, F., Sutendra, G., and Ussher, J.R. (2021). FoxOl inhibition alleviates type 2 diabetes-related diastolic dysfunction by increasing myocardial pyruvate dehydrogenase activity. Cell Rep 35, 108935. 10.1016 / j.celrep.2021.108935.59. Gopal, K., Almutairi, M., Al Batran, R., Eaton, F., Gandhi, M., and Ussher, J.R. (2018). Cardiac-Specific Deletion of Pyruvate Dehydrogenase Impairs Glucose Oxidation Rates and Induces Diastolic Dysfunction. Front Cardiovasc Med 5, 17. 10.3389 / fcvm.2018.00017.60. Hue, L., and Taegtneyer, H. (2009). The Randle cycle revisited: a new head for an old hat. Am J Physiol Endocrinol Metab 297, E578-591. 10.1152 / ajpendo.00093.2009.61. McVeigh, J.J., and Lopaschuk, G.D. (1990). Dichloroacetate stimulation of glucose oxidation improves recovery of ischemic rat hearts. Am J Physiol 259, H1079-1085.10.1152 / ajpheart.1990.259.4.H1079.62. Taniguchi, M., Wilson, C., Hunter, C.A., Pehowich, D.J., Clanachan, A.S., and Lopaschuk, G.D. (2001). Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak. Am J Physiol Heart Circ Physiol 280, H1762-1769.10.1152 / ajpheart.2001.280.4.H1762.63WSLEGAL\055326\00505\42539390v663. Wambolt, R.B., Lopaschuk, G.D., Brownsey, R.W., and Allard, M.F. (2000). Dichloroacetate improves postischemic function of hypertrophied rat hearts. J Am Coll Cardiol 36, 1378-1385.10.1016 / s0735-1097(00)00856-l.64. Abe, J., Vujic, A., Prag, H.A., Murphy, M.P., and Krieg, T. (2024). Malonate given at reperfusion prevents post-myocardial infarction heart failure by decreasing ischemia / reperfusion injury. Basic Res Cardiol 119, 691-697. 10.1007 / s00395-024-01063-z.65. Siraj, M.A., Mundil, D., Beca, S., Momen, A., Shikatani, E.A., Afiroze, T., Sun, X., Liu, Y., Ghaffari, S., Lee, W., et al. (2020). Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-alpha. J Clin Invest 130, 1392-1404. 10.1172 / JCI99934.66. Gao, T., Furnari, F., and Newton, A.C. (2005). PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth. Mol Cell 18, 13-24.10.1016 / j .molcel .2005.03.008.67. Kovacic, S., Soltys, C.L., Barr, A.J., Shiojima, I., Walsh, K., and Dyck, J.R. (2003). Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278, 39422-39427. 10.1074 / jbc.M305371200.68. Chan, A.Y., Soltys, C.L., Young, M.E., Proud, C.G., and Dyck, J.R. (2004). Activation of AMP-activated protein kinase inhibits protein synthesis associated with hypertrophy in the cardiac myocyte. J Biol Chem 279, 32771-32779. 10.1074 / jbc.M403528200.69. Hua, Y., Zhang, Y., Ceylan-Isik, A.F., Wold, L.E., Nunn, J.M., and Ren, J. (2011). Chronic Akt activation accentuates aging-induced cardiac hypertrophy and myocardial contractile dysfunction: role of autophagy. Basic Res Cardiol 106, 1173-1191. 10.1007 / s00395-011-0222-8.70. Marino, A., Hausenloy, D.J., Andreadou, I., Horman, S., Bertrand, L., and Beauloye, C. (2021). AMP-activated protein kinase: A remarkable contributor to preserve a healthy heart against ROS injury. Free Radic Biol Med 166, 238-254. 10.1016 / j.freeradbiomed.2021.02.047.71. Shi, S.Y., Lu, S.Y., Sivasubramaniyam, T., Revelo, X.S., Cai, E.P., Luk, C.T., Schroer, S.A., Patel, P., Kim, R.H., Bombardier, E., et al. (2015). DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice. Nat Commun 6, 7415. 10.1038 / ncomms8415.72. Pinho, G.M., Martin, J.G.A., Farrell, C., Haghani, A., Zoller, J.A., Zhang, J., Snir, S., Pellegrini, M., Wayne, R.K., Blumstein, D.T., and Horvath, S. (2022). Hibernation slows epigenetic ageing in yellow-bellied marmots. Nat Ecol Evol 6, 418-426. 10.1038 / s41559-022-01679-1.73. Sullivan, I.R., Adams, D.M., Greville, L.J.S., Fame, P.A., and Wilkinson, G.S. (2022). Big brown bats experience slower epigenetic ageing during hibernation. Proc Biol Sci 289, 20220635.10.1098 / rspb.2022.0635.74. Keiser, M.J., Roth, B.L., Armbruster, B.N., Emsberger, P., Irwin, J.J., and Shoichet, B.K. (2007). Relating protein pharmacology by ligand chemistry. Nat Biotechnol 25, 197-206. 10.1038 / nbtl284. 75. Ho, K.L., Zhang, L., Wagg, C., Al Batran, R., Gopal, K., Levasseur, J., Leone, T., Dyck, J.R.B., Ussher, J.R., Muoio, D.M., et al. (2019). Increased ketone body oxidation provides additional energy for the failing heart without improving cardiac efficiency. Cardiovasc Res 115, 1606-1616.10.1093 / cvr / cvz045.64WSLEGAL\055326\00505\42539390v676. Saleme, B., Gurtu, V., Zhang, Y., Kinnaird, A., Boukouris, A.E., Gopal, K., Ussher, J.R., and Sutendra, G. (2019). Tissue-specific regulation of p53 by PKM2 is redox dependent and provides a therapeutic target for anthracy cline -induced cardiotoxicity. Sci Transl Med 11.10.1126 / scitranslmed.aau8866.65WSLEGAL\055326\00505\42539390v6

Claims

CLAIMS1. A compound of Formula (I):RfH;(I)wherein:R1 is cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; branched or unbranched acyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; R2 is hydrogen, hydroxy, methoxy, halogen, or CX-O-S(=O)2-OH, where x is zero or 1, 2, 3, 4 or 5;R3, R4 and R5 are each, independently of each occurrence, hydrogen; hydroxy, methoxy, or halogen; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl;or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1 wherein R1 is ethanoic, propanoic or butanoic acid.

3. The compound of claim 1 or 2 wherein R3, R4 and R5 are each independently H, F or Cx-O-S(=O)2-OH, where x is zero or 1, 2, 3, 4 or 5.

4. The compound of claim 1, which is selected from the group consisting of SNAP001 to SNAP020:WSLEGAL\055326\00505\42539390v6WSLEGAL\055326\00505\42539390v65. The compound of claim 1 which is SNAP001 (3-(2-methyl-8-(2-((oxidanidylsulfonyl)oxy)ethyl)-3,4-dihydro-2H-chromen-2-yl) propanoic acid)) or a pharmaceutically acceptable salt thereof.

6. The compound of claim 5 which is the potassium, sodium, calcium, magnesium, or pyridinium salt of the compound.

7. A compound of Formula (II):wherein R1 is hydrogen; hydroxy; methoxy, halogen; oxygen; nitrogen; sulphur; sulfonyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; or cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; or a pharmaceutically acceptable salt thereof.68WSLEGAL\055326\00505\42539390v68. The compound of claim 7, which is one of SNAP021 to SNAP024:>9. A pharmaceutical composition comprising at least one SNAP compound and pharmaceutically acceptable carrier.

10. Use of a compound of Formula I or Formula II to protect an in vivo or ex vivo organ which is at risk of ischemia or to treat a disease or a condition in a patient in need thereof, wherein the disease or condition is marked by a risk of organ ischemia.

11. The use of claim 10, wherein the organ which is at risk of ischemia is a heart.

12. A method of protecting or treating an in vivo or ex vivo organ from injury comprising administering a SNAP compound of Formula I or Formula II, or a pharmaceutically acceptable salt, in an effective amount, to the organ.

13. The method of claim 12 which is selected from the group consisting of:(a) protecting a solid organ from IR injury that occurs while in transport from donor to recipient.(b) treating a heart that is suffering IR injury upon thrombolysis or coronary intervention during a myocardial infarction;69WSLEGAL\055326\00505\42539390v6(c) treating a heart that is suffering IR injury during coronary artery bypass surgery (CABG); and(d) treating an internal organ, such as a heart, suffering injury during septic or hemorrhagic shock.

14. The use of claim 10 or 11, or the method of claim 12 or 13, wherein the compound is selected from the group consisting of SNAP001 to SNAP024.

15. A method of inducing a hibernation-like state in a subject comprising administering a SNAP compound of Formula I or Formula II, or a pharmaceutically acceptable salt, in an effective amount, to the subject.

16. A method treating a subject having cancer, comprising administering a SNAP compound of Formula I or Formula II, or a pharmaceutically acceptable salt, in an effective amount, to the subject in need of such treatment.

17. A method of promoting longevity of a subject, or slowing the rate of aging of the subject, comprising administering a SNAP compound of Formula I or Formula II, or a pharmaceutically acceptable salt, in an effective amount, to the subject.

18. A method of inducing quiescence of human cells, comprising administering a SNAP compound of Formula I or Formula II, or a pharmaceutically acceptable salt, in an effective amount, to the cells.

19. The method of claim 18 wherein the cells are hemopoietic stem cells or human ovarian oocytes (eggs).

20. The method of any one of claims 15-17, wherein the subject is a human.

21. The method of any one of claims 15-17, wherein the subject is an animal.

22. The method of any one of claims 19-21, wherein the compound is selected from the group consisting of SN AP001 to SNAP024.

23. The use of claim 14 or the method of claim 22, wherein the compound is SNAP001.70WSLEGAL\055326\00505\42539390v6

Images