Therapeutic targeting of hypdh / prodh2 and prodh1 to suppress 4-hydroxyproline and proline catabolism

Abstract

The small molecule N-propargylglycine (N-PPG), a covalent inactivator of the L-proline catabolic enzyme PRODH1, has been found to promote liver and kidney degradation of its mitochondrial paralog, HYPDH / PRODH2, resulting in systemwide increases in hydroxyproline (Hyp) levels after administration. The availability of N-PPG, and chemical analogs thereof, as well-tolerated suicide inhibitors of HYPDH / PRODH2 and / or PRODH1 provide evaluation of their efficacy as new therapeutic agents. Provided are compositions, methods of treating, and methods of targeting PRODH2, PRODH1, or a combination thereof.

Description

Docket No.: 94127.2.WOU1 THERAPEUTIC TARGETING OF HYPDH / PRODH2 AND PRODH1 TO SUPPRESS 4-HYDROXYPROLINE AND PROLINE CATABOLISM CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 510,031, filed June 23, 2023, and entitled “Therapeutic Targeting of HYPDH / PRODH2 to Suppress 4- Hydroxyproline Catabolism”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The various embodiments herein relate to treatments for primary hyperoxaluria, neurodegeneration, cancer, and other disorders involving excess endogenous production of oxalate, and more specifically to compositions and methods of use containing N- Propargylglycine (N-PPG) or a stereoisomer of methyl-PPG for suppressing 4-hydroxyproline and / or proline catabolism, and for preventing and / or treating disorders mediated by HYPDH / PRODH2 and / or PRODH1. BACKGROUND

[0003] HYPDH / PRODH2 is a mitochondrially located enzyme that is solely responsible for the first step in the catabolism of 4-hydroxyproline (Hyp). Collagen turnover and dietary intake results in a daily load of Hyp estimated to exceed 500 mg in normal individuals. The liver— and to a lesser degree, the kidneys—are responsible for the catabolism of Hyp. During the catabolism of Hyp, glyoxylate (a highly reactive aldehyde) is generated. In normal circumstances, glyoxylate is rapidly processed, with the majority being converted to glycine in peroxisomes or reduced to glycolate in the mitochondrial or cytosol. The portion of glyoxylate that does become oxidized to oxalate, which is an unusable end product of mammalian metabolism, must be secreted by the kidneys to avoid the buildup of tissue-damaging calcium oxalate crystals. The failure to properly catabolize glyoxylate underlies primary hyperoxaluria (PH), a rare but debilitating set of mono-genetic diseases where abnormally high oxalate levels lead to calcium oxalate stone developing in the kidney, with ensuing nephrocalcinosis, and end stage kidney failure in severe cases. Occurring in three genetic subtypes (PH1, PH2 and PH3), PH has been well characterized at the molecular level, however, effective treatment options are lacking, with kidney / liver transplants being required in extreme cases. Only recently, lumasiran—an siRNA agent directed against glycolate oxidase—was approved for PH1 treatment.Docket No.: 94127.2.WOU1

[0004] Molecular studies examining oxalate production in the context of PH, identified Hyp catabolism by HYPDH / PRODH2 as contributing significantly to oxalate production. In normal individuals, Hyp catabolism accounts for approximately 15% of urine oxalate while in PH1 patients, PH2 patients, and PH3 patients, Hyp catabolism accounts for approximately 18%, approximately 57%, and approximately 33% respectively, of urine oxalate levels. Studies with the HYPDH / PRODH2 knockout mouse have demonstrated that while having plasma Hyp levels approximately 20-fold higher than controls, they exhibited a normal phenotype with urine oxalate levels in males reduced 18% relative to control males. Most importantly, when the HYPDH / PRODH2 knockout mouse was crossed with a GRHPR knockout mouse (which develop calcium oxalate crystals in the kidney as a result of failure to reduce glyoxylate to glycolate and models PH2), the double knockout mouse was spared the development of calcium oxalate crystal.

[0005] The consequence of inheriting a failure of glyoxylate metabolism as characterized by the different PH conditions has been studied clinically with the underlying molecular defects comprehensively detailed, however, effective pharmacological interventions to deal with the accumulating calcium oxalate crystals characteristic of PH and responsible for the ensuing nephrocalcinosis are lacking.

[0006] There is a need in the art for improved treatments of diseases, such as hyperoxaluria, as well as a need for treatments which target PRODH2 and / or PRODH1, and reduce the accumulation of oxalate. SUMMARY

[0007] Provided are compositions, methods of treating, and methods of targeting PRODH2.

[0008] In Example 1, method of preventing and / or treating a disorder mediated by HYPDH / PRODH2, PRODH1, or a combination thereof comprises administering to a subject in need thereof a therapeutic agent comprising N-propargylglycine (N-PPG) or a chemical analog thereof, and inhibiting HYPDH / PRODH2, PRODH1, or a combination thereof.

[0009] Example 2 relates to the method according to Example 1, wherein the disorder comprises a primary hyperoxaluria (PH), a hyperoxaluria disorder, oxalosis, a disorder involving the production and renal excretion of excess levels of oxalate, a disorder involving the production of kidney stones, or a combination thereof.

[0010] Example 3 relates to the method according to Example 1 or 2, wherein the administering prevents or reduces the occurrence of nephrocalcinosis, kidney stone formation, bladder stone formation, renal failure, or a combination thereof.Docket No.: 94127.2.WOU1

[0011] Example 4 relates to the method according to any one of Example 1 to 3, wherein the chemical analog provides target specificity to PRODH2 over PRODH1.

[0012] Example 5 relates to the method according to Example 1, wherein the disorder comprises a neurodegenerative disorder or cancer, and wherein the N-PPG or chemical analog inhibits a combination of HYPDH / PRODH2 and PRODH1.

[0013] Example 6 relates to the method according to any one of Examples 1 to 5, wherein the chemical analog thereof comprises 2-[(but-3-yn-2-yl)amino]acetic acid having the following formula: . method according to Examples 5 or 6, wherein the chemicalanalog thereof comprises the following formula: .the method according to any one of Examples 1 to 7, wherein 4- hydroxyproline (Hyp) catabolism is suppressed.

[0016] In Example 9, a method of targeting PRODH2 comprises administering to a subject a therapeutic agent comprising N-propargylglycine (N-PPG) or a chemical analog thereof.

[0017] Example 10 relates to the method according to Example 9, wherein PRODH2 is targeted in addition to PRODH1.

[0018] Example 11 relates to the method according to Example 9, wherein the chemical analog provides target specificity to PRODH2 over PRODH1.

[0019] Example 12 relates to the method according to any one of Examples 9 to 11, wherein the chemical analog thereof comprises the following formula: .to the method according to any one of Examples 9 to 12, wherein the therapeutic agent prevents or treats a primary hyperoxaluria (PH), a hyperoxaluria disorder,Docket No.: 94127.2.WOU1 oxalosis, a disorder involving the production and renal excretion of excess levels of oxalate, a disorder involving the production of kidney stones, or a combination thereof.

[0021] In Example 14, a pharmaceutical composition for use in preventing and / or treating a disorder mediated by HYPDH / PRODH2, PRODH1, or a combination thereof comprises a therapeutic agent comprising N-propargylglycine (N-PPG) or a chemical analog thereof, and a pharmaceutically effective carrier.

[0022] Example 15 relates to the pharmaceutical composition according to Example 14, wherein the N-PPG or chemical analog provides target specificity to both PRODH2 and PRODH1.

[0023] Example 16 relates to the pharmaceutical composition according to Example 12 or 13, wherein the chemical analog thereof comprises 2-[(but-3-yn-2-yl)amino]acetic acid having the following formula: .the pharmaceutical composition of any one of Examples 14 to 16, wherein the composition prevents or treats a primary hyperoxaluria (PH), a hyperoxaluria disorder, oxalosis, a disorder involving the production and renal excretion of excess levels of oxalate, a disorder involving the production of kidney stones, or a combination thereof.

[0025] Example 18 relates to the pharmaceutical composition of Example 14, wherein the composition prevents or treats a disorder comprising a neurodegenerative disorder or cancer, and wherein the N-PPG or chemical analog inhibits a combination of HYPDH / PRODH2 and PRODH1.

[0026] Example 19 relates to the pharmaceutical composition of Example 18, wherein the chemical analog thereof comprises the following formula: .to the pharmaceutical composition of any one of Examples 14 to 19, wherein the composition is provided as a powder and dissolved in saline.Docket No.: 94127.2.WOU1

[0028] Example 21 relates to the pharmaceutical composition of any one of Examples 14 to 21, wherein the composition is provided at a dose of between about 20 mg / kg to about 300 mg / kg.

[0029] While multiple embodiments are disclosed, still other embodiments of the present disclosure will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the disclosure. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1A shows a heatmap of N-PPG targets HYPDH / PRODH2 modulating 4- hydroxyproline levels of unsupervised clustering of 20 amino acids and 21 amino acids–related metabolites using kidney samples from N-PPG-treated (50 mg / kg) and vehicle-treated wild- type (WT) and R6 / 2 mice (N3 mice per group). Heatmap of metabolite levels using log10- transformed concentrations determined using the Biocrates MxP Quant 500 kit. Color scale bar represents z-score values after normalization. Red box highlights proline and 4-hydroxproline (Hyp) as the most differentially altered levels between N-PPG-treated and vehicle mice. Right, supervised clustering of hydroxyproline and proline also shows that these metabolites were up- regulated in N-PPG-treated vs vehicle-treated wild-type (WT) and R6 / 2 mice. Differences were significant for both hydroxyproline and proline, with a false-discovery rate (FDR) of 3.34 e-2 as determined using one-way ANOVA analysis and Fisher's LSD post-hoc testing. Metabolites include ß-aminobutyric acid, taurine, 5-aminovaleric acid, phenylacetylglycine, citrulline, Trp, anserine, a-aminobutyric acid, betaine, carnosine, proline betaine, Asn, Ser, Leu, Ile, Phe, Val, sarcosine, Pro, t-OH-pro, a-aminoadipic acid, ornithine, Met, methionine sulfoxide, homoarginine, Asp, Arg, Lys, Thr, Glu, Tyr, homocysteine, Ala, Cys, Cystine, 3- methylhistidine, symmetric dimethylarginine, 1-methylhistidine, His, Gln, and Gly. The ultra- performance liquid chromatography–tandem mass spectrometry (UPLC-MS / MS) output data were imported into Skyline-daily for peak integration, calibration, and concentration calculations [K.J Adams et al., Alzheimer’s Disease Metabolomics C. Skyline for Small Molecules: A Unifying Software Package for Quantitative Metabolomics, J. Proteome Res.19 (4) (2020) 1447–1458, https: / / doi.org / 10.1021 / acs.jproteome.9b00640 (Epub 20200326. PubMed PMID: 31984744; PMCID:PMC7127945) ]. Exported data from Skyline were processed using a custom visual basic macro for importing into MetIDQ software (Biocrates AG) and MetaboAnalyst 5.0 [Z. Pang et al., MetaboAnalyst 5.0: narrowing the gap betweenDocket No.: 94127.2.WOU1 raw spectra and functional insights, Nucleic Acids Res. 49 (W1) (2021), https: / / doi.org / 10.1093 / nar / gkab382 (W388-W96. PubMed PMID: 34019663;PMCID: PMC8265181) ].

[0031] FIG. 1B shows a metabolomic analysis comparing blood and whole brains from R6 / 2 mice treated for 14 days with 50 mg / kg N-PPG (on right side) or vehicle (VEH, on left side) as in panel A. Bar plots display the averaged metabolite concentration obtained in N-PPG- treated mice (N5) and VEH-treated mice (N4). For blood, only 4-hydroxyproline was significantly up-regulated upon N-PPG treatment, while in brain lysates, both proline and hydroxyproline were significantly up-regulated. Statistical analysis was performed using two- sample t-test followed by p-value correction for multiple testing. *FDR = 0.05, ** FDR = 0.01, *** FDR = 0.001. The error bars correspond to the standard deviation. Metabolite concentrations are expressed in µM.

[0032] FIG. 2A shows the AlphaFold model of HYPDH / PRODH2 superimposed onto the crystal structure of an N-PPG inactivated PutA PRODH domain to determine if the predicted active site structure of HYPDH / PRODH2 is consistent with the covalent inactivation mechanism of N-PPG [D.A. Korasick et al, Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure, FEBS J. 284 (18) (2017) 3029–3049, https: / / doi.org / 10.1111 / febs.14165 (Epub 20170801. PubMed PMID: 28710792; PMCID: PMC5603418)]. This modeling showed that HYPDH / PRODH2 is predicted to have a structure needed for both binding of N-PPG in the substrate pocket and subsequent mechanistic steps (bond breaking and formation), leading to covalent linking of the FAD N5 atom to ε-amino group of the conserved active site lysine (Lys236 in HYPDH / PRODH2) [T.A.White et al., Structural basis for theinactivation of Thermus thermophilus proline dehydrogenase by N- propargylglycine, Biochemistry 47 (20) (2008) 5573–5580, https: / / doi.org / 10.1021 / bi800055w (Epub 20080422. PubMed PMID: 18426222; PMCID: PMC2708978); D. Srivastava et al., The structure of the proline utilization a proline dehydrogenase domain inactivated by N-propargylglycine provides insight into conformational changes induced by substrate binding and flavin reduction, Biochemistry 49 (3) (2010) 560– 569, https: / / doi.org / 10.1021 / bi901717s (PubMed PMID: 19994913; PMCID:PMC3727237) ]. HYPDH / PRODH2 molecular structure accommodates N-PPG binding and inhibition. Structural model of HYPDH / PRODH2 from AlphaFold2 viewed looking down the enzymatic (βα)8 barrel structure using a rainbow coloring scheme with blue at the N-terminus and red at the C-terminus. The N-PPG covalently interacting elements FAD (yellow) and Lys236 are noted. Two unique α-helices, α8 (red) and α5a (yellow), distinguish the HYPDH / PRODH2Docket No.: 94127.2.WOU1 barrel from other (βα)8 barrel proteins. Superposition of the HYPDH / PRODH2 model (cyan) with the crystal structure of an N-PPG-inactivated bacterial PRODH (PDB ID 5UR2, gray) showing that Lys236 has the potential for covalent linkage to the FAD upon inactivation by N- PPG with N-PPG's 3 carbon link (3-C link) between the bacterial PRODH's FAD and lysine shown in red.

[0033] FIG. 2B shows a Western blot probed with HYPDH / PRODH2 and Rieske (for normalization) of mitochondria (mito) and cytoplasmic(cyto) fractions from HepG2 cells treated for 48 h with 5 mM N-PPG (+N-PPG) or untreated. Arrows indicate HYPDH / PRODH2 and Rieske bands. Nonspecific bands are seen only in the cytoplasmic fraction that contains no signal from the mitochondrial HYPDH / PRODH2 (left). Western blot using mouse WT lysates from whole kidney and whole liver probed for Hypdh / Prodh2 with normalization to Rieske (right).

[0034] FIG. 2C shows densistometry results from the whole liver lysate Western blot PRDOH1 and PRODH2 signals shown in FIG 2B and normalized to Rieske signals. Quantification of the PRODH1 and PRODH2levels normalized to Rieske are shown in the lower panel. **, p = 0.0025 and 0.0076 for Prodh1 and Hypdh / Prodh2, respectively.

[0035] FIG. 3 shows N-PPG treatment induced increases in hydroxyproline (hyp) levels in WT and HD (R6 / 2) mouse kidneys. Kidney samples (n = 12) from the N-PPG- and Veh-treated mice from a study in which 5-week-old mice (R6 / 2 HD = 8, B6 WT = 8) receive daily gavage treatments (N-PPG at 50 mg / kg or Veh) for 9 days before sacrifice and snap freezing of whole brain and kidney tissue samples for later immunoblotting, RNA sequencing, and metabolomic analyses, where said mice were profiled for proline, hydroxyproline and sarcosine (not shown). Box-whisker plots of kidney hydroxyproline levels for the four different groups (n = 3 mice / group) show significant NPPG induced increases in hyp with significance calculated by Fisher’s LSD F, p, and FDR values. Not fully shown, WT samples relative to treated HD samples showed consistently greater N-PPG inductions of proline (2.4- vs. 1.4-fold), hydroxyproline (4.6- vs.2.8-fold), and sarcosine (2.5- vs.1.9-fold).

[0036] FIG.4 shows a metabolomic profiling of brain (cerebellum), blood and kidney samples collected at 14 days showed as displayed on the heat map the impact of N-PPG treatment on various clusters of quantified amino acid-like metabolites, including the smaller clade of proline, hydroxyproline and sarcosine. Box-whisker plots show the N-PPG induced group and average increases in brain proline (2.6-fold) and hydroxyproline (2.7-fold), blood proline (3.6- fold), and kidney hydroxyproline (3.6- fold) and sarcosine (5.0-fold), with Q-values indicating significant p-values adjusted for FDR.Docket No.: 94127.2.WOU1

[0037] FIG.5 shows a graph of a statistical analysis using an unpaired 2-tailed t-test comparing percent differences between early and late time urinary oxalate / creatinine (µM / µM) ratios for Control and N-PPG treated mice having a p-value = 0.0095. Day 9 urinary oxalate concentrations under 1% hyp diet are all increased relative to the Day 0 oxalate concentration under normal diets. The Day 9 N-PPG treatment of GRHPR KO mice significantly reduces (by about 50%) the urinary oxalate concentration relative to vehicle (VEH) treated GRHPR KO mice.

[0038] FIG. 6A shows a bright-field image with 0% polarization showing oxalate crystal formation in kidney tissue from a 10-month-old male GRHPR KO mouse on a a 1% hydroxyproline (hyp) diet treated daily with only the oral drug vehicle (saline). On this single 5 micron H&E kidney section, both larger (>25 micron, 11 circled in green) and smaller (<25 micron) crystals can be visualized and counted, together yielding a total crystal count per kidney section (total = 37 crystals seen in this section).

[0039] FIG. 6B shows a bright-field image with 0% polarization showing oxalate crystal formation in kidney tissue from a 10-month-old male GRHPR KO mouse on a 1% hydroxyproline diet (1% hyp) orally treated for 21 days with 200 mg / kg N-PPG. On this single 5 micron H&E kidney section, both larger (>25 micron, 2 circled in green) and smaller (<25 micron) crystals can be visualized and counted, together yielding a total crystal count per section (total = 6 crystals seen in this section).

[0040] FIG. 6C shows a bar graph of the mean total crystal counts of all Ca-oxalate kidney crystals (<25 microns in size + >25 microns in size) visualized by birefringent (double refractant) polarizing microscopy (as exemplified in FIG 6A and FIG 6B) enumerated and averaged over 4 H&E stained adjacent 5 micron sagittal sections from each of 4 different sets of orally treated mice: 3 wildtype (WT) mice treated for 21 days with vehicle (VEH), 4 WT mice treated with 22 days of N-PPG (200 mg / kg), 4 GRHPR KO mice treated with 22 days of VEH, and 5 GRHPR KO mice treated with 22 days of N-PPG (200 mg / kg). Significant differences (P<0.05) shown for mean count distributions between all 4 experimental mouse groups denoted by * brackets.

[0041] FIG. 7A shows a Western blot of mitochondria from in vitro cultured HepG2 cells treated for 48 hours in two separate experiments with 5 mM of N-PPG, its S-(mPPG) or R- (mPPG) stereoisomers. These immortalized human liver cancer cells express PRODH2 but not PRODH1. The S-methyl isomer shows comparable ability as N-PPG at inhibiting and degrading mitochondrial PRODH2 (relative to Rieske), while the R-methyl isomer shows much less potency in its anti-PRODH mitochondrial effects.Docket No.: 94127.2.WOU1

[0042] FIG. 7B shows Western blots of mitochondria from in vitro cultured ZR-75-1 cells treated for 48 hours in multiple studies using either 5 mM of N-PPG, its S-(mPPG) or R- (mPPG) stereoisomers. ZR-75-1 human breast cancer cells express PRODH1 but not PRODH2. The S-methyl isomer shows comparable ability as N-PPG at inhibiting and degrading mitochondrial PRODH1 (relative to Rieske, AOC1, or AOC2), while the R-methyl isomer shows minimal ability to reduce mitochondrial PRODH1.

[0043] FIG. 7C shows a Western blot of livers isolated from mice treated orally for 5 days with VEH (control), or 200 mg / kg of N-PPG, its S-(mPPG) or R-(mPPG) isomers. The S- methyl isomer is about as potent as N-PPG at reducing PRODH1 / Rieske and PRODH2 / Rieske protein ratios, while the R-methyl isomer is much less effective at reducing mitochondrial PRODH1 or PRODH2.

[0044] Various embodiments of the present disclosure will be described in detail with reference to the figures. Reference to various embodiments does not limit the scope of the disclosure. Figures represented herein are not limitations to the various embodiments according to the disclosure and are presented for exemplary illustration of the disclosure. DETAILED DESCRIPTION

[0045] The present disclosure relates to compositions and methods related to the targeting of HYPDH / PRODH2, leading to increases in Hyp levels and garnering use as a therapeutic. Studies with the compound N-propargylglycine (N-PPG), initially undertaken for its targeting of the proline catabolizing enzyme PRODH (also referred to as PRODH1), surprisingly revealed that N-PPG not only targets PRODH1 but further targets HYPDH / PRODH2 with even greater efficiency, thus offering possibilities for its use as a therapeutic agent. In further embodiments, alternative chemical analogs may be used to target PRODH2, including, but not limited to 2-[(but-3-yn-2-yl)amino]acetic acid (C6H9NO2). In even further embodiments, alternative chemical analogs may be used to target both PRODH1 and PRODH2, including, but not limited to methyl-PPG analogs, particularly the S-methyl-PPG isomer. In some aspects, the alternative chemical analogs having effects on both PRODH1 and PRODH2 may be further used as a therapeutic for neurodegeneration disorders and cancer.

[0046] In some aspects, the present disclosure provides for use of a novel cancer therapeutic for prevention of neurodegeneration and kidney stone diseases. N-PPG has been previously described as an orally bioavailable suicide inhibitor of mitochondrial proline dehydrogenase (PRODH), capable of starving malignant cells of anaplerotic glutamate and ATP production essential for their metastatic spread. Without being limited to any particular mechanism ofDocket No.: 94127.2.WOU1 action or theory, as N-PPG is able to distort PRODH structure and serendipitously induce the mitochondrial unfolded protein response (UPRmt) in normal cells, Applicant uncovered its beneficial brain mitohormesis response, including its ability to partially reverse the abnormal transcriptome of Huntington mouse (R6 / 2) brains. In some further aspects, N-PPG can increase systemwide proline as well as hydroxyproline levels, leading to mouse studies of the disclosure demonstrating that N-PPG’s suicide inhibition of the mitochondrial paralog, PRODH2, may also offer novel therapeutics against Primary Hyperoxaluria, a lethal oxalate stone-forming kidney disease. Definitions

[0047] The term "about" or "approximately" as used herein refers to being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, e.g., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of ±20%, preferably ±10%, more preferably ±5% and more preferably still ±1% of a given value.

[0048] The terms "subject," "individual," and "patient" may be used interchangeably and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, rodents, lagomorphs, and the like). In various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.

[0049] As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of theDocket No.: 94127.2.WOU1 art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0050] As used herein, the term “pharmaceutically acceptable carrier” or “carrier” refers to sterile aqueous or nonaqueous solutions, colloids, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers canDocket No.: 94127.2.WOU1 include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0051] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0052] The term “treat” or “treatment” when used with reference to treating, e.g., a pathology or disease refers to the mitigation and / or elimination of one or more symptoms of that pathology or disease, and / or a delay in the progression and / or a reduction in the rate of onset or severity of one or more symptoms of that disorder or disease, and / or the prevention of that pathology or disease. The term “treat” can refer to prophylactic treatment which includes a delay or in the onset or the prevention of the onset of a pathology or disease.

[0053] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, administration to specific organs through invasion, intramuscular administration, intratumoral administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0054] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbersDocket No.: 94127.2.WOU1 within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1½, and 4¾ This applies regardless of the breadth of the range.

[0055] The present disclosure describes compositions of matter and methods for their use. In aspects, the methods involve the suppression of Hyp catabolism. In certain aspects, compounds and pharmaceutical compositions disclosed herein include a therapeutic agent comprising N- PPG or a chemical analog thereof in therapeutically effective amounts. In some embodiments, the chemical analog may comprise 2-[(but-3-yn-2-yl)amino]acetic acid (C6H9NO2). N-PPG is a unique mechanism-based inactivator of proline dehydrogenase enzymes that installs an irreversible covalent link between the flavin adenine dinucleotide (FAD) cofactor and a conserved active site lysine residue. Previous studies with PRODH1 demonstrated that this covalent modification promoted degradation of the enzyme, whereas classical, reversible, competitive inhibitors of PRODH1, such as proline analogs, had no impact on protein levels.

[0056] However, in embodiments of the present disclosure, the administration of N-PPG beneficially promotes liver and kidney degradation of HYPDH / PRODH2, resulting in systemwide increases in Hyp levels after administration. In aspects, the inactivation of HYPDH / PRODH2 results in a reduction of Hyp catabolism, leading to an increase in Hyp levels. In some embodiments, a chemical analog, may comprise a compound having the following formula: , wherein R1 is H or a C1-C4 alkyl. In further embodiments, thebut is not limited to, a methyl-PPH, such as, for example, 2-[(but-3-yn-2-yl)amino]acetic acid (C6H9NO2), and may be used to target HYPDH / PRODH2 and / or PRODH1. In some aspects, the chemical analog may be more specific in inhibiting PRODH2, providing more target selectivity to PRODH2. In other aspects, the chemical analog may be more specific in inhibiting both PRODH2 and PRODH1. In some aspects, the methyl-PPH may comprise 2-[(but-3-yn-2-yl)amino]acetic acid (C6H9NO2) having the following chemical . In certain embodiments, the chemical analog, or 2-[(but-3-, may be provided as a racemic mixture. In other embodiments, the chemical analog or 2-[(but-Docket No.: 94127.2.WOU1 3-yn-2-yl)amino]acetic acid (C6H9NO2) is provided as a predominantly L-enantiomer. In further embodiments, the chemical analog or methyl-PPH may be provided as an R-methyl- PPH stereoisomer having the following chemical In other embodiments, the chemical analog or methyl-PPHstereoisomer having the following chemical . In aspects, N-PPG is an irreversible, suicide inhibitor, of both aspects,the S-methyl-PPH stereoisomer is an irreversible, suicide inhibitor, of both PRODH1 and PRODH2. Beneficially, the suicide inhibition mechanism of N-PPG and S-methyl-PPH stereoisomer causes mitochondrial decay as well as induction of the mitochondrial unfolded protein response (UPRmt) which leads to mitohormesis. In some aspects, and at least within the entire brain of a subject, the mechanism induces protection against neurodegeneration- associated proteotoxicity.

[0057] In some aspects, the S-methyl-PPH stereoisomer has similar or more potent activity than N-PPG in reducing or inhibiting PRODH1 and PRODH2. The S-methyl-PPH stereoisomer has significantly more activity than the R-methyl-PPH stereoisomer against both PRODH1 and PRODH2. In further aspects, the R-methyl-PPH stereoisomer has low effect in reducing or inhibiting PRODH1, and therefore, may be beneficially used in methods where the inhibition of PRODH2 is preferred. As competitive inhibitors of PRODH1 and PRODH2 do not reduce mitochondrial levels of these enzymes, the irreversible, suicide inhibiting mechanism of action on PRODH2 and / or PRODH1 is important for its maximum suppression of hydroxyproline and / or proline catabolism.

[0058] According to certain embodiments, the therapeutic agent comprising N-PPG or a chemical analog thereof is further provided in a therapeutic or pharmaceutical composition. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. In embodiments, the pharmaceutically acceptable carrier employed may be, for example, a solid, liquid, or gas. Examples of solid carriers include, but are not limited to, lactose, mannitol, microcrystalline cellulose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers include, but are not limited to, sugarDocket No.: 94127.2.WOU1 syrup, peanut oil, olive oil, ethanol, propylene glycol, saline and water. Examples of gaseous carriers include, but are not limited to, carbon dioxide, nitrogen, and compressed air.

[0059] In preparing the compositions into a dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs, and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules, and tablets. Because of their ease of administration, tablets and capsules are possible oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. In certain aspects, the compositions may be administered orally. In embodiments, the therapeutic agent may be provided in a solid form, such as a powder, and dissolved into a pharmaceutically acceptable carrier to form a pharmaceutical composition. The pharmaceutical composition may be further neutralized as needed.

[0060] In embodiments, the therapeutic agent may be present in any therapeutically effective amount as would be appreciated by those skilled in the art. In embodiments, the therapeutic agent may be present in an amount of between about 20 mg / kg to about 300 mg / kg, between about 30 mg / kg to about 275 mg / kg, between about 40 mg / kg to about 250 mg / kg, or between about 50 mg / kg to about 200 mg / kg.

[0061] The disclosed compounds, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds. In some aspects, the one or more other therapeutically active compounds may include, but are not limited to, salicylic acid derivatives, high dose vitamin B6, siRNA, small molecular inhibitors of oxalate producing enzymes, small molecular inhibitors of oxalate producing pathways, agents that treat primary hyperoxaluria, agents that prevent oxalate kidney stone formation, or a combination thereof. In further embodiments, the pharmaceutical composition may be used in conjunction with dietary restriction protocols.

[0062] According to certain embodiments, disclosed is a method of preventing and / or treating a disorder mediated by HYPDH / PRODH2. In further embodiments, a method of targeting PRODH2 is provided. In embodiments, the compounds and pharmaceutical compositions described herein target both PRODH1 and PRODH2. In other embodiments, the compounds and pharmaceutical composition described herein target only PRODH2, providing more target selectivity to PRODH2. In some aspects, the inhibition of PRODH1, PRODH2, or theDocket No.: 94127.2.WOU1 combination of PRODH1 and PRODH2 is irreversible. In embodiments, the compositions as described herein may be used in the methods of the disclosure.

[0063] In some implementations, a method of preventing and / or treating a disorder mediated by HYPDH / PRODH2 is provided, comprising a step of administering to a subject in need thereof N-propargylglycine (N-PPG). In some aspects, the administration of the N-PPG inhibits HYPDH / PRODH2. In some examples, the administration of a chemical analog, such as, for example, the R-methyl-PPH stereoisomer, inhibits HYPDH / PRODH2. In further embodiments, the methods target PRODH2, where the administration of N-PPG results in the targeting of PRODH2 in addition to PRODH1. In even further embodiments, the administration of a chemical analog of N-PPG results in the targeting of both PRODH2 in addition to PRODH1. In some aspects, the chemical analog comprises a methyl-PPH. In further aspects, the methyl-PPH comprises the S-methyl-PPH stereoisomer.

[0064] In embodiments, the disorder comprises a primary hyperoxaluria (PH), hyperoxaluria disorders, oxalosis, disorders involving the production and renal excretion of excess levels of oxalate, or disorders involving the production of kidney stones (including subjects whose kidney stones are composed of oxalate crystals). In some aspects, the PH may include PH1, PH2, PH3, or a combination thereof. Beneficially, the administration of the disclosed compositions prevent and / or reduce the occurrence of nephrocalcinosis, kidney stone formation, bladder stone formation, renal failure, or a combination thereof. In preferred embodiments, the composition comprises N-PPG or a chemical analog that targets PRODH2, with or without further targeting PRODH (PRODH1).

[0065] In further embodiments, the disorder comprises a neurodegenerative disease or cancer. In preferred embodiments, the composition comprises N-PPG or a chemical analog that targets both PRODH2 and PRODH1. In some aspects, N-PPG, even when orally administered, can adequately penetrate the blood-brain-barrier of mammals in an amount sufficient to induce UPRmt and mitohormesis in the brain and also prevent the earliest (e.g., presymptomatic) pathogenic subcellular manifestations of a lethal neurodegenerative condition such as Huntington's Disease (HD), and the like. Accordingly, in various embodiments, the methods in the treatment and / or prophylaxis of a neurodegenerative disorder in a subject (e.g., a mammal) include, but are not limited to Huntington's disease, Alzheimer's disease, Parkinson's disease, age-related dementia, mild cognitive impairment (MCI), amyotrophic lateral sclerosis (ALS), an ischemic event (e.g., stroke and / or traumatic brain injury), and the like. In a prophylactic context, the compounds and compositions of the disclosure may be administered in a subject identified as having one or more risk factors for a neurodegenerative disorder. SuchDocket No.: 94127.2.WOU1 risk factors can include, but is not limited to, family history, genetic markers, and / or biochemical markers.

[0066] The methods described herein can be used to treat any subject susceptible to cancers that rely on the production of glutamate to fuel mitochondrial ATP production. Examples of suitable subjects include mammals, e.g., research animals or pets, such as mice, rats, guinea pigs, rabbits, cats, dogs, as well as monkeys and other primates. In certain embodiments, the subject is human.

[0067] Cancers that are amenable to treatment using the methods described herein include those that rely on the production of glutamate to fuel mitochondrial ATP production. Generally, cancers amenable to such treatment contain the proline catabolic enzyme targeted by the method, e.g., at a detectable level. In some embodiments, the cancer is tested to determine whether it contains detectable levels of PRODH (PRODH1), PRODH2, PC5DH, and / or GLDH. If PRODH, PC5DH, or GFDH is detected, the cancer can be treated by inhibiting PRODH, PC5DH, or GFDH, respectively. If two or all three of these enzymes are detected, the cancer can be treated by administering the inhibitors for the combination of enzymes detected. PRODH1, PRODH2, is the rate-limiting step for anaplerotic use of proline (commonly derived from collagen in the extracellular matrix) to generate mitochondrial ATP under nutrient stress and hypoxic conditions. Because most, if not all, cancers are expected to use this pathway, at least to some extent, its inhibition is expected to be effective, either alone or in a combination therapy described herein, against a broad range of cancers.

[0068] In certain embodiments, the cancer is a cancer known to be amenable to p53 restoration therapy, in which case, treatment can include inhibition of proline catabolism in combination with p53 restoration. As noted above, a cancer need not have any functional p53 alleles to be treatable by p53 restoration therapy; however, the presence or absence of at least one functional p53 allele will inform treatment. Therefore, in some embodiments, the cancer can be tested to determine whether it includes cells having at least one functional p53 allele. If so, the cancer can be treated, e.g., by administering a p53-reactivating drug to the subject. If not, p53 function must be provided, e.g., using gene therapy. The vast majority of human cancers are characterized by some disruption in the p53 network, and therefore, p53 restoration in combination with inhibition of proline catabolism is expected to be broadly effective.

[0069] In certain embodiments, the cancer is a cancer known to be amenable to inhibition of glutaminase (e.g., GLS1), such as one that is amenable to treatment using CB-839 / Calithera, for example. In particular embodiments in which inhibition of proline catabolism is combined with inhibition of GLS, the cancer is one that contains the proline catabolic enzyme targetedDocket No.: 94127.2.WOU1 by the method as well as GLS, e.g., both at a detectable levels. In some embodiments, the cancer is tested to determine whether it contains detectable levels of GLS and, optionally to determine whether it contains detectable levels of PRODH, PC5DH, and / or GLDH. The cancer can be treated by administering the inhibitor(s) for the enzyme(s) detected.

[0070] In various embodiments, a subject treated according to one of the methods described herein has one or more of the following cancers: adrenal, anal, bile duct, bladder, bone, brain / central nervous system, breast, cervical, colon, endometrial, esophageal, eye, gallbladder, gastrointestinal, hypopharyngeal, kidney (renal cell and renal pelvis), laryngeal, leukemia, liver, lung, melanoma, lymphoma, malignant mesothelioma, multiple myeloma, nasal cavity, pancreatic, paranasal sinus, nasopharyngeal, neuroblastoma, oral cavity, oropharyngeal, osteosarcoma, ovarian, pituitary, prostate, rectal, retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin (e.g., basal cell, squamous cell, melanoma), small intestine, stomach, testicular, thymus, thyroid, uterine, vaginal, and vulvar cancer.

[0071] In various embodiments, a subject treated according to one of the methods described herein has one or more of the following cancers: breast cancer, prostate cancer, colon cancer, cervical cancer, ovarian cancer, pancreatic cancer, renal cell (kidney) cancer, glioblastoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Adrenocortical carcinoma, AIDS-related cancers (e.g., kaposi sarcoma, lymphoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid / rhabdoid tumor, bile duct cancer, extrahepatic cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain stem glioma, brain tumors (e.g., astrocytomas, brain and spinal cord tumors, brain stem glioma, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumors (e.g., childhood, gastrointestinal), cardiac tumors, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, duct cancers e.g. (bile, extrahepatic), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma), fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., ovarian cancer, testicular cancer, extracranial cancers, extragonadal cancers, central nervous system), gestational trophoblastic tumor, brainDocket No.: 94127.2.WOU1 stem cancer, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kaposi sarcoma, kidney cancer (e.g., renal cell, Wilm's tumor, and other kidney tumors), langerhans cell histiocytosis, laryngeal cancer, leukemia, acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myelogenous (CML), hairy cell, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., childhood, non- small cell, small cell), lymphoma (e.g., AIDS-related, Burkitt (e.g., non-Hodgkin lymphoma), cutaneous T-Cell (e.g., mycosis fungoides, Sézary syndrome), Hodgkin, non-Hodgkin, primary central nervous system (CNS)), macroglobulinemia, Waldenström, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma (e.g., childhood, intraocular (eye)), merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, Myelogenous Leukemia, Chronic (CML), multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity cancer, lip and oropharyngeal cancer, osteosarcoma, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., Ewing, Kaposi, osteosarcoma, rhadomyosarcoma, soft tissue, uterine), Sézary syndrome, skin cancer (e.g., melanoma, merkel cell carcinoma, basal cell carcinoma, nonmelanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer with occult primary, stomach (gastric) cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and / or Wilm's tumor.

[0072] In embodiments, the pharmaceutical compositions and compounds described herein may be administered in one or more doses per day. In some embodiments, the composition is administered only once per day. In other embodiments, the composition is administered in multiple doses per day, such as at least two times per day, at least three times per day, at least four times per day, or at least five times per day, or more. In other embodiments, the composition is administered at least once per week, at least twice per week, at least three timesDocket No.: 94127.2.WOU1 per week, at least four times per week, at least five times per week, at least six times per week, or at least seven times per week.

[0073] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated as incorporated by reference. EXAMPLES

[0074] Embodiments of the present disclosure are further defined in the following non-limiting Examples. It should be understood that these Examples, while indicating certain embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the disclosure to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Materials:

[0075] Drug and Antibodies: N-propargylglycine (N-PPG; #58160-95-5) was purchased from BOC Sciences (Shirley, NY). Antibodies used included mouse monoclonals for PRODH1 (A- 11 sc-3764 and Rieske FeS IgG (A-5 sc-271609) from Santa Cruz Biotechnology (Santa Cruz, CA)); PRODH2 / HYPDH rabbit polyclonal (PA5-62366) from Thermo-Fisher (Ashville, NC); HRP-conjugated goat anti-mouse secondary (#1706516) from BioRad Laboratories, Inc. (Hercules, CA); and HRP-conjugated mouse anti-rabbit monoclonal (#211-032-171)from Jackson ImmunoResearch Laboratories (West Grove, PA).

[0076] Cell Culture: The HepG2 human hepatoblastoma cell line was obtained from American Type Culture Collection (ATCC; Rockville, MD) and cultured under 5% CO2 at 37 °C using McCoy’s 5A Media (Corning; Manassas, VA) supplement with 10% fetal bovine serum (Corning; Manassas, VA). Routine mycoplasma checks were performed using the MycoAlert detection kit from Lonza (Basel, Switzerland).

[0077] Vehicle and N-PPG treatment of B6 wild type and R6 / 2 mice: The Buck Institute for Research on Aging animal facility is an AAALAC International accredited institution (Unit Number 001070). All protocols and procedures described herein were approved by the Buck’sDocket No.: 94127.2.WOU1 Institutional Animal and Use Committee. Male R6 / 2 (Huntington’s Disease model) and B6 wild type (WT) mice were purchased from Jackson labs (Bar Harbor, ME) and received at ~4 weeks of age. CAG length was confirmed by PCR. Commercially obtained N-PPG (150 mg) was dissolved in 1 mL of 0.9% saline and the solution neutralized to pH 6.9 with 1M NaOH to achieve final solution volumes of N-PPG. An initial study administrated N-PPG at 50 mg / kg daily for 9 days to 4 WT and 4 R6 / 2 mice by oral gavage with identical numbers of Vehicle control mice similarly gavaged with 200 µl volumes of saline. A second study administered N-PPG at 50 mg / kg by oral gavage daily for 14 days to 5 R6 / 2 mice with 5 Vehicle control R6 / 2 mice gavaged with 200 µl volumes of saline. A third study used 4 WT mice administered with N-PPG at 200 mg / kg daily for 5 days by oral gavage with 4 WT mice gavaged daily for 5 days with 200 µl saline. All mice were weighed every other day and all drug doses adjusted according to mouse weight. From 1-4 hours after their final oral gavage treatment, mice were anesthetized with isofluorane (Butler Schein) and cervically dislocated; and mouse tissues (brain, kidney, and / or blood) were immediately resected and snap frozen into liquid nitrogen. Example 1

[0078] Previous mouse studies using N-PPG delivered by oral gavage at 50 mg / kg demonstrated not only good tolerance but also the ability to penetrate the blood-brain barrier and promote the decay of the L-proline catabolic enzyme, PRODH1, with the subsequent induction of a beneficial mitohormesis effect. Therefore, with the object of confirming that N- PPG treatment produced enhanced proline levels as reflected in PRODH1 protein reduction, metabolomic analysis of kidney lysates from mice treated at 50 mg / kg daily for nine days was performed.

[0079] As used throughout the Examples, the following methods were followed for metabolomic analysis. Quantitative metabolomics profiling was performed using 50 mg aliquots of pulverized whole kidney plus 200 µl of blood collected in EDTA tubes that was transferred into soft tissue homogenizing CK14 tubes (Bertin Corp, Rockville, MD) for analysis using the Biocrates MxP Quant 500 Kit (Biocrates Life Sciences AG) at the Duke University School of Medicine’s Proteomics and Metabolomics Core Facility at the Center for Genomics and Computational Biology (Durham, NC). The UPLC-MS / MS output data was imported into Skyline-daily (ref-see Joanna) for peak integration, calibration, and concentration calculations (Adams et al., 2020). Exported data from Skyline was processed using a custom visual basic macro for importing into MetIDQ software (Biocrates AG) and MetaboAnalyst 5.0 (ref see JOanna). Of the 20 amino acid and 30 amino acid relatedDocket No.: 94127.2.WOU1 metabolites targeted for detection, 20 amino acid and 21 amino acid related metabolites satisfied the filtering criterions.

[0080] While the anticipated upregulation in proline levels were observed, it was startling to see that the most significantly upregulated metabolite was Hyp in both B6 wild type as well as Huntington R6 / 2 mice (FIG. 1A). In another metabolic study, R6 / 2 mice treated at 50mg / kg daily by oral gavage for 14 days found only Hyp as significantly upregulated in blood showing a log21.84-fold increase, while in the brain, both Hyp and proline were found significantly upregulated with Hyp and proline showing a log2 1.45- and log2 1.36-fold increase respectively (FIG.1B). These results suggested that N-PPG targets both PRODH2 in addition to PRODH1. Example 2

[0081] Analysis was conducted comparing an AlphaFold model of PRODH2 obtained from UniProt (Q9UF12) with PutA PRODH crystal structures, revealing that the canonical PRODH ( ^ ^)8 fold and FAD binding site are conserved in HYPDH / PRODH, consistent with HYPDH / PRODH being a potential target of N-PPG (FIG. 2A). Importantly, Lys236 of PRODH2 occupied an active site location analogous to PRODH1’s Lys234 with structural modeling, predicting the covalent linkage of N-PPG with PRODH2’s FAD and Lys236 (Fig 2A right panel).

[0082] Structural modeling was completed in order to make the comparison. Comparison of the AlphaFold model of PRODH2 obtained from UniProt (Q9UF12) with PutA PRODH crystal structures revealed residues 160-516 of PRODH2 were predicted to form the same ( ^ ^)8fold and FAD cofactor environment as the PutA PRODH domain. Notably, the PRODH2 fold includes two unique ^-helices, denoted ^8 and ^5a, that distinguish the PRODH barrel from other ( ^ ^)8barrel proteins (FIG. 2A). To determine if the predicted active site structure of PRODH2 was consistent with the covalent inactivation mechanism of N-PPG, the AlphaFold model of PRODH2 was superimposed onto the crystal structure of an N-PPG inactivated PutA PRODH domain (PMID: 28710792). This modeling showed that PRODH2 is predicted to have the constellation of amino acids needed for both binding of N-PPG in the substrate pocket and subsequent mechanistic steps (bond breaking and formation) leading to covalent linking of the FAD N5 atom to epison-amino group of the conserved active site lysine (Lys236 in PRODH2).

[0083] Western Analysis was further conducts, where aliquots from whole liver and kidney pulverized in liquid nitrogen were sonicated in a low salt buffer supplemented with detergentDocket No.: 94127.2.WOU1 (10 mM Tris pH 7.5, 50 mM NaCl, 1% SDS). Prior to western blotting, protein content was determined by Bradford Coomassie Assay (BCA) kit (Pierce, Rockford, IL) and then diluted into 2X Laemmli sample buffer. Western blotting was performed as previously described using polyvinylidene fluoride (PVDF) membranes blocked with 5% non-fat milk in TBST (tris- buffered saline with 0.1% tween-20) incubated with primary and then secondary antibodies conjugated to horse radish peroxidase; the resulting western blots were developed using chemiluminescence with autoradiographs scanned for densitometry using ImageJ. HepG2 cultures of control or treated (48 hours with 5 mM N-PPG) cells were lysed in mitochondrial isolation buffer (250 mM sucrose 10 mM Tris Ph 7.5, 4 mM EDTA) followed by a low speed centrifugation (1000 × g, 10 min, 4 °C) to remove nuclei followed by a high speed centrifugation (10,000 × g, 10 min, 4 °C) of remaining supernatant to obtain the mitochondrial pellet and cytosolic fraction. The mitochondrial pellet was resuspended in Laemmli buffer with aliquots of the mitochondrial fractions together with aliquots of the cytosolic fractions used for western analysis as described above for the tissue lysates.

[0084] To explicitly examine the influence of N-PPG on HYPDH / PRODH2 protein levels as explanation for the observed increase in HYP levels in mice treated with N-PPG, the hepatoblastoma cell line HepG2, a cell line well characterized as recapitulating Hyp catabolism by HYPDH / PRODH2, was treated with N-PPG. As shown in FIG. 2C (left side), N-PPG treatment at 5 mM for 48hr produced a ~40% reduction in the mitochondrially located HYPDH / PRODH2. It is noteworthy that the mitochondrially located PRODH2 bands at ~55 kDa are consistent with the PRODH2 knockout value while the strong bands at ~75 kDa in the HepG2 cytoplasmic fraction are insensitive to N-PPG treatment and indicative of nonspecific bands detected by this antibody. This N-PPG induced loss of HYPDH / PRODH2 in HepG2 cells suggests that, similarly to PRODH1, the covalent interaction between N-PPG and HYPDH / PRODH2 promotes protein structural distortion with subsequent protein degradation.

[0085] The increased levels of Hyp induced by N-PPG treatment suggest a reduction in the liver’s HYPDH / PRODH2 catabolizing capacity. It is noteworthy that while both liver and kidney catabolize Hyp, the liver assumes major Hyp catabolizing role and in this regard HYPDH / PRODH2 liver levels assuming the liver 4 time the mass of two kidneys exceed those of kidney by at least a factor of 40 (Fig 2C, right side). To assess N-PPG’s impact on HYPDH / PRODH2 liver levels B6 mice were treated at 200mg / kg daily by oral gavage for 5 days. Significantly this 5-day N-PPG treatment was sufficient to produce reduction of HYPDH / PRODH2 liver levels to approximately 73% of control (FIG. 2C). Additionally, as PRODH1 is also expressed in the liver, N-PPG reduced PRODH1 to approximately 66% ofDocket No.: 94127.2.WOU1 control (FIG. 2C), a result consistent with previous observations where N-PPG induced PRODH1 reduction in the brain.

[0086] The results demonstrate that the orally available and well tolerated drug N-PPG was found to produce elevated Hyp levels as a consequence of reducing the liver’s content of HYDH / PRODH2, a well-documented PH target. In studies underscoring N-PPG’s safety, wild type B6 mice gavaged with 100 mg / kg and 200 mg / kg daily for 8 weeks followed by 8 weeks of observation revealed no health or phenotypic difference relative to control.

[0087] As the GRHPR knockout (KO) and AGT KO (PH 1 model) mouse models were developed as tools for the development of PH therapeutics. Interestingly, while having approximately 3-fold elevated urine oxalate levels relative to control with males more impacted than females, only ~25% of GRHPR KO male mice developed nephrocalcinosis with symptoms remaining mild (ref Salido). The AGT KO mouse also exhibit urine hyperoxaluria with males more impacted than females with approximately 10% of the male mice developing mild nephrocalcinosis. However, when challenged with a 1% Hyp diet all GRHPH KO mice presented with server nephrocalcinosis, a condition that could be rescued by crossing into a HYDH / PRODH2 KO background. It is thus anticipated given its safety and capacity to suppress Hyp catabolism, N-PPG will prove effective in mitigating nephrocalcinosis in Hyp fed GRHPR KO and AGT KO mice, establishing a major step forward in the development of an effective PH therapeutic. Example 3

[0088] To assess the effect of consuming a 1% hydroxyproline (hyp) rich diet on urinary oxalate levels and Ca-Oxalate kidney stone formation, 10-month-old GRHPR-KO male and female mice (an animal model for primary hyperoxaluria type 2) were fed a 1% hyp or control diet for 21 days ad libitum (control diet, n=3, 1% hyp, n=3). Mice were singly housed in Techniplast mouse metabolic cages for collection of three 8-hour urine samples on the following timepoints: Day 0 (before starting the diet), Day 16, and Day 21. Urine was immediately stored at 80 ^C and used later to determine oxalate concentration using a commercially available oxalate kit (EOXA-100 Oxalate Assay Kit, Bioassay Systems). After the completion of the study, the animals were anesthetized with isoflurane and sacrificed via cervical dislocation. Liver and kidney tissues were harvested immediately, flash-frozen, and stored at 80 ^C. One kidney from each animal was fixed in 4% paraformaldehyde and embedded in paraffin for sectioning. Kidney sections were stained with hematoxylin and eosin for examination of oxalate crystals as shown in FIG.6A and FIG.6B.Docket No.: 94127.2.WOU1

[0089] The results demonstrated strong if not significant trends toward an increase in urinary oxalate levels (FIG.5) in all four treatment groups associated with significant increases in Ca- Oxalate kidney stones (FIGS. 6A-6C) in animals fed the 1% hyp diet relative to mice on a normal diet. These results are consistent with prior reports indicatingthat a 1% hydroxyproline diet exacerbates Ca-Oxalate kidney stones and hyperoxaluria. Example 4

[0090] To assess the effect of N-PPG on urinary oxalate levels, 4-month-old male WT and GRHPR-KO mice were fed a 1% hyp diet, ad libitum, and treated at 200mg / kg daily via oral gavage for 21 days (WT untreated, n=3; WT treated, n=4; GRHPR-KO untreated, n=4; GRHPR-KO treated, n=5).

[0091] Mice were singly housed in Techniplast mouse metabolic cages for collection of 8-hour urine samples on the following timepoints: Day 0 (before starting the diet), and Day 9(on the 1% hyp diet). Urine was immediately stored at 80°C and used later to determine oxalate concentration using a commercially available oxalate kit (EOXA-100 Oxalate Assay Kit, Bioassay Systems). After the completion of the study on Day 21, the animals were anesthetized with isoflurane and sacrificed via cervical dislocation. Liver and kidney tissues were harvested immediately, flash-frozen, and stored at 80°C. One kidney from each animal was fixed in 4% paraformaldehyde and embedded in paraffin for sectioning. Multiple adjacent kidney sections from each mouse were stained with hematoxylin and eosin (H&E) for examination (FIGS.6A and 6B) and enumeration of Ca-Oxalate crystals (FIG.6C).

[0092] The results demonstrated significant reductions in urinary oxalate levels (Day 9) in animals treated with N-PPG (FIG 5 p-values show significant differences between box plot comparisons); more impressively, this same experiment demonstrated complete suppression of total mean Ca-oxalate kidney stone counts across H&E sections (counting both larger and smaller crystals, FIG 6B and 6C) across all of the GRHPRO KO mice receiving 21 days of oral treatment with N-PPG (FIG 6C, with p-values showing significant total count distributions between bracketed mouse groups). Throughout the duration of the Example, urinary creatinine did not change between N-PPG treated WT and GRHPR KO mice, indicating that it does not affect overall renal function between all N-PPG comparison groups. These FIG. 5 results supported the hypothesis that short term (e.g. 9 days) treatment with N-PPG significantly reduces urinary oxalate secretion in WT and GRHPR KO mice on a 1% Hyp diet. The FIG. 6 results show support the hypothesis that a longer duration of oral N-PPG treatment (e.g. 21 days) can completely suppress GRHPRO KO mouse kidney stone formation, despite their intake of a 1% Hyp diet, down to baseline levels observed in the kidneys of VEH and N-PPGDocket No.: 94127.2.WOU1 treated WT mice receiving a 1% Hyp diet. The complete suppression of kidney stone formation after 3 weeks of oral N-PPG treatment relative to the significant but incomplete suppression of urinary oxalate section after only 9 days of oral N-PPG treatment could represent a treatment duration effect (e.g. more complete hepatic inhibition of oxalate production) or an additional impact on kidney tissue directly suppressing implantation and growth of Ca-oxalate crystals. Example 5

[0093] Comparisons between R and S stereoisomers of methyl-PPG were further assessed. The R and S stereoisomers of methyl-PPG were synthesized and tested in vivo and in vitro for their mitochondrial activities as suicide inhibitors of PRODH1 and PRODH2 (FIGS.7A, 7B, and 7C).

[0094] Livers isolated from mice treated orally for 5 days with N-PPG, S-methyl-PPG, or R- methyl-PPG stereoisomers were assessed. A western blot of the liver probed for PRODH1 and PRODH2 shows levels normalized to Rieske normalization for the Control, N-PPG, S-methyl- PPG (S isomer), and R-methyl-PPG (R isomer) (FIG. 7C). As shown by the results, the S- methyl-PPG stereoisomer is more potent than N-PPG at reducing PRODH1 / Rieske and PRODH2 / Rieske protein ratios, while the R-methyl-PPG stereoisomer was much less potent than N-PPG in its anti-PRODH mitochondrial effects (FIG.7C).

[0095] Further, mitochondria from in vitro cultured HepG2 (liver) cells treated twice (experiment #1, experiment #2) for 48 hours each with either 5 mM of N-PPG, S-methyl-PPG, or R-methyl-PPG stereoisomers were assessed. A western blot of the HepG2 mitochondria from both experiments probed for PRODH2 shows levels normalized to Rieske for Control, N- PPG, S-methyl-PPG (S isomer), and R-methyl-PPG (R isomer) treatment conditions (FIG.7A). The experiments were performed about 13 days apart. As shown by the results, the immortalized human liver cancer cells express PRODH2, but not PRODH1. Similar to the in vivo mouse liver study (FIG.7C), the S-methyl-PPG stereoisomer showed comparable ability to N-PPG at inhibiting and degrading mitochondrial PRODH2 (relative to Rieske), while the R-methyl-PPG stereoisomer showed much less potency in its anti-PRODH2 mitochondrial effects. Likewise, mitochondria from in vitro cultured ZR-75-1 (human breast cancer) cells treated in three different experiments (experiment #1, experiment #2, experiment #3) for 48 hours each with either 5 mM of N-PPG, S-methyl-PPG, or R-methyl-PPG stereoisomers were assessed. Western blots of the ZR-75-1 mitochondria from each experiment were probed for PRODH1 and signal levels normalized to Rieske (and / or mitochondrial aconitase, AOC) for Control, N-PPG, S-methyl-PPG (S isomer), and R-methyl-PPG (R isomer) treatment conditions (FIG. 7B). The three experiments were performed several weeks apart. As shownDocket No.: 94127.2.WOU1 by the results (FIG. 7B), the human breast cancer cells express PRODH1 but not PRODH2. Similar to the in vivo mouse liver study (FIG. 7C), the S-methyl-PPG stereoisomer showed comparable ability to N-PPG at inhibiting and degrading mitochondrial PRODH1, while the R-methyl-PPG stereoisomer showed much less potency in its anti-PRODH2 mitochondrial effects against the ZR-75-1 cells.

[0096] The disclosures being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the disclosures and all such modifications are intended to be included within the scope of the following claims.

Claims

Docket No.: 94127.2.WOU1 CLAIMS What is claimed is:

1. A method of preventing and / or treating a disorder mediated by HYPDH / PRODH2, PRODH1, or a combination thereof, comprising: administering to a subject in need thereof a therapeutic agent comprising N- propargylglycine (N-PPG) or a chemical analog thereof; and inhibiting HYPDH / PRODH2, PRODH1, or a combination thereof.

2. The method of claim 1, wherein the disorder comprises a primary hyperoxaluria (PH), a hyperoxaluria disorder, oxalosis, a disorder involving the production and renal excretion of excess levels of oxalate, a disorder involving the production of kidney stones, or a combination thereof.

3. The method of claim 1 or 2, wherein the administering prevents or reduces the occurrence of nephrocalcinosis, kidney stone formation, bladder stone formation, renal failure, or a combination thereof.

4. The method of any one of claims 1-3, wherein the N-PPG or chemical analog thereof provides target specificity to PRODH2 over PRODH1.

5. The method of claim 1, wherein the disorder comprises a neurodegenerative disorder or cancer, and wherein the N-PPG or chemical analog inhibits a combination of HYPDH / PRODH2 and PRODH1.

6. The method of any one of claims 1-5, wherein the chemical analog thereof comprises 2-[(but-3-yn-2-yl)amino]acetic acid having the following formula: .

7. The method of claim 5 or 6, wherein the chemical analog thereof comprises the following formula:Docket No.: 94127.2.WOU1 .

8. any one claims 1-7, wherein 4-hydroxyproline (Hyp) catabolism is suppressed.

9. A method of targeting PRODH2, comprising: administering to a subject a therapeutic agent comprising N-propargylglycine (N-PPG) or a chemical analog thereof.

10. The method of claim 9, wherein PRODH2 is targeted in addition to PRODH1.

11. The method of claim 9, wherein the chemical analog provides target specificity to PRODH2 over PRODH1.

12. The method of any one of claims 9-11, wherein the chemical analog thereof comprises the following formula: .

13. The method of any one of claims 9-12, wherein the therapeutic agent prevents or treats a primary hyperoxaluria (PH), a hyperoxaluria disorder, oxalosis, a disorder involving the production and renal excretion of excess levels of oxalate, a disorder involving the production of kidney stones, or a combination thereof.

14. A pharmaceutical composition for use in preventing and / or treating a disorder mediated by HYPDH / PRODH2, PRODH1, or a combination thereof, comprising: a therapeutic agent comprising N-propargylglycine (N-PPG) or a chemical analog thereof; andDocket No.: 94127.2.WOU1 a pharmaceutically effective carrier.

15. The pharmaceutical composition of claim 14, wherein the N-PPG or chemical analog provides target specificity to both PRODH2 and PRODH1.

16. The pharmaceutical composition of claim 14 or 15, wherein the chemical analog thereof comprises 2-[(but-3-yn-2-yl)amino]acetic acid having the following formula: .

17. The pharmaceutical composition of any one of claims 14-16, wherein the composition prevents or treats a primary hyperoxaluria (PH), a hyperoxaluria disorder, oxalosis, a disorder involving the production and renal excretion of excess levels of oxalate, a disorder involving the production of kidney stones, or a combination thereof.

18. The pharmaceutical composition of claim 14, wherein the composition prevents or treats a disorder comprising a neurodegenerative disorder or cancer, and wherein the N-PPG or chemical analog inhibits a combination of HYPDH / PRODH2 and PRODH1.

19. The pharmaceutical composition of claim 18, wherein the chemical analog thereof comprises the following formula: .

20. The pharmaceutical composition of any one of claims 14-19, wherein the composition is provided as a powder and dissolved in saline.

21. The pharmaceutical composition of any one of claims 14-21, wherein the composition is provided at a dose of between about 20 mg / kg to about 300 mg / kg.

Images