Castration-resistant prostate cancer

Abstract

This invention relates to inhibitors of UDP-glucose dehydrogenase, and more particularly to UDP-glucose dehydrogenase inhibitors that are useful in the treatment of prostate cancer. Methods of inhibiting UDP-glucose dehydrogenase and improving the efficacy of additional prostate cancer therapies are also provided.

Claims

1. A method of treating prostate cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a compound selected from the group consisting of: ##STR00271## ##STR00272## ##STR00273## ##STR00274## ##STR00275## ##STR00276## ##STR00277## or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the compound is administered in combination with one or more additional therapies comprising administration of a chemotherapeutic agent, androgen deprivation therapy, or a combination thereof.

3. The method of claim 1, wherein the compound is selected from the group consisting of 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof, and 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein the prostate cancer is mediated by UDP-glucose dehydrogenase (UGDH).

5. The method of claim 4, wherein the compound is selected from the group consisting of 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof, and 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

6. The method of claim 1, wherein the prostate cancer is castration resistant prostate cancer (CRPC).

7. The method of claim 1, wherein the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof.

8. The method of claim 1, wherein the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

9. The method of claim 2, wherein the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof.

10. The method of claim 2, wherein the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

11. The method of claim 4, wherein the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof.

12. The method of claim 4, wherein the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

13. A method of treating castration resistant prostate cancer (CRPC), comprising administering to a patient in need thereof a therapeutically effective amount a compound which is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof.

14. The method of claim 13, wherein the compound is administered in combination with one or more additional therapies comprising administration of a chemotherapeutic agent, androgen deprivation therapy, or a combination thereof.

15. A method of treating castration resistant prostate cancer (CRPC), comprising administering to a patient in need thereof a therapeutically effective amount a compound which is 345-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

16. The method of claim 15, wherein the compound is administered in combination with one or more additional therapies comprising administration of a chemotherapeutic agent, androgen deprivation therapy, or a combination thereof.

17. A method of treating castration resistant prostate cancer (CRPC) mediated by UDP-glucose dehydrogenase (UGDH), comprising administering to a patient in need thereof a therapeutically effective amount a compound selected from the group consisting of 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof, and 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein the compound is administered in combination with one or more additional therapies comprising administration of a chemotherapeutic agent, androgen deprivation therapy, or a combination thereof.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a Gantt chart providing representative timing of the assay of Example 1.

(2) FIG. 2 is a diagram showing a mechanism wherein UGDH provides precursors for androgen inactivation by UGT-mediated glucuronidation.

(3) FIGS. 3A-3C shows results of a simulated androgen deprivation therapy assay. For FIG. 3A: Left Bar=C33; Right Bar=C81. For FIG. 3B: Left Bar=3 days; Right Bar=15 days. For FIG. 3C: Left Bar=Basal; Right Bar=DHT 24 h.

(4) FIG. 4. shows modulation of function of AR using a luciferase reporter assay driven by the AR-stimulated PSA promoter/enhancer region.

(5) FIG. 5A is a diagram showing a mechanism wherein UGDH loss of activity may allow cells to sustain sensitivity to androgen deprivation.

(6) FIG. 5B shows data wherein UGDH knockdown lowers steroid dose required for AR gene expression.

(7) FIGS. 6A-6B shows a mixed-model inhibition fit for UDP-xylose used for determination of K.sub.i.

(8) FIG. 7A-7E show kinetic characterization of inhibitors (1) (i.e. 5210344) and (31) (i.e., 6847944).

(9) FIGS. 8A-8D show UDP-xylose effects on the thermal stability of WT, T325A (inducible hexamer), and T325D (obligate dimer) UGDH.

(10) FIGS. 9A-9D shows inhibitor (1) effects on the thermal stability of WT, T325A (inducible hexamer), and T325D (obligate dimer) UGDH.

(11) FIG. 10A-10C shows inhibitor (31) effects on the thermal stability of WT, T325A (inducible hexamer), and T325D (obligate dimer) UGDH.

(12) FIG. 11 shows UDP-xylose stabilizing UGDH T325A and T325D mutants against limited trypsin proteolysis.

(13) FIG. 12 illustrates the effects of inhibitor (1) and (31) on trypsin digestion of WT and mutant UGDH.

DETAILED DESCRIPTION

(14) UDP-glucose dehydrogenase (UGDH) catalyzes the NAD.sup.+-dependent, two-step oxidation of UDP-glucose to UDP-glucuronic acid, an essential precursor for hyaluronan synthesis by HAS enzymes, other glycosaminoglycan/proteoglycan production in the Golgi, and glucuronidation of steroid hormones by UGTs for solubilization and excretion (see, e.g., Prydz et al., J. Cell Sci. 2000, 113, 193-205; Fraser et al., J. Intern. Med. 1997, 242, 27-33; Guillemette C., Pharmacogenomics J. 2003, 3, 136-158; and King et al., Toxicol. Sci. 2001, 61, 49-53). High levels of UGDH expression are specific to the liver and prostate in males, and prostate tumor progression has been correlated with a loss of UGDH regulation. Accordingly, the present application provides inhibitors of UGDH that are useful in the treatment of prostate cancer and methods for predicting the efficacy of androgen deprivation therapy.

Definitions

(15) For the terms “for example” and “such as” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. As used herein, the term “about” is meant to account for variations due to experimental error. All measurements reported herein are understood to be modified by the term “about”, whether or not the term is explicitly used, unless explicitly stated otherwise. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

(16) A “therapeutically effective amount” of a conjugate with respect to the subject method of treatment, refers to an amount of the conjugate(s) in a preparation which, when administered as part of a desired dosage regimen (to a patient, e.g., a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.

(17) As used herein, the term “treating” or “treatment” includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in manner to improve or stabilize a patient's condition.

(18) Compounds and Pharmaceutical Compositions

(19) The present application provides, inter alia, compounds that are useful as UDP-glucose dehydrogenase (UGDH) inhibitors. In some embodiments, the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1) (i.e., compound (1) or inhibitor (1)) is also referred to as inhibitor 5210344. In some embodiments, the compound 3-[5-(2-thienyl)-2-furyl]propanoic acid (31) (i.e. compound (31) or inhibitor (31)) is also referred to as inhibitor 6847944.

(20) In some embodiments, a compound is selected from the group provided in Table 1.

(21) TABLE-US-00001 TABLE 1 List of Compounds Compound # Name Structure 1 2,2′-[1-(4-amino-1,2,5- oxadiazol-3- yl)-1H-1,2,3- triazole-4,5-diyl]di(2- butanol) 2 2-[1-(4-amino-1,2,5- oxadiazol-3-yl)-5-(2- hydroxypropan-2-yl)- 1,2,3-triazol- 4-yl]propan- 2-ol 3 1-[1-(4-amino-1,2,5- oxadiazol-3- yl)-5-methyl- 1,2,3-triazol- 4-yl]ethanol 4 [3-(4-amino-1,2,5- oxadiazol-3-yl)-1,2,3- triazol-4-yl]methanol 5 [1-(4-amino-1,2,5- oxadiazol-3- yl)-1H-1,2,3- triazol-4-yl]methanol 6 1-[1-(4-amino-1,2,5- oxadiazol-3-yl)-1,2,3- triazol-4-yl]ethanol 7 2-[1-(4-amino-1,2,5- oxadiazol-3-yl)-1,2,3- triazol-4-yl] propan-2-ol 8 4-(5-ethyl-1,2,3- triazol-1-yl)- 1,2,5-oxadiazol-3- amine 9 methyl 1-(4- amino-1,2,5- oxadiazol-3-yl)-5-tert- butyl-1,2,3-triazole-4- carboxylate 10 methyl 1-(4- amino-1,2,5- oxadiazol-3- yl)-5-ethyl- 1,2,3-triazole-4- carboxylate 0 11 ethyl 1-(4- amino-1,2,5- oxadiazol-3- yl)-5-ethyl- 1,2,3-triazole-4- carboxylate 12 1-(4-amino-1,2,5- oxadiazol-3- yl)-5-propyl- 1,2,3-triazole-4- carboxylic acid 13 tert-butyl 1-(4-amino- 1,2,5-oxadiazol- 3-yl)-5- methyl-1,2,3- triazole-4- carboxylate 14 isopropyl 1-(4-amino- 1,2,5-oxadiazol- 3-yl)-5- methyl-1,2,3- triazole-4- carboxylate 15 1-(4-amino-1,2,5- oxadiazol-3-yl)-5- (methoxymethyl)-1H- 1,2,3-triazole-4- carboxylic acid 16 methyl 1-(4- amino-1,2,5- oxadiazol-3-yl)-5- (methoxymethyl)-1H- 1,2,3-triazole-4- carboxylate 17 1-(4-amino-1,2,5- oxadiazol-3-yl)-5- [(dimethylamino) methyl]- 1,2,3-triazole-4- carboxylic acid 18 1-(4-amino-1,2,5- oxadiazol-3- yl)-5-phenyl- 1,2,3-triazole-4- carboxylic acid 19 prop-2-en-1-yl 1-(4- amino-1,2,5- oxadiazol- yl)-5-methyl- 1H-1,2,3- triazole-4- carboxylate 20 2-methoxyethyl 1-(4- amino-1,2,5- oxadiazol-3- yl)-5-methyl-1,2,3- triazole-4-carboxylate 0 21 ethyl 5- (adamantan-1-yl)- 1-(4-amino-1,2,5- oxadiazol-3- yl)-1H-1,2,3- triazole-4-carboxylate 22 (2R,2′S)-2,2′-(1-(4- amino-1,2,5- oxadiazol-3- yl)-1H-1,2,3- triazole-4,5- diyl)bis(butan-2-ol) 23 (S)-1-(1-(4- amino-1,2,5- oxadiazol-3- yl)-5-methyl- 1H-1,2,3-triazol-4- yl)ethanol 24 2-[1-(4- amino-1,2,5- oxadiazol-3- yl)-1H-1,2,3- triazol-5-yl] propan-2-ol 25 [3-(4-amino-1,2,5- oxadiazol-3-yl)-5- (hydroxymethyl)- 1,2,3-triazol- 4-yl]methanol 26 1-[3-(4-amino-1,2,5- oxadiazol- 3-yl)-1,2,3- triazol-4-yl]ethanol 27 1-(4-amino-1,2,5- oxadiazol-3- yl)-5-tert- butyl-1,2,3- triazole-4- carboxylic acid 28 1-(4-amino-1,2,5- oxadiazol-3-yl)-5- (propan-2-yl)- 1H-1,2,3- triazole-4- carboxylic acid 29 methyl 1-(4- amino-1,2,5- oxadiazol-3-yl)-5- isopropyl-1H-1,2,3- triazole-4- carboxylate 30 1-(4-amino-1,2,5- oxadiazol-3-yl)-5-(4- methylphenyl)-1,2,3- triazole-4- carboxylic acid 0 31 3-[5-(2-thienyl)-2- furyl]propanoic acid 32 N-methyl-N- phenylglycine hydrochloride 33 3-(5-ethyl-2- thienyl)acrylic acid 34 2-[(carboxymethyl) thio]-3-methyl-1,3- benzothiazol-3-ium bromide 35 3-[5-(4- fluorophenyl)-2- furyl]acrylic acid 36 3-phenyl-1,3- thiazolidine-2- carboxylic acid 37 3-(5-phenyl-2- furyl)propanoic acid 38 3-[5-(4- fluorophenyl)-2- furyl]propanoic acid 39 (E)-3-(5- phenylfuran-2- yl)prop-2-enoic acid 40 3-(5-phenyl- 1,3-oxazol-2- yl)propanoic acid 0 41 3-[5-(4-chlorophenyl)- 1,3-oxazol-2- yl]propanoic acid 42 3-(5-phenyl-1,3,4- oxadiazol-2-yl) propanoic acid 43 2-[(4-phenyl- 1,3-thiazol- 2-yl)sulfanyl] acetic acid 44 3-[5-(4- bromophenyl)- 1,3-oxazol-2- yl]propanoic acid 45 3-(5-thiophen- 2-yl-1H- pyrrol-2-yl) propanoic acid 46 3-(5-phenyl-3,4- dihydropyrazol-2- yl)propanoic acid 47 (E)-3-[5-(2- chlorophenyl) furan-2- yl]prop-2- enoic acid 48 2-[(E)-2- nitroethenyl]-5- phenylfuran 49 3-(6-oxo-3- phenylpyridazin-1- yl)propanoic acid 50 3-(3-phenyl-1,2,4- oxadiazol-5-yl) propanoic acid 0 51 2-quinolin-2- ylsulfanylacetate 52 2-[(5-phenyl-1,3,4- oxadiazol-2- yl)sulfanyl] acetic acid 53 3-(5-phenyl- 1H-pyrrol-2- yl)propanoic acid 54 3-[3-(4- chlorophenyl)-6- oxopyridazin-1- yl]propanoic acid 55 3-[3-(4- fluorophenyl)-6- oxopyridazin-1- yl]propanoic acid 56 2-[3-(tetrazol-1- yl)phenoxy]acetic acid 57 (E)-3-[5-(2- bromophenyl)furan-2- yl]prop-2-enoic acid 58 4-phenyl- methoxybutanoic acid 59 3-[3-(4- methylphenyl)-6- oxopyridazin-1- yl]propanoic acid 60 3-[3-(2-fluorophenyl)- 1,2,4-oxadiazol-5- yl]propanoic acid 0 61 6-nitro-2- phenylindazole 62 2-[1-(2,2,2- trifluoroethyl) pyrazol-3- yl]acetic acid 63 3-(5-thiophen- 2-ylfuran- 2-yl)propanoate 64 3-(5-thiophen- 2-ylfuran- 2-yl)prop-2- enoic acid 65 methyl 3- (5-thiophen-2- ylfuran-2-yl) propanoate 66 (E)-3-(5-thiophen-2- ylfuran-2-yl) prop-2-enoic acid 67 5-(5-methylfuran-2- yl)thiophene- 2-carboxylic acid 68 2-(5-thiophen- 2-ylfuran- 2-yl)acetic acid 69 methyl 3- (5-thiophen-2- ylfuran-2-yl)prop-2- enoate 70 thiophen-2- ylmethyl 3- (furan-2-yl) propanoate 0 71 72 73 74 75 76 77 78 79 80 0 81 82 83 84 85 86 87 88 89 90 0 91 92 93 94 95 96 97 98 99 100 00 101 01 102 02 103 03 104 04 105 05 106 06 107 07 108 08 109 09 110 0 111 112 113 114 115 116 117 118 119 120 0 121 122 123 124 125 126 127 128 129 130 0 131 132 133 134 135 136 137 138 139 140 0 141 142 143 144 145 146 147 148 149 150 0 151 152 153 154 155 156 157 158 159 160 0 161 162 163 164 165 166 167 168 169 170 0

(22) The present application further provides a pharmaceutical composition comprising a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

(23) When employed as pharmaceuticals, the compounds provided herein can be administered in the form of pharmaceutical compositions; thus, the methods described herein can include administering pharmaceutical compositions provided herein.

(24) These compositions can be prepared as described herein or elsewhere, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral, or parenteral. Parenteral administration may include, but is not limited to intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; or intracranial, (e.g., intrathecal, intraocular, or intraventricular) administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

(25) In making the compositions provided herein, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

(26) Some examples of suitable excipients include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include, without limitation, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; flavoring agents, or combinations thereof.

(27) The active compounds can be effective over a wide dosage range and are generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered and the schedule of administration will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

(28) Methods of Use and Combination Therapies

(29) The present application further provides methods of treating prostate cancer in a patient in need thereof. As used herein, the term “patient” refers to any animal, including mammals, for example, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the patient is a human. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

(30) In some embodiments, the prostate cancer comprises a cancer selected from the group consisting of acinar adenocarcinoma, atropic adenocarcinoma, foamy adenocarcinoma, colloid adenocarcinoma, signet ring carcinoma, ductal adenocarcinoma transitional cell (or urothelial) cancer, squamous cell cancer, carcinoid, small cell cancer, sarcoma cancer, sarcomatoid cancer, and castration resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is castration resistant prostate cancer (CRPC).

(31) In some embodiments, a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof is administered in combination with one or more additional therapies. In some embodiments, at least one of the one or more additional therapies is selected from the group consisting of administration of a chemotherapeutic agent, radiation therapy, a surgical procedure, androgen deprivation therapy, or any combination thereof. In some embodiments, at least one of the one or more additional therapies comprises administration of at least one chemotherapeutic agent. In some embodiments, at least one of the one or more additional therapies comprises androgen deprivation therapy. In some embodiments, at least one of the one or more additional therapies is androgen deprivation therapy. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, is administered prior to the one or more additional therapies. In some embodiments, a compound provided herein, or pharmaceutically acceptable salt thereof, is administered concurrently with the one or more additional therapies. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, is administered after the one or more additional therapies.

(32) In some embodiments, the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof, and at least one of the one or more additional therapies is androgen deprivation therapy. In some embodiments, the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof, and at least one of the one or more additional therapies is androgen deprivation therapy.

(33) In some embodiments, the method comprises:

(34) i) administering to the patient a therapeutically effective amount of 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof; and

(35) ii) administering androgen deprivation therapy.

(36) In some embodiments, the method comprises:

(37) i) administering to the patient a therapeutically effective amount of 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof; and

(38) ii) administering androgen deprivation therapy.

(39) The present application further provides a method of modulating an activity of UDP-glucose dehydrogenase (UGDH) in a cell, the method comprising contacting the cell with an effective amount of a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof. In some embodiments, the modulating an activity of UDP-glucose dehydrogenase (UGDH) comprises inhibiting UDP-glucose dehydrogenase (UGDH). In some embodiments, the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

(40) The present application further provides a method of treating a prostate cancer mediated by UDP-glucose dehydrogenase (UGDH) in a patient in need thereof, the method comprising administering a therapeutically effective amount of a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof.

(41) In some embodiments, the prostate cancer comprises a cancer selected from the group consisting of acinar adenocarcinoma, atropic adenocarcinoma, foamy adenocarcinoma, colloid adenocarcinoma, signet ring carcinoma, ductal adenocarcinoma transitional cell (or urothelial) cancer, squamous cell cancer, carcinoid, small cell cancer, sarcoma cancer, sarcomatoid cancer, and castration resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is castration resistant prostate cancer (CRPC).

(42) In some embodiments, the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

(43) The present application further provides a method of predicting patient response to prostate cancer therapy, comprising:

(44) a) obtaining a biopsy sample from the patient, wherein the biopsy sample comprises prostate cancer cells and non-cancerous tissue cells; and

(45) b) comparing the UDP-glucose dehydrogenase (UGDH) expression in the prostate cancer cells and the non-cancerous tissue cells;

(46) wherein when the UDP-glucose dehydrogenase (UGDH) expression is greater in the prostate cancer cells compared to the UDP-glucose dehydrogenase (UGDH) expression in the non-cancerous tissue cells, then the patient is more likely to respond to the prostate cancer therapy.

(47) In some embodiments, the prostate cancer cells comprise a prostate cancer selected from the group consisting of acinar adenocarcinoma cells, atropic adenocarcinoma cells, foamy adenocarcinoma cells, colloid adenocarcinoma cells, signet ring carcinoma cells, ductal adenocarcinoma transitional cell (or urothelial) cancer cells, squamous cell cancer cells, carcinoid cells, small cell cancer cells, sarcoma cancer cells, sarcomatoid cancer cells, and castration resistant prostate cancer (CRPC) cells. In some embodiments, the prostate cancer cells comprise castration resistant prostate cancer (CRPC) cells. In some embodiments, the prostate cancer is castration resistant prostate cancer (CRPC).

(48) As used herein, the term “non-cancerous tissue cells” refers to non-cancerous tissue cells in the area surrounding the prostate cancer cells. For example, non-cancerous tissue cells may refer to non-cancerous prostate tissue cells, non-cancerous acini, and normal-appearing acini (NAA). In some embodiments, the non-cancerous or normal-appearing acini are selected from the group consisting of acini of the stomach, acini of the sebaceous gland of the scalp, acini of the liver, acini of the lung, acini of the lacrimal gland, acini of mammary gland, acini of the pancreas, and acini of the prostate. In some embodiments, the non-cancerous tissue cells comprise prostate tissue cells. In some embodiments, the non-cancerous tissue cells are prostate tissue cells. In some embodiments, the non-cancerous tissue cells comprise non-cancerous acini or normal-appearing acini (NAA). In some embodiments, the non-cancerous cells comprise normal-appearing acini. Examples of normal-appearing acini that may be used in the method provided herein may be found, for example, in Huang et al., Int. J. Cancer, 2010, 126(5), 315-327, the disclosure of which is incorporated herein in its entirety.

(49) In some embodiments, the comparing comprises determining the ratio of UDP-glucose dehydrogenase (UGDH) expression in the prostate cancer cells and UDP-glucose dehydrogenase (UGDH) expression in the non-cancerous tissue cells. In some embodiments, the comparing comprises:

(50) a) immunofluorescence staining of the biopsy sample; and

(51) b) quantifying the fluorescence pixel intensity of acini within the prostate cancer cells and the non-cancerous tissue cells.

(52) In some embodiments, the quantifying comprises determining the average mean pixel intensity acini within the prostate cancer cells and acini within the non-cancerous tissue cells (e.g., non-cancerous acini or normal-appearing acini (NAA)).

(53) In some embodiments, the patient is more likely to respond to prostate cancer therapy when the average mean pixel intensity of acini within the prostate cancer cells is at least about 10% greater than the average mean pixel intensity of acini within the non-cancerous tissue cells (e.g., non-cancerous acini or normal-appearing acini (NAA)), for example, at least about 10%, at least about 15% at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, the patient is more likely to respond to prostate cancer therapy when the average mean pixel intensity of acini within the prostate cancer cells is at least about 15% greater than the average mean pixel intensity of acini within the non-cancerous tissue cells (e.g., non-cancerous acini or normal-appearing acini (NAA)).

(54) In some embodiments, the prostate cancer therapy is selected from the group consisting of administration of a chemotherapeutic agent, radiation therapy, a surgical procedure, androgen deprivation therapy, or any combination thereof. In some embodiments, a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof, is administered prior to the prostate cancer therapy. In some embodiments, a compound provided herein, or pharmaceutically acceptable salt thereof, is administered concurrently with the prostate cancer therapy. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, is administered after the prostate cancer therapy. In some embodiments, the prostate cancer therapy comprises administration of at least one chemotherapeutic agent. In some embodiments, the prostate cancer therapy comprises androgen deprivation therapy. In some embodiments, the prostate cancer therapy is androgen deprivation therapy.

(55) The present application further provides a method of treating a prostate cancer mediated by UDP-glucose dehydrogenase (UGDH) in a patient in need thereof, the method comprising:

(56) a) obtaining a biopsy sample from the patient, wherein the biopsy sample comprises prostate cancer cells and non-cancerous tissue cells;

(57) b) comparing the UDP-glucose dehydrogenase (UGDH) expression in the prostate cancer cells and the non-cancerous tissue cells; and

(58) c) if the prostate cancer is determined to be associated with one or more of overexpression and amplification of UDP-glucose dehydrogenase (UGDH) in the prostate cancer cells compared to the non-cancerous tissue cells, administering a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof.

(59) In some embodiments, the prostate cancer is determined to be associated with one or more of overexpression and amplification of UDP-glucose dehydrogenase (UGDH) in the prostate cancer cells compared to the non-cancerous tissue cells when the average mean pixel intensity of acini within the prostate cancer cells is at least about 10% greater than the average mean pixel intensity of acini within the non-cancerous tissue cells (e.g., non-cancerous acini or normal-appearing acini (NAA)), for example, at least about 10%, at least about 15% at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, the patient is more likely to respond to prostate cancer therapy when the average mean pixel intensity of acini within the prostate cancer cells is at least about 15% greater than the average mean pixel intensity of acini within the non-cancerous tissue cells (e.g., non-cancerous acini or normal-appearing acini (NAA)).

(60) In some embodiments, the prostate cancer is determined to be associated with one or more of overexpression and amplification of UDP-glucose dehydrogenase (UGDH) in the prostate cancer cells compared to the non-cancerous tissue cells when the expression of UGDH within the prostate cancer cells is at least about 10% greater than the expression of UGDH within the non-cancerous tissue cells (e.g., non-cancerous acini or normal-appearing acini (NAA)), for example, at least about 10%, at least about 15% at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, the patient is more likely to respond to prostate cancer therapy when the expression of UGDH is within the prostate cancer cells is at least about 15% greater than the expression of UGDH within the non-cancerous tissue cells (e.g., non-cancerous acini or normal-appearing acini (NAA)).

(61) In some embodiments, the method further comprises administration of one or more additional therapies. In some embodiments, at least one of the one or more additional therapies is selected from the group consisting of administration of a chemotherapeutic agent, radiation therapy, a surgical procedure, androgen deprivation therapy, or any combination thereof. In some embodiments, at least one of the one or more additional therapies administering at least one chemotherapeutic agent. In some embodiments, at least one of the one or more additional therapies comprises androgen deprivation therapy. In some embodiments, at least one of the one or more additional therapies is androgen deprivation therapy. In some embodiments, a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof, is administered prior to the one or more additional therapies. In some embodiments, a compound provided herein, or pharmaceutically acceptable salt thereof, is administered concurrently with the one or more additional therapies. In some embodiments, a compound provided herein, or a pharmaceutically acceptable salt thereof, is administered after the one or more additional therapies.

(62) In some embodiments, the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

(63) The present application further provides a method of improving the efficacy of androgen deprivation therapy in a patient, comprising administering to the patient a therapeutically effective amount of a UDP-glucose dehydrogenase (UGDH) inhibitor.

(64) In some embodiments, the prostate cancer comprises a cancer selected from the group consisting of acinar adenocarcinoma, atropic adenocarcinoma, foamy adenocarcinoma, colloid adenocarcinoma, signet ring carcinoma, ductal adenocarcinoma transitional cell (or urothelial) cancer, squamous cell cancer, carcinoid, small cell cancer, sarcoma cancer, sarcomatoid cancer, and castration resistant prostate cancer (CRPC). In some embodiments, the prostate cancer is castration resistant prostate cancer (CRPC).

(65) In some embodiments, the UDP-glucose dehydrogenase (UGDH) inhibitor is selected from a compound provided herein (e.g., a compound provided in Table 1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 2,2′-[1-(4-amino-1,2,5-oxadiazol-3-yl)-1H-1,2,3-triazole-4,5-diyl]di(2-butanol) (1), or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is 3-[5-(2-thienyl)-2-furyl]propanoic acid (31), or a pharmaceutically acceptable salt thereof.

EXAMPLES

(66) The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.

Example 1

High Throughput Screening for Inhibitors of UDP-Glucose Dehydrogenase

(67) i. Reagents

(68) TABLE-US-00002 UDP-glucose Dehydrogenase Stock 2.5 mg/ml Stored at −80°, as aliquots of 2 mL, don't refreeze Storage buffer: 20 mM Tris pH 7.4, 1 mM DTT NAD.sup.+ (MW = 663.4), Sigma N1511 Make 50 mM stock in PBS fresh each day. Do not store overnight, even if frozen. UDP-glucose (MW = 610), Sigma U4625 Stock 100 mM in water Stored at −20°. Do not refreeze. BSA, Meso Scale Discovery, Blocker A, catalog# R93BA-1 Stock 5.6% in PBS (W/V). Alliquot and store at −20° UDP-xylose, lot VX0707 (MW = 554.3), supplied by CaraboSource Services, CCRC/University of Georgia Stock 1 mM in PBS Stored at −20°, as aliquots of 1 ml. Don't re-freeze DMSO (MW = 78.1), Fisher D136-1 PBS, pH 7.4, Sigma #P3813 Dissolve in 1 L water and store at room temp. NADH (MW = 742), Sigma N4505 Stock 10 mM in PBS Store at −20° as aliquots of 100 μl. Do not re-freeze.
ii. Cocktails
Enzyme Buffer 2.8×

(69) 14 μg/mL enzyme;

(70) 0.56% BSA; and

(71) In PBS, pH 7.4.

(72) Preparation of 1 liter: Prepare enzyme in polypropylene. Do not let enzyme touch glass or polystyrene. Because the enzyme is the least stable of all reagents used in the assay, it should be prepared last. Enzyme buffer should be kept cold if any time passes before use. 1. 900 mL PBS; 2. 100 mL 5.6% BSA stock; and 3. 5.6 mL of 2500 μg/mL enzyme stock
Substrate Solution 2.8×

(73) 700 μM NAD+;

(74) 92.4 μM UDP-glucose; and

(75) In PBS, pH 7.4.

(76) Preparation of 1 L: 1. 1 liter PBS; 2. 14 mL of 50 mM NAD+ stock; and 3. 9.24 mL of 10 mM UDP-glucose stock or 924 μL of 100 mM UDP-glucose stock.
Positive Control Solution 3.5×

(77) 70 μM UDP-xylose;

(78) 2.5% DMSO; and

(79) In PBS, pH 7.4

(80) Preparation of 100 mL: 1. 97.5 mL PBS; 2. 2.5 mL DMSO; and 3. 7 mL 1 mM UDP-xylose stock
Negative Control Solution 3.5×

(81) 2.5% DMSO; and

(82) In PBS, pH 7.4.

(83) Preparation of 100 mL: 1. 97.5 mL PBS 2. 2.5 mL DMSO
Instruments and Lab-Equipment 16-channel Finnpipette 5-50 μl 2 Thermo Scientific Multidrop 384 & cassettes. Tecan Safire.sup.2. Microtiterplates: Greiner FLUOTRAC 200
iii. Methodology

(84) 1. Preparation of the Compound-Plates: Add Positive & Negative Controls Plates are stored at −20° with 20 μL/well of 25 μg/mL compound in 2.5% DMSO in water. a. Dispense, with the 16channel Finnpipette, 20 μL of the negative control solution (2.5% DMSO) in column 1. b. Dispense, with the 16channel Finnpipette, 20 μL of the positive control solution (2.5% DMSO & 70 μM UDP-glucose) in column 2.

(85) 2. First Addition: Add Enzyme to Compounds a. Dispense, with the Multidrop 384, 25 μL/well Enzyme solution. b. Incubate for 5 minutes at room temperature. Incubation starts as soon as the Multidrop Micro starts adding Enzyme Solution. Incubation has ended when the Multidrop 384 starts adding Substrate solution. So the next step is done while the plates are incubating.

(86) 3. First Read: Measure Baseline (Compound) Fluorescence.

(87) 4. Second Addition: Add Substrates to Enzyme & Compounds a. Dispense, with the Multidrop 384, 25 μL/well Substrate solution. b. Incubate again for 30 minutes at RT. Again, incubation starts as soon as the Multidrop Micro starts adding Substrate Solution. However, incubation has ended when the Safire.sup.2 starts reading the plate. Keep plates stacked with an empty plate on top of the stack to prevent photobleaching and evaporation.

(88) 5. Second Read: Measure NADH Levels

(89) 6. Standard Curves: In a separate plate, prepare an 11-point, 2× serial dilution of NADH in 0.2% BSA in PBS. Leave one “no NADH” point. Use BSA from the same stock used to make the Enzyme Solution. Start the serial dilution at 100 μM NADH. Dispense 70 μL/well in quadruplicate

(90) Plates can be run in batches up to 25. FIG. 1. provides a gantt chart which shows how the assay timing would work.

(91) iv. Data-Analysis

(92) All data is converted to NADH concentration using the standard curve generated each day. All data is converted to: [second read−the first read]. Because NADH is generated in the reaction, an increase in NADH signals enzyme activity. Percent of Controls (POC) expresses the activity of a compound relative to the positive and negative controls (Equation 1).

(93) $\begin{array}{cc}\%\mathrm{of}\mathrm{controls}=\frac{\left(C_{\mathrm{CMPD}}-C_{\mathrm{NEG}}\right)}{\left(C_{\mathrm{POS}}-C_{\mathrm{NEG}}\right)}.& \mathrm{Equation}1\end{array}$ C.sub.CMPD=NADH concentration measured in a well containing compound C.sub.NEG=NADH concentration measured in a negative control well (DMSO only) C.sub.POS=NADH concentration measured in a positive control well (UDP-xylose)

(94) Note that the positive control wells (with UDP-xylose) will have lower value (less NADH) than will negative control wells. So the denominator in this equation will be a negative number. An active (inhibitory) compound will also result in a lower value, so the numerator will also be negative. Thus, the higher the POC, the more inhibitory the compound is (see e.g., Table 2).

(95) TABLE-US-00003 TABLE 2 POC Values POC Value Compound Effect <0% Test compound speeds up the reaction =0% Test compound is not active >0% Test compound is inhibitory =100%  Test compound is as inhibitory as is 20 μM UDP-glucose >100%  Test compound is more inhibitory than is 20 μM UDP-glucose

(96) Table 3 shows POC values for compounds screened in the high throughput assay.

(97) TABLE-US-00004 TABLE 3 Compound Data Compound Structure POC  61%  73%  86%  46%  83%  81% 106% 101%  59% 0  74%  47%  80%  88% 148%  54%  62% 126%  72%  50% 0  88%  46%  62% 105%  61%  63%  86% 103%  65%  61% 00  75% 01  63% 02  56% 03  55% 04  71% 05  66% 06  55% 07  51% 08  51% 09  58% 0  50% 103%  74%  46%  65%  50%  52%  58%  70%  56% 0  66%  46%  57%  46%  57%  62%  82%  87%  49%  57% 0  51%  47%  46%  58%  47%  85%  68%  47%  45%  67% 0  46%  45%  47%  58%  45%  50%  48%  51%  73%  73% 0  59%  83%  67%  83%  76%  76%  71%  73%  98%  98% 0  99%  53%  97%  58%  61%  73%  91%  96%  69%  79% 0  66%

Example 2

Androgen Deprivation Model and UGDH Knockdown

(98) FIG. 2 shows a diagram of UGDH providing precursors for androgen inactivation by UGT-mediated glucuronidation. To model androgen deprivation therapy in cell culture, cells were treated for two weeks in the absence or presence of androgen decrements. Cells were then treated with 10 nM DHT and analyzed by western blot for UGDH, PSA and AR, as shown in FIG. 3. AR promoter binding was modulated, so we further confirmed modulation of the AR using a luciferase reporter assay driven by the AR-stimulated PSA promoter/enhancer region, as shown in FIG. 4. FIG. 5A-5B shows that a loss of activity of UGDH may allow cells to sustain sensitivity to androgen deprivation.

Example 3

UGDH Kinetic Characterizations

(99) Kinetic parameters of WT and mutant UGDH are shown below in Table 4. The K.sub.m and V.sub.max for the substrate (UDP-glucose) and cofactor (NAD+) was determined by a nonlinear regression fit of initial velocity vs. substrate/cofactor concentration. T325A and T325D are engineered inducible hexameric and obligate dimeric UGDH species, respectively.

(100) TABLE-US-00005 TABLE 4 Kinetic parameters of WT and mutant UGDH UDP-glucose NAD+ K.sub.m V.sub.MAX K.sub.m V.sub.MAX (μM) (nmol/min/mg) (μM) (nmol/min/mg) WT-UGDH 48.8 ± 5.5 240.9 ± 8.0 1031 ± 215 206.1 ± 13.0 T325A- 82.2 ± 9.6 260.8 ± 9.8 1682 ± 455  103 ± 8.5 UGDH T325D- 25.3 ± 4.0  63.1 ± 3.2 1203 ± 290 38.0 ± 2.5 UGDH

Example 4

UGDH Inhibition Assay

(101) Table 5 shows inhibition data for UDP-xylose, inhibitor (1) (i.e., 5210344), and inhibitor (31) (i.e., 6847944). IC.sub.50 values for UDP-xylose and inhibitor (1) were determined using Km concentrations of UDP-glucose and NAD+ (50 μM UDP-glc and 1 mM NAD.sup.+). Ki values were determined by varying [UDP-glc] and holding NAD+ at saturating concentrations. UDP-xylose is a more potent inhibitor than the other compounds. Inhibitor (1) may have greatest effect on the dimer. Inhibitor (31) appears to require the ability for hexamer formation to inhibit which may suggest interference at the dimer-dimer interface as mechanism of action.

(102) TABLE-US-00006 TABLE 5 Inhibition Data UDP-xylose Inh #5210344 Inh #6847944 IC.sub.50 (μM) K.sub.i (μM) IC.sub.50 (μM) K.sub.i (μM) IC.sub.50 (μM) K.sub.i (μM) WT-UGDH 0.58 ± 0.09 2.67 ± 0.54 260.7 ± 6.2 ND* 146.8 ± 1.05 ND*** T325A-UGDH ND ND ND 421.4 ± 117.6 86.98 ± 0.03 ND T325D-UGDH ND ND ND ND  799.4 ± 0.05** ND *Inhibitor (1) caused a synergistic product inhibition curve (very high Ki) **Required 4 μM T325D in order to see effect instead of 1 μM ***Fit to allosteric sigmoidal curve which led to increased Hill coefficients

Example 5

UDP-xylose K.SUB.i .and IC.SUB.50 .Determination

(103) FIGS. 6A-6B show a mixed-model inhibition fit for UDP-xylose used for determination of K.sub.i. UDP-xylose is a competitive inhibitor of UGDH (K.sub.i=2.67±0.54 μM). IC.sub.50 curve was performed as described above (see, e.g., Example 2) and was found to be 0.58±0.09 μM.

Example 6

Inhibitors K.SUB.i .and IC.SUB.50 .Comparisons

(104) FIG. 7A-7E show kinetic characterization of inhibitors (1) and (31). IC.sub.50 experiments were performed as described above (see e.g., Example 2). Calculated IC.sub.50 and K.sub.i values can be found in Table 5. Inhibitor (1) did not show inhibition in the WT K curve, but did with T325A which may support requirement of the dimer for inhibition. Inhibitor (31) is an allosteric inhibitor that may bind in the dimer-dimer interface or alter the interface to disrupt hexamer formation and inhibit the enzyme.

Example 7

Thermal Stability

(105) UDP-Xylose Enhances Thermal Stability

(106) FIG. 8A-8D show UDP-xylose effects on the thermal stability of WT, T325A (inducible hexamer), and T325D (obligate dimer) UGDH. The UDP-xylose inhibitor triggered multiple unfolding events of UGDH, and increased the thermal stability of apo WT and T325A, similarly to the effect of the UDP-sugar substrate and cofactor. UDP-xylose appears to significantly increase thermal stability of NAD+ complexes with WT, T325A, and T325D, similarly to the effect of the productive holo complexes. Statistical analyses were performed using a two-way ANOVA with Bonferroni post tests on PRISM.

(107) Inhibitor (1) Selectively Affects Thermal Stability

(108) FIG. 9A-9D shows inhibitor (1) (i.e., 5210344) effects on the thermal stability of WT, T325A (inducible hexamer), and T325D (obligate dimer) UGDH. The previously validated inhibitor appears to decrease thermal stability and cause multiple unfolding events of UDP-glcA complexes with WT, T325A, and T325D. This inhibitor also causes multiple unfolding events with the apo T325D and T325A mutants, which may indicate that this inhibitor can only bind to the dimeric form of UGDH in order to affect activity (supports the high IC.sub.50 value with WT-UGDH).

(109) Inhibitor (31) has Negligible Effect on Thermal Stability

(110) FIG. 10A-10C shows inhibitor (31) (i.e., 6847944) effects on the thermal stability of WT, T325A (inducible hexamer), and T325D (obligate dimer) UGDH. This modestly affects the T325A and T325D mutants by decreasing thermal stability of all binary complexes except UDP-glucose.

Example 8

Trypsin Sensitivity

(111) UDP-xylose stabilizes UGDH T325A and T325D mutants against limited trypsin proteolysis, as shown in FIG. 11. UDP-xylose [20 μM] protected T325A apo from trypsin digestion (red star) and only slightly affected the other forms. UDP-xylose [20 μM] also significantly protected T325D complexes with NAD+, NADH, and UDP-glcA, but did not affect the apo and UDP-glc forms. Each assay contained 10 μg of enzyme, 10 ng Trypsin, and combinations of substrate, cofactor, and UDP-xylose, which was incubated 2.5 h, followed by SDS-PAGE.

Example 9

Proteolysis

(112) FIG. 12 shows inhibitor (1) and (31) effects on trypsin digestion of WT and mutant UGDH. Inhibitor (1) [750 μM] significantly affects trypsin digestion of T325A with UDP-glcA and UDP-glcA with NADH. 6847944 [500 μM] significantly affects ternary complexes for both WT and T325A. The same procedures from above (see e.g., Example 7) were followed.

OTHER EMBODIMENTS

(113) It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.