LIN WENWEI (US)
LI YONGTAO (US)
US20190255109A1 | 2019-08-22 |
KANG ET AL.: "Design of a fluorescence polarization assay platform for the study of human Hsp70", BIOORG MED CHEM LETT, vol. 18, no. 13, 2008, pages 3749 - 3751, XP022716291, DOI: 10.1016/j.bmcl.2008.05.046
CHAN ET AL.: "Impact of Target Warhead and Linkage Vector on Inducing Protein Degradation: Comparison of Bromodomain and Extra-Terminal (BET) Degraders Derived from Triazolodiazepine (JQ1) and Tetrahydroquinoline (I-BET726) BET Inhibitor Scaffolds", J. MED. CHEM., vol. 61, 2018, pages 504 - 513, XP055930443, DOI: 10.1021/acs.jmedchem.6b01912
CLAIMS What is claimed is: 1. A compound having a structure represented by a formula: , wherein L is a linker; wherein R1 is a residue of a fluorophore, a residue of biotin, or a residue of a biotin derivative; and wherein R2 is a residue of a von Hippel-Lindau protein (pVHL) ligand. 2. The compound of claim 1, wherein R1 is a residue of biotin. 3. The compound of claim 1, wherein R1 is a residue of a biotin derivative. 4. The compound of claim 3, wherein the biotin derivative is biocytin or desthiobiotin. 5. The compound of claim 1, wherein R1 is a residue of a fluorophore. 6. The compound of claim 5, wherein the fluorophore is fluorescein, Oregon green, rhodamine (e.g., TAMRA dye), eosin, Texas red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, a squaraine derivative, a naphthalene derivative (e.g., a dansyl or prodan derivative), a coumarin derivative, an oxadiazole derivative (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole), an anthracene derivative (e.g., an anthraquinone such as DRAQ5, DRAQ7, and CyTRAK Orange), cascade blue, Nile red, Nile blue, cresyl violate, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, prophin, phthalocyanine, an alexa fluor series dye, or bilirubin. 7. The compound of claim 5, wherein the fluorophore is a 4,4-difluoro-4-bora-3a,4a- diaza-s-indacene (BODIPY) fluorophore. 8. The compound of claim 7, wherein the BODIPY fluorophore is selected from: , , , , and . 9. The compound of claim 7, wherein the BODIPY fluorophore is: 10. The compound of of any one of claims 1 to 9, wherein L is selected from C2-C15 alkyl and ‒(CH2CH2O)n; and wherein n is selected from 1, 2, 3, 4, 5, 6, 7, and 8. 11. The compound of claim 10, wherein L is C2-C15 alkyl. 12. The compound of claim 10, wherein L is C2-C12 alkyl. 13. The compound of claim 10, wherein L is C2-C8 alkyl. 14. The compound of claim 10, wherein L is C5 alkyl. 15. The compound of claim 10, wherein L is ‒(CH2CH2O)n‒. 16. The compound of claim 10, wherein L is ‒(CH2CH2O)4‒. 17. The compound of any one of claims 1 to 16, wherein the residue of the pVHL ligand has a structure represented by a formula: , wherein m is 0 or 1; wherein Q, when present, is ‒OC(O)‒, ‒C(R10a)(R10b)C(O)‒, ‒OC(R10a)(R10b)C(O)‒, ‒ C(R10a)(R10b)C(O)C(cyclopropyl)C(O)‒, ‒C(R10a)(R10b)C(O)N(R11a)CH2CH(R11b)C(O)‒, ‒ C(C3-C4 cycloalkyl)C(O)‒, ‒NH(CH2CH2O)qCH2C(O)‒, ‒NHCH2C(cyclopropyl)C(O)‒, or ‒CH2C(O)N(R12)CH(R13)C(O)‒; wherein q, when present, is 1, 2, 3, 4, 5, or 6; wherein each of R10a and R10b, when present, is independently hydrogen or C1-C4 alkyl; or wherein each of R10a and R10b, when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl or a C2-C3 heterocycloalkyl; or wherein R10, when present, is covalently bound to R3, and, together with the intermediate atoms, comprises a 5-membered heterocycle; wherein each of R11a and R11b, when present, are covalently bound, and, together with the intermediate atoms, comprise a 4-membered heterocycle; wherein R12, when present, is hydrogen; and wherein R13, when present, is C1-C4 alkyl, ‒CH2C6H5, or ‒C6H5; or wherein each of R12 and R13, when present, are covalently bound, and, together with the intermediate atoms, comprise an 10-membered heterocycloalkyl; wherein R3 is hydrogen or C1-C4 alkyl; and wherein R4 is a C1-C4 alkyl, C1-C4 hydroxyalkyl, or C6H5; or wherein each of R3 and R4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 0 or 1 ‒OH group; or wherein each of R3 and R10, when present, are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle; wherein R5 is hydrogen or methyl; and wherein R6 is hydrogen, ‒OH, or C1-C4 alkyl halide. 18. The compound of claim 17, wherein each of R3, R5, and R6 is hydrogen. 19. The compound of claim 17 or claim 18, wherein R4 is tert-butyl. 20. The compound of claim 17 or claim 19, wherein R6 is hydrogen. 21. The compound of claim 17 or claim 19, wherein R6 is C1-C4 alkyl. 22. The compound of claim 21, wherein R6 is ‒CH2Br or ‒CH2Cl. 23. The compound of claim 17, wherein the residue of the pVHL ligand has a structure represented by a formula: . 24. The compound of claim 17, wherein the residue of the pVHL ligand has a structure represented by a formula: . 25. The compound of claim 17, wherein the residue of the pVHL ligand has a structure represented by a formula: . 26. The compound of claim 17, wherein the residue of the pVHL ligand has a structure selected from: , , , , and . 27. The compound of claim 17, wherein the residue of the pVHL ligand has a structure: 28. The compound of any one of claims 1 to 27, wherein the compound has a structure represented by a formula: wherein m is 0 or 1; wherein Q, when present, is ‒OC(O)‒, ‒C(R10a)(R10b)C(O)‒, ‒OC(R10a)(R10b)C(O)‒, ‒ C(R10a)(R10b)C(O)C(cyclopropyl)C(O)‒, ‒C(R10a)(R10b)C(O)N(R11a)CH2CH(R11b)C(O)‒, ‒ C(C3-C4 cycloalkyl)C(O)‒, ‒NH(CH2CH2O)qCH2C(O)‒, ‒NHCH2C(cyclopropyl)C(O)‒, or ‒CH2C(O)N(R12)CH(R13)C(O)‒; wherein q, when present, is 1, 2, 3, 4, 5, or 6; wherein each of R10a and R10b, when present, is independently hydrogen or C1-C4 alkyl; or wherein each of R10a and R10b, when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl or a C2-C3 heterocycloalkyl; or wherein R10, when present, is covalently bound to R3, and, together with the intermediate atoms, comprises a 5-membered heterocycle; wherein each of R11a and R11b, when present, are covalently bound, and, together with the intermediate atoms, comprise a 4-membered heterocycle; wherein R12, when present, is hydrogen; and wherein R13, when present, is C1-C4 alkyl, ‒CH2C6H5, or ‒C6H5; or wherein each of R12 and R13, when present, are covalently bound, and, together with the intermediate atoms, comprise an 10-membered heterocycloalkyl; wherein R1 is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore; wherein R3 is hydrogen or C1-C4 alkyl; and wherein R4 is a C1-C4 alkyl, C1-C4 hydroxyalkyl, or C6H5; or wherein each of R3 and R4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 0 or 1 ‒OH group; or wherein each of R3 and R10, when present, are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle; wherein R5 is hydrogen or methyl; and wherein R6 is hydrogen, ‒OH, or C1-C4 alkyl halide. 29. The compound of claim 28, wherein the compound has a structure represented by a formula: . 30. The compound of claim 28, wherein the compound has a structure represented by a formula: . 31. The compound of claim 28, wherein the compound has a structure represented by a formula: . 32. The compound of claim 28, wherein the compound has a structure represented by a formula: N 5 O H . 33. The compound of claim 28, wherein the compound has a structure represented by a formula: . 34. The compound of claim 1, wherein the compound is: . 35. The compound of claim 1, wherein the compound is: . 36. A method of modulating von Hippel-Lindau protein (pVHL) in a sample, the method comprising contacting the sample with an effective amount of the compound of any one of claims 1 to 35, thereby modulating VHL protein in the sample. 37. The method of claim 36, wherein modulating is decreasing. 38. The method of claim 36, wherein modulating is inhibiting. 39. The method of claim 36, wherein contacting is in the presence of an antibody. 40. The method of claim 39, wherein the antibody is Tb-anti-GST, Tb-anti-HIS, Tb-anti- FLAG, Tb-anti-HA, Eu-anti-GST, Eu-anti-HIS, Eu-anti-FLAG, or Eu-anti-HA. 41. The method of claim 36, wherein contacting is in the presence of a pVHL ligand. 42. The method of claim 36, wherein contacting is in the presence of a non-pVHL ligand. 43. The method of claim 36, wherein the sample is a buffer. 44. The method of claim 36, wherein the sample is a cell. 45. The method of claim 44, wherein the cell is mammalian. 46. The method of claim 36, wherein the effective amount is within ± 30% of a Kd concentration of the compound. 47. The method of claim 46, wherein the effective amount is within ± 25% of a Kd concentration of the compound. 48. The method of claim 46, wherein the Kd concentration is of from about 2.0 nM to about 5.0 nM. 49. The method of claim 46, wherein the Kd concentration is about 3.0 nM. 50. The method of claim 46, wherein the effective amount is of from about 2.0 nM to about 5.0 nM. 51. The method of claim 46, wherein the effective amount is about 4.0 nM. 52. The method of claim 36, wherein contacting is for a time period of from about 90 minutes to about 300 minutes. 53. A method of identifying a von Hippel-Lindau protein (pVHL) ligand in a library, the method comprising: (a) providing a library that contains a plurality of ligands; (b) combining the compound of any one of claims 1 to 35 and a sample having pVHL, thereby forming a mixture; (c) exposing each ligand to the mixture; and (d) detecting a fluorescence emission of the mixture after exposure to each ligand, wherein a decrease in fluorescence emission indicates that the ligand is a pVHL ligand, and wherein a lack of decrease in fluorescence emission indicates that the ligand is a non- pVHL ligand. 54. A kit comprising the compound of any one of claims 1 to 35, and one or more of: (a) a sample that contains von Hippel-Lindau protein (pVHL); (b) a library that contains a plurality of ligands; (c) instructions for modulating pVHL; (d) instructions for identifying a pVHL ligand and/or a non-pVHL ligand; and (e) instructions for performing a fluorescence-based assay. 55. The kit of claim 54, wherein the fluorescence-based assay is a time-resolved fluorescence energy transfer (TR-FRET) assay, a fluorescence polarization (FP) assay, an enzyme-linked immunosorbent assay (ELISA), western blot analysis, an immunohistochemistry (IHC) assay, an immunoprecipitation (IP) assay, or a fluorescence- activated cell sorting (FACS) assay. |
,
,
. [00153] In yet a further aspect, the BODIPY fluorophore is: . d. R 2 GROUPS [00154] In one aspect, R 2 is a residue of a von Hippel-Lindau protein (pVHL) ligand. [00155] In a further aspect, the residue of the pVHL ligand has a structure represented by a formula: . [00156] In a further aspect, the residue of the pVHL ligand has a structure represented by a formula: . [00157] In a further aspect, the residue of the pVHL ligand has a structure represented by a formula: . [00158] In a further aspect, the residue of the pVHL ligand has a structure represented by a formula: . [00159] In a further aspect, the residue of the pVHL ligand has a structure selected from: ,
, ,
,
, , , OH , and . [00160] In a further aspect, the residue of the pVHL ligand has a structure:
. e. R 3 R 4 AND GROUPS [00161] In one aspect, R 3 is hydrogen or C1-C4 alkyl; and wherein R 4 is a C1-C4 alkyl, C1-C4 hydroxyalkyl, or C 6 H 5 ; or wherein each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 0 or 1 ‒OH group; or wherein each of R 3 and R 10a , when present, are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle. [00162] In a further aspect, R 3 is hydrogen or C1-C4 alkyl. In a still further aspect, R 3 is hydrogen, methyl, ethyl, n-propyl, or isopropyl. In yet a further aspect, R 3 is hydrogen, methyl, or ethyl. In an even further aspect, R 3 is hydrogen or ethyl. In a still further aspect, R 3 is hydrogen or methyl. [00163] In a further aspect, R 3 is C1-C4 alkyl. In a still further aspect, R 3 is methyl, ethyl, n-propyl, or isopropyl. In yet a further aspect, R 3 is methyl or ethyl. In an even further aspect, R 3 is ethyl. In a still further aspect, R 3 is methyl. [00164] In a further aspect, R 3 is hydrogen. [00165] In a further aspect, R 4 is a C1-C4 alkyl, C1-C4 hydroxyalkyl, or C 6 H 5 . In a still further aspect, R 4 is methyl, ethyl, n-propyl, isopropyl, ‒CH 2 OH, ‒CH 2 CH 2 OH, ‒ CH 2 CH 2 CH 2 OH, ‒CH(CH3)CH 2 OH, or C 6 H 5 . In yet a further aspect, R 4 is methyl, ethyl, ‒ CH 2 OH, ‒CH 2 CH 2 OH, or C 6 H 5 . In an even further aspect, R 4 is methyl, ‒CH 2 OH, or C 6 H 5 . [00166] In a further aspect, R 4 is a C1-C4 alkyl. In a still further aspect, R 4 is methyl, ethyl, n-propyl, or isopropyl. In yet a further aspect, R 4 is methyl or ethyl. In an even further aspect, R 4 is methyl. In a still further aspect, R 4 is a C4 alkyl. In yet a further aspect, R 4 is isobutyl, sec-butyl, or tert-butyl. In an even further aspect, R 4 is tert-butyl. [00167] In a further aspect, R 4 is a C1-C4 hydroxyalkyl. In a still further aspect, R 4 is ‒CH 2 OH, ‒CH 2 CH 2 OH, ‒CH 2 CH 2 CH 2 OH, or ‒CH(CH 3 )CH 2 OH. In yet a further aspect, R 4 is ‒CH 2 OH or ‒CH 2 CH 2 OH. In an even further aspect, R 4 is ‒CH 2 OH. [00168] In a further aspect, R 4 is C 6 H 5 . [00169] In a further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 0 or 1 ‒OH group. Examples of 5- and 6-membered heterocycles include, but are not limited to, pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, and pyranyl. In a still further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 1 ‒OH group. In yet a further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 0 ‒ OH groups. In an even further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise an unsubstituted 5- or 6-membered heterocycle. [00170] In a further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle having 0 or 1 ‒OH group. In a still further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle having 1 ‒OH group. In yet a further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle having 0 ‒OH groups. [00171] In a further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 6-membered heterocycle having 0 or 1 ‒OH group. In a still further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 6-membered heterocycle having 1 ‒OH group. In yet a further aspect, each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 6-membered heterocycle having 0 ‒OH groups. [00172] In a further aspect, each of R 3 and R 10a , when present, are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle. Examples of 5-membered heterocycles include, but are not limited to, pyrrolidinyl, pyrrolidino, thiolanyl, and tetrahydrofuranyl. In a still further aspect, each of R 3 and R 10a , when present, are covalently bound, and, together with the intermediate atoms, comprise an unsubstituted 5- membered heterocycle. f. R 5 GROUPS [00173] In one aspect, R 5 is hydrogen or methyl. In a further aspect, R 5 is hydrogen. In a still further aspect, R 5 is methyl. g. R 6 GROUPS [00174] In one aspect, R 6 is hydrogen, ‒OH, or C1-C4 alkyl halide. In a further aspect, R 6 is hydrogen, ‒OH, ‒CH 2 F, ‒CH 2 Cl, ‒CH 2 CH 2 F, ‒CH 2 CH 2 Cl, ‒CH 2 CH 2 CH 2 F, ‒ CH 2 CH 2 CH 2 Cl, ‒CH(CH3)CH 2 F, or ‒CH(CH3)CH 2 Cl. In a still further aspect, R 6 is hydrogen, ‒OH, ‒CH 2 F, ‒CH 2 Cl, ‒CH 2 CH 2 F, or ‒CH 2 CH 2 Cl. In yet a further aspect, R 6 is hydrogen, ‒OH, ‒CH 2 F, or ‒CH 2 Cl. [00175] In a further aspect, R 6 is ‒OH. [00176] In a further aspect, R 6 is C1-C4 alkyl halide. In a still further aspect, R 6 is ‒ CH 2 F, ‒CH 2 Cl, ‒CH 2 Br, ‒CH 2 CH 2 F, ‒CH 2 CH 2 Cl, ‒CH 2 CH 2 Br, ‒CH 2 CH 2 CH 2 F, ‒ CH 2 CH 2 CH 2 Cl, ‒CH 2 CH 2 CH 2 Br, ‒CH(CH3)CH 2 F, ‒CH(CH3)CH 2 Cl, or ‒CH(CH3)CH 2 Br. In yet a further aspect, R 6 is ‒CH 2 F, ‒CH 2 Cl, ‒CH 2 Br, ‒CH 2 CH 2 F, ‒CH 2 CH 2 Cl, or ‒ CH 2 CH 2 Br. In an even further aspect, R 6 is ‒CH 2 F, ‒CH 2 Cl, or ‒CH 2 Br. [00177] In a further aspect, R 6 is hydrogen. h. R10A AND R10B GROUPS [00178] In one aspect, each of R 10a and R 10b , when present, is independently hydrogen or C1-C4 alkyl; or wherein each of R 10a and R 10b , when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl or a C2-C3 heterocycloalkyl; or wherein R 10a , when present, is covalently bound to R 3 , and, together with the intermediate atoms, comprises a 5- membered heterocycle. [00179] In a further aspect, each of R 10a and R 10b , when present, is independently hydrogen or C1-C4 alkyl. In a still further aspect, each of R 10a and R 10b , when present, is independently hydrogen, methyl, ethyl, n-propyl, or isopropyl. In yet a further aspect, each of R 10a and R 10b , when present, is independently hydrogen, methyl, or ethyl. In an even further aspect, each of R 10a and R 10b , when present, is independently hydrogen or methyl. [00180] In a further aspect, each of R 10a and R 10b , when present, is independently C1- C4 alkyl. In a still further aspect, each of R 10a and R 10b , when present, is independently methyl, ethyl, n-propyl, or isopropyl. In yet a further aspect, each of R 10a and R 10b , when present, is independently methyl or ethyl. In an even further aspect, each of R 10a and R 10b , when present, is methyl. [00181] In a further aspect, each of R 10a and R 10b , when present, is hydrogen. [00182] In a further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl or a C2-C3 heterocycloalkyl. In a still further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl or a C2-C3 heterocycloalkyl, and are unsubstituted. [00183] In a further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl. In a still further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise a cyclopropyl. In yet a further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise a cyclobutyl. In an even further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise an unsubstituted C3-C4 cycloalkyl. [00184] In a further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise a C2-C3 heterocycloalkyl. Examples of C2-C3 heterocycloalkyls include, but are not limited to, oxirane, aziridine, and thiirane. In a still further aspect, each of R 10a and R 10b , when present, are covalently bound, and, together comprise an unsubstituted C2-C3 heterocycloalkyl. [00185] In a further aspect, R 10a , when present, is covalently bound to R 3 , and, together with the intermediate atoms, comprises a 5-membered heterocycle. Examples of 5- membered heterocycles include, but are not limited to, pyrrolidinyl, pyrrolidino, thiolanyl, and tetrahydrofuranyl. In a still further aspect, R 10a , when present, is covalently bound to R 3 , and, together with the intermediate atoms, comprises an unsubstituted 5-membered heterocycle. i. R 11A AND R 11B GROUPS [00186] In one aspect, each of R 11a and R 11b , when present, are covalently bound, and, together with the intermediate atoms, comprise a 4-membered heterocycle. Examples of 4- membered heterocycles include, but are not limited to, trimethylene oxide, thietane, 1,3- diazetidine, and azetidine. In a further aspect, each of R 11a and R 11b , when present, are covalently bound, and, together with the intermediate atoms, comprise an unsubstituted 4- membered heterocycle. j. R 12 AND R 13 GROUPS [00187] In one aspect, R 12 , when present, is hydrogen; and wherein R 13 , when present, is C1-C4 alkyl, ‒CH 2 C 6 H 5 , or ‒C 6 H 5 ; or wherein each of R 12 and R 13 , when present, are covalently bound, and, together with the intermediate atoms, comprise a 10-membered heterocycloalkyl. [00188] In a further aspect, R 12 , when present, is hydrogen. [00189] In a further aspect, R 13 , when present, is C1-C4 alkyl, ‒CH 2 C 6 H 5 , or ‒C 6 H 5 . In a still further aspect, R 13 , when present, is methyl, ethyl, n-propyl, isopropyl, ‒CH 2 C 6 H 5 , or ‒C 6 H 5 . In yet a further aspect, R 13 , when present, is methyl, ethyl, ‒CH 2 C 6 H 5 , or ‒C 6 H 5 . In an even further aspect, R 13 , when present, is methyl, ‒CH 2 C 6 H 5 , or ‒C 6 H 5 . [00190] In a further aspect, R 13 , when present, is C1-C4 alkyl. In a still further aspect, R 13 , when present, is methyl, ethyl, n-propyl, or isopropyl. In yet a further aspect, R 13 , when present, is methyl or ethyl. In an even further aspect, R 13 , when present, is methyl. [00191] In a further aspect, R 13 , when present, is ‒CH 2 C 6 H 5 or ‒C 6 H 5 . In a still further aspect, R 13 , when present, is ‒CH 2 C 6 H 5 . In yet a further aspect, R 13 , when present, is ‒C 6 H 5 . [00192] In a further aspect, each of R 12 and R 13 , when present, are covalently bound, and, together with the intermediate atoms, comprise a 10-membered heterocycloalkyl. Examples of 10-membered heterocycloalkyls include, but are not limited to, tetrahydroisoquinolinyl and decahydroisoquinolinyl. 2. EXAMPLE COMPOUNDS [00193] In one aspect, a compound can be present as: . [00194] In one aspect, a compound can be present as: . C. METHODS OF MAKING A COMPOUND [00195] The compounds of this invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein. [00196] Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the following Reaction Schemes, as described and exemplified below. In certain specific examples, the disclosed compounds can be prepared by Routes I-III, as described and exemplified below. The following examples are provided so that the invention might be more fully understood, are illustrative only, and should not be construed as limiting. 1. ROUTE I [00197] In one aspect, the compounds disclosed herein can be prepared as shown below. SCHEME 1A. [00198] Compounds are represented in generic form, where R is ‒OH, ‒NH 2 , or ‒O‒ acetate such that 1.2 is a carboxylic acid, amide, or anhydride, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below. SCHEME 1B. [00199] In one aspect, compounds of type 1.6, and similar compounds, can be prepared according to reaction Scheme 1B above. Thus, compounds of type 1.6 can be prepared by a coupling reaction between an appropriate amine, e.g., 1.4 as shown above, and an appropriate carboxylic acid, amide, or anhydride, e.g., 1.5 as shown above. Appropriate amines and appropriate carboxylic acids, amines, and anhydrides are commercially available or prepared by methods known to one skilled in the art. The coupling reaction is carried out in the presence of an appropriate base, e.g., diisopropylethylamine (DIPEA), in an appropriate solvent, e.g., dichloromethane, at an appropriate temperature, e.g., 4 ⁰C to room temperature, for an appropriate period of time, e.g., 30 minutes. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 1.1 and 1.2), can be substituted in the reaction to provide pVHL ligands similar to Formula 1.3. 2. ROUTE II [00200] In one aspect, the compounds disclosed herein can be prepared as shown below. SCHEME 2A. [00201] Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below. SCHEME 2B. [00202] In one aspect, compounds of type 2.6, and similar compounds, can be prepared according to reaction Scheme 2B above. Thus, compounds of type 2.6 can be prepared by a coupling reaction between an appropriate carboxyl or amine analog, e.g., 2.4 as shown above, and an appropriate alcohol or amine, e.g., 2.5 as shown above. Appropriate amines and appropriate alcohols are commercially available or prepared by methods known to one skilled in the art. The coupling reaction is carried out in the presence of an appropriate coupling agent, e.g., hydroxybenzotriazole (HOBt), an appropriate activating agent, e.g., 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI), and an appropriate base, e.g., diisopropylethylamine (DIPEA), at an appropriate temperature, e.g., room temperature,. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 2.1 and 2.2), can be substituted in the reaction to provide compounds similar to Formula 2.6. 3. ROUTE III [00203] In one aspect, the compounds disclosed herein can be prepared as shown below. SCHEME 3A. [00204] Compounds are represented in generic form, where R and R’ are independently groups capable of coupling with one another such as, for example, carboxylic acids and amines, and with other substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below. SCHEME 3B. [00205] In one aspect, compounds of type 3.6, and similar compounds, can be prepared according to reaction Scheme 3B above. Thus, compounds of type 3.6 can be prepared by a coupling reaction between an appropriate alcohol or amine analog, e.g., 3.4 as shown above, and an appropriate carboxylic acid, e.g., 3.5 as shown above. Appropriate carboxylic acids are commercially available or prepared by methods known to one skilled in the art. The coupling reaction is carried out in the presence of an appropriate coupling agent, e.g., hydroxybenzotriazole (HOBt), an appropriate activating agent, e.g., 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI), and an appropriate base, e.g., diisopropylethylamine (DIPEA), in an appropriate solvent, e.g., dimethylsulfoxide, at an appropriate temperature, e.g., room temperature,. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 3.1 and 3.2), can be substituted in the reaction to provide compounds similar to Formula 3.6. D. METHODS OF MODULATING VON HIPPEL-LINDAU PROTEIN IN A SAMPLE [00206] In one aspect, disclosed are methods of modulating von Hippel-Lindau protein (pVHL) in a sample, the method comprising contacting the sample with an effective amount of a disclosed compound, thereby modulating VHL protein in the sample. [00207] In various aspects, disclosed are methods of modulating von Hippel-Lindau protein (pVHL) in a sample, the method comprising contacting the sample with an effective amount of a compound,having a structure represented by a formula: wherein L is a linker; wherein R 1 is a residue of a fluorophore, a residue of biotin, or a residue of a biotin derivative; and wherein R 2 is a residue of a von Hippel-Lindau protein (pVHL) ligand, thereby modulating VHL protein in the sample. [00208] In various aspects, the compound has a structure represented by a formula: wherein m is 0 or 1; wherein Q, when present, is ‒OC(O)‒, ‒C(R 10a )(R 10b )C(O)‒, ‒ OC(R 10a )(R 10b )C(O)‒, ‒C(R 10a )(R 10b )C(O)C(cyclopropyl)C(O)‒, ‒ C(R 10a )(R 10b )C(O)N(R 11a )CH 2 CH(R 11b )C(O)‒, ‒C(C3-C4 cycloalkyl)C(O)‒, ‒ NH(CH 2 CH 2 O)qCH 2 C(O)‒, ‒NHCH 2 C(cyclopropyl)C(O)‒, or ‒ CH 2 C(O)N(R 12 )CH(R 13 )C(O)‒; wherein q, when present, is 1, 2, 3, 4, 5, or 6; wherein each of R 10a and R 10b , when present, is independently hydrogen or C1-C4 alkyl; or wherein each of R 10a and R 10b , when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl or a C2-C3 heterocycloalkyl; or wherein R 10 , when present, is covalently bound to R 3 , and, together with the intermediate atoms, comprises a 5-membered heterocycle; wherein each of R 11a and R 11b , when present, are covalently bound, and, together with the intermediate atoms, comprise a 4-membered heterocycle; wherein R 12 , when present, is hydrogen; and wherein R 13 , when present, is C1-C4 alkyl, ‒CH 2 C 6 H 5 , or ‒C 6 H 5 ; or wherein each of R 12 and R 13 , when present, are covalently bound, and, together with the intermediate atoms, comprise an 10-membered heterocycloalkyl; wherein R 1 is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore; wherein R 3 is hydrogen or C1-C4 alkyl; and wherein R 4 is a C1-C4 alkyl, C1-C4 hydroxyalkyl, or C 6 H 5 ; or wherein each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 0 or 1 ‒OH group; or wherein each of R 3 and R 10 , when present, are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle; wherein R 5 is hydrogen or methyl; and wherein R 6 is hydrogen, ‒OH, or C1-C4 alkyl halide. [00209] In a further aspect, the compound has a structure represented by a formula: . [00210] In a further aspect, the compound has a structure represented by a formula: . [00211] In a further aspect, the compound has a structure represented by a formula: . [00212] In a further aspect, the compound has a structure represented by a formula: N 5 O H . [00213] In a further aspect, the compound has a structure represented by a formula: . [00214] In a further aspect, the compound is:
. [00215] In a further aspect, the compound is: . [00216] In various aspects, modulating is decreasing. In various further aspects, modulating is inhibiting. [00217] In various aspects, contacting is in the presence of an antibody. As would be appreciated by one of ordinary skill in the art, the antibody selected can be dependent on the assay being used. Exemplary antibodies are well-known by those of ordinary skill, and include, for example, Tb-anti-GST, Tb-anti-HIS, Tb-anti-FLAG, Tb-anti-HA, Eu-anti-GST, Eu-anti-HIS, Eu-anti-FLAG, and Eu-anti-HA. [00218] In various aspects, contacting is in the presence of a pVHL ligand. Examples of pVHL ligands include, but are not limited to, VH032, VH298, MZ1, VH032, Me-VH032 amine, BOC-VH032, VH032 Phenol, and VH032-PEG4-amine. Additional VHL ligands and residues thereof are disclosed herein. See also Galdeano, et al. (2014) Journal of Medicinal Chemistry 57: 8657-8663 and Soares, et al. (2018) Journal of Medicinal Chemistry 61: 599- 618. [00219] In various aspects, contacting is in the presence of a non-pVHL ligand. Examples of non-VHL ligands include, but are not limited to, (+)-JQ-1, thalidomide 4’- oxyacetamido-alkylC4-amine, and dBET1. [00220] In various aspects, contacting is for a time period of from about 90 minutes to about 300 minutes, from about 90 minutes to about 250 minutes, from about 90 minutes to about 200 minutes, from about 90 minutes to about 150 minutes, from about 90 minutes to about 100 minutes, from about 10 minutes to about 300 minutes, from about 150 minutes to about 300 minutes, from about 200 minutes to about 300 minutes, from about 250 minutes to about 300 minutes, from about 100 minutes to about 250 minutes, or from about 150 minutes to about 200 minutes. [00221] In various aspects, the sample is a buffer. In a further aspect, the sample is a cell. In a still further aspect, the cell is mammalian. [00222] In various aspects, the effective amount is within ± 30% of a K d concentration of the compound. In a further aspect, the effective amount is within ± 25% of a K d concentration of the compound. In a still further aspect, the effective amount is within ± 20% of a K d concentration of the compound. [00223] In various aspects, the K d concentration is of from about 2.0 nM to about 5.0 nM, from about 2.0 nM to about 4.5 nM, from about 2.0 nM to about 4.0 nM, from about 2.0 nM to about 3.5 nM, from about 2.0 nM to about 3.0 nM, from about 2.0 nM to about 2.5 nM, from about 2.5 nM to about 5.0 nM, from about 3.0 nM to about 5.0 nM, from about 3.5 nM to about 5.0 nM, from about 4.0 nM to about 5.0 nM, from about 4.0 nM to about 5.0 nM, or from about 2.5 nM to about 4.5 nM. In a further aspect, the K d concentration is about 3.0 nM. [00224] In various aspects, the effective concentration is of from about 2.0 nM to about 5.0 nM, from about 2.0 nM to about 4.5 nM, from about 2.0 nM to about 4.0 nM, from about 2.0 nM to about 3.5 nM, from about 2.0 nM to about 3.0 nM, from about 2.0 nM to about 2.5 nM, from about 2.5 nM to about 5.0 nM, from about 3.0 nM to about 5.0 nM, from about 3.5 nM to about 5.0 nM, from about 4.0 nM to about 5.0 nM, from about 4.0 nM to about 5.0 nM, or from about 2.5 nM to about 4.5 nM. In a further aspect, the effective amount concentration is about 4.0 nM. E. METHODS OF IDENTIFYING A VON HIPPEL-LINDAU PROTEIN (PVHL) LIGAND [00225] In one aspect, disclosed are methods of identifying a von Hippel-Lindau protein (pVHL) ligand in a library, the method comprising: (a) providing a library that contains a plurality of ligands; (b) combining a disclosed compound and a sample having pVHL, thereby forming a mixture; (c) exposing each ligand to the mixture; and (d) detecting a fluorescence emission of the mixture after exposure to each ligand, wherein a decrease in fluorescence emission indicates that the ligand is a pVHL ligand, and wherein a lack of decrease in fluorescence emission indicates that the ligand is a non-pVHL ligand. [00226] In various aspects, disclosed are methods of identifying a von Hippel-Lindau protein (pVHL) ligand in a library, the method comprising: (a) providing a library that contains a plurality of ligands; (b) combining a compound and a sample having pVHL, thereby forming a mixture; (c) exposing each ligand to the mixture; and (d) detecting a fluorescence emission of the mixture after exposure to each ligand, wherein a decrease in fluorescence emission indicates that the ligand is a pVHL ligand, wherein a lack of decrease in fluorescence emission indicates that the ligand is a non-pVHL ligand, and wherein the compound has a structure represented by a formula: wherein L is a linker; wherein R 1 is a residue of a fluorophore, a residue of biotin, or a residue of a biotin derivative; and wherein R 2 is a residue of a von Hippel-Lindau protein (pVHL) ligand, thereby modulating VHL protein in the sample. [00227] In various aspects, the compound has a structure represented by a formula: wherein m is 0 or 1; wherein Q, when present, is ‒OC(O)‒, ‒C(R 10a )(R 10b )C(O)‒, ‒ OC(R 10a )(R 10b )C(O)‒, ‒C(R 10a )(R 10b )C(O)C(cyclopropyl)C(O)‒, ‒ C(R 10a )(R 10b )C(O)N(R 11a )CH 2 CH(R 11b )C(O)‒, ‒C(C3-C4 cycloalkyl)C(O)‒, ‒ NH(CH 2 CH 2 O)qCH 2 C(O)‒, ‒NHCH 2 C(cyclopropyl)C(O)‒, or ‒ CH 2 C(O)N(R 12 )CH(R 13 )C(O)‒; wherein q, when present, is 1, 2, 3, 4, 5, or 6; wherein each of R 10a and R 10b , when present, is independently hydrogen or C1-C4 alkyl; or wherein each of R 10a and R 10b , when present, are covalently bound, and, together comprise a C3-C4 cycloalkyl or a C2-C3 heterocycloalkyl; or wherein R 10 , when present, is covalently bound to R 3 , and, together with the intermediate atoms, comprises a 5-membered heterocycle; wherein each of R 11a and R 11b , when present, are covalently bound, and, together with the intermediate atoms, comprise a 4-membered heterocycle; wherein R 12 , when present, is hydrogen; and wherein R 13 , when present, is C1-C4 alkyl, ‒CH 2 C 6 H 5 , or ‒C 6 H 5 ; or wherein each of R 12 and R 13 , when present, are covalently bound, and, together with the intermediate atoms, comprise an 10-membered heterocycloalkyl; wherein R 1 is a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophore; wherein R 3 is hydrogen or C1-C4 alkyl; and wherein R 4 is a C1-C4 alkyl, C1-C4 hydroxyalkyl, or C 6 H 5 ; or wherein each of R 3 and R 4 are covalently bound, and, together with the intermediate atoms, comprise a 5- or 6-membered heterocycle having 0 or 1 ‒OH group; or wherein each of R 3 and R 10 , when present, are covalently bound, and, together with the intermediate atoms, comprise a 5-membered heterocycle; wherein R 5 is hydrogen or methyl; and wherein R 6 is hydrogen, ‒OH, or C1-C4 alkyl halide. [00228] In a further aspect, the compound has a structure represented by a formula: . [00229] In a further aspect, the compound has a structure represented by a formula: . [00230] In a further aspect, the compound has a structure represented by a formula: . [00231] In a further aspect, the compound has a structure represented by a formula: N 5 O H . [00232] In a further aspect, the compound has a structure represented by a formula: . [00233] In a further aspect, the compound is:
. [00234] In a further aspect, the compound is: . [00235] In various aspects, exposing is in the presence of an antibody. As would be appreciated by one of ordinary skill in the art, the antibody selected can be dependent on the assay being used. Exemplary antibodies are well-known by those of ordinary skill, and include, for example, Tb-anti-GST, Tb-anti-HIS, Tb-anti-FLAG, Tb-anti-HA, Eu-anti-GST, Eu-anti-HIS, Eu-anti-FLAG, and Eu-anti-HA. [00236] In various aspects, the library contains at least one pVHL ligand. In a further aspect, the library contains a plurality of pVHL ligands. Examples of pVHL ligands include, but are not limited to, VH032, VH298, MZ1, VH032, Me-VH032 amine, BOC-VH032, VH032 Phenol, and VH032-PEG4-amine. Additional VHL ligands and residues thereof are disclosed herein. See also Galdeano, et al. (2014) Journal of Medicinal Chemistry 57: 8657- 8663 and Soares, et al. (2018) Journal of Medicinal Chemistry 61: 599-618. [00237] In various aspects, the library contains at least one non-pVHL ligand. In a further aspect, the library contains a plurality of non-pVHL ligands. Examples of non-VHL ligands include, but are not limited to, (+)-JQ-1, thalidomide 4’-oxyacetamido-alkylC4- amine, and dBET1. [00238] In various aspects, exposing is for a time period of from about 90 minutes to about 300 minutes, from about 90 minutes to about 250 minutes, from about 90 minutes to about 200 minutes, from about 90 minutes to about 150 minutes, from about 90 minutes to about 100 minutes, from about 10 minutes to about 300 minutes, from about 150 minutes to about 300 minutes, from about 200 minutes to about 300 minutes, from about 250 minutes to about 300 minutes, from about 100 minutes to about 250 minutes, or from about 150 minutes to about 200 minutes. F. ADDITIONAL METHODS OF USING THE COMPOSITIONS [00239] Provided are methods of using of a disclosed compound. In one aspect, the method of use is as a probe. The probe can be useful in, for example, identifying a von Hippel-Lindau protein ligand in a library. In a further aspect, the method of use is in modulatiing of von Hippel-Lindau protein in a sample such as, for example, a cell or a buffer. In a further aspect, the disclosed compounds can be used as single agents or in combination with one or more other probes, pVHL ligands, non-pVHL ligands, and antibodies in the aforementioned uses. [00240] The samples, mixtures, and methods of the present invention can further comprise other agents as noted herein, which are usually applied in biological assays such as, for example, fluorescence assays. 1. USE OF COMPOUNDS, SAMPLES, AMD MIXTURES [00241] Also provided are the uses of the disclosed compounds, samples, and mixtures. Thus, in one aspect, the invention relates to the use of von Hippel-Lindau (VHL) small molecule probes. [00242] In a further aspect, the use relates to a process for preparing a sample or mixture comprising an effective amount of a disclosed compound or a product of a disclosed method, and one or more of an antibody, a pVHL ligand, a non-pVHL ligand, a buffer, and a solvent, for use as in a fluorescence-based assay. [00243] In a further aspect, the use relates to a process for preparing a sample or mixture comprising an effective amount of a disclosed compound or a product of a disclosed method, wherein one or more of an antibody, a pVHL ligand, a non-pVHL ligand, a buffer, and a solvent is intimately mixed with an effective amount of the disclosed compound or the product of a disclosed method. [00244] In various aspects, the use relates to the modulation of von Hippel-Lindau protein in a sample. In a further aspect, the use relates to the modulation of von Hippel- Lindau protein in a buffer. In a further aspect, the use relates to the modulation of von Hippel-Lindau protein in a cell. [00245] In various aspects, the use relates to the identification of a von Hippel-Lindau protein ligand in a library. [00246] It is understood that the disclosed uses can be employed in connection with the disclosed compounds, methods, samples, mixtures, and kits. In a further aspect, the invention relates to the use of a disclosed compound, sample, or mixture for a fluorescence-based assay. 2. KITS [00247] In one aspect, disclosed are kits comprising a disclosed compound, and one or more of: (a) a sample that contains von Hippel-Lindau protein (pVHL); (b) a library that contains a plurality of ligands; (c) instructions for modulating pVHL; (d) instructions for identifying a pVHL ligand and/or a non-pVHL ligand; and (e) instructions for performing a fluorescence-based assay. [00248] [00249] In various aspects, the compounds, samples, and/or libraries described herein can be provided in a kit. The kit can also include combinations of the compounds, samples, and/or libraries. [00250] In various aspects, the informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or to the use of the agents for the methods described herein. For example, the informational material may relate to the use of the compounds to modulate pVHL, to identify pVHL ligands and/or non- pVHL ligands, or to perform a fluorescence-based assay. The kits can also include paraphernalia for administering the compounds of this invention to a sample such as, for example, a buffer or a cell (e.g., in culture). [00251] In various aspects, the compounds of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a fragrance or other assay ingredient (e.g., an antibody). In such aspects, the kit can include instructions for admixing the compound and the other ingredients, or for using one or more compounds together with the other ingredients. [00252] In a further aspect, the compound and the sample are co-formulated. In a still further aspect, the compound and the sample are co-packaged. [00253] The foregoing description illustrates and describes the disclosure. Additionally, the disclosure shows and describes only the preferred embodiments but, as mentioned above, it is to be understood that it is capable to use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the invention concepts as expressed herein, commensurate with the above teachings and/or the skill or knowledge of the relevant art. The embodiments described herein above are further intended to explain best modes known by applicant and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses thereof. Accordingly, the description is not intended to limit the invention to the form disclosed herein. Also, it is intended to the appended claims be construed to include alternative embodiments. [00254] All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls. G. EXAMPLES [00255] Herein, the first small molecule-based VHL fluorescent probe, BODIPY FL VH032 (5), is described, which is high affinity with a K d value of 3.01 nM to a VCB protein complex in a TR-FRET binding assay. In a VHL FP assay, the BODIPY FL VH032 (5) has a K d value of 100.8 nM to the VCB protein complex. Then, the newly developed BODIPY FL VH032 (5)-based TR-FRET and FP assays were used to test a panel of reported VHL ligands FIG.1) including VH032 (1), VH298 (2), MZ1 (3), VH032 amine (6), Me-VH032 amine (7), BOC-VH032 (8), VH032 phenol (9), and VH032-PEG4-amine (10) (Gadd, et al. (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514-521; Soares, et al. (2018) Group-Based Optimization of Potent and Cell- Active Inhibitors of the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Structure-Activity Relationships Leading to the Chemical Probe (2S,4R)-1-((S)-2-(1- Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydrox y-N-(4-(4-methylthiazol- 5-yl)benzyl)pyrrolidine-2-carboxamide (VH298). J Med Chem 61, 599-618; Lai, et al. (2016) Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL. Angew Chem Int Ed Engl 55, 807-810; Raina, et al.. (2016) PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc Natl Acad Sci U S A 113, 7124-7129; Maniaci, et al. (2017) Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat Commun 8, 830) and non-VHL ligands (FIG. 2) including (+)-JQ1 (4), Thalidomide-4’-oxyacetamido-alkylC4-amine (11, a cereblon E3 ligase ligand), and dBET1 (12, a bivalent BRD-CRBN PROTAC) (Winter, et al. (2015) DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376-1381). Only VHL ligands showed binding, demonstrating the specificity of both the BODIPY FL VH032 (5)-mediated TR-FRET and FP assays. Whereas the BODIPY FL VH032 (5)-mediated FP assay has similar sensitivity to the reported FP assay based on a FAM-labeled HIF-1α peptide (FAM-DEALAHyp- YIPMDDDFQLRSF, 19-mer), the BODIPY FL VH032 (5)-mediated TR-FRET assay was more sensitive and consumed less VCB protein than the reported FP assay. In addition, The BODIPY FL VH032 (5)-mediated TR-FRET assay was resistant to assay interference and capable of detecting VHL ligands with a wide range of binding affinity. In summary, a new and high-affinity VHL fluorescent probe BODIPY FL VH032 (5) has been developed, and has been used develop a TR-FRET assay that is sensitive, selective, resistant to assay interference, and suitable for VHL ligand identification and characterization through large scale screening, as further described herein below. [00256] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. [00257] The Examples are provided herein to illustrate the invention, and should not be construed as limiting the invention in any way. Examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way. 1. CHEMISTRY METHODS [00258] VH032 amine and VH032-PEG4-amine hydrochloride salt were purchased from MedChemExpress LLC (Monmouth Junction, NJ). BODIPY FL propionic acid was purchased from BroadPharm (San Diego, CA). Acetic anhydride, N,N-diisopropyl ethylamine (DIPEA), dichloromethane (DCM), dimethyl sulfoxide (DMSO), 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI), Hydroxybenzotriazole (HOBt) and all other basic chemical reagents and solvent were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide-d6 (DMSO-d6) and chloroform-d were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). [00259] Reported protocols (Lin, et al. (2014) Development of BODIPY FL vindoline as a novel and high-affinity pregnane X receptor fluorescent probe. Bioconjug Chem 25, 1664-1677) were adopted to assess or verify reaction progress, product purity and identity; to determine high-resolution mass spectra and to record 1 H and 13 C NMR spectra. a. (2S,4R)-1-((S)-2-ACETAMIDO-3,3-DIMETHYLBUTANOYL)-4- HYDROXY-N-(4-(4-METHYLTHIAZOL-5-YL)BENZYL)PYRROLIDINE-2- C ARBOXAMIDE (1, VH032) [00260] VH032 amine (6, 215 mg, 0.5 mmol) was solubilized in a stirred solution of DCM (5 mL) and DIPEA (263 µL, 1.5 mmol) with an ice-water batch. Ac2O (60 µL, 0.635 mmol) was then added. The ice-water bath was removed after 5 min and the reaction was continued for another 25 min under room temperature (RT). The reaction mixture was diluted with DCM (20 mL) and quenched with brine (30 mL). After separation from the brine, the DCM solution was washed with brine (20 mL × 2) and dried with anhydrous Na2SO4. White raw powder product was obtained after the solvent was removed from the DCM solution with an IKA RV 10 digital rotavapor (IKA Works, Inc., Wilmington, NC) and was further purified with an Acquity prep-UPLC system (Waters Corporation, Milford, MA) equipped with an Acquity UPLC BEH C181.7 µm, 2.1 × 50 mm column to yield the product VH032 (1, 143 mg, 60.6% yield and 98.0% purity) as a white solid. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.98 (s, 1H), 8.57 (t, J = 6.1 Hz, 1H), 7.95 (d, J = 9.3 Hz, 1H), 7.43 – 7.36 (m, 4H), 5.12 (d, J = 3.5 Hz, 1H), 4.54 (d, J = 9.4 Hz, 1H), 4.48 – 4.39 (m, 2H), 4.36 – 4.32 (m, 1H), 4.21 (dd, J = 15.9, 5.4 Hz, 1H), 3.72 – 3.60 (m, 2H), 2.44 (s, 3H), 2.08 – 2.00 (m, 1H), 1.93 – 1.89 (m, 1H), 1.88 (s, 3H), 0.93 (s, 9H). 13 C NMR (126 MHz, DMSO-d 6 ) δ 172.42, 170.14, 169.55, 151.94, 148.19, 139.99, 131.64, 130.10, 129.10, 127.88, 69.34, 60.23, 59.14, 56.86, 42.10, 38.43, 35.67, 26.83, 22.80, 16.42. ESI-TOF HRMS m/z: [M + H] + Calcd for C 24 H 33 N 4 O 4 S + 473.2217; Found 473.2225. b. (2S,4R)-1-((S)-2-(TERT-BUTYL)-21-(5,5-DIFLUORO-7,9-DIMETHYL- 5H-5Λ 4 ,6Λ 4 -DIPYRROLO[1,2-C:2',1'-F][1,3,2]DIAZABORININ-3-YL)- 4,19-DIOXO-6,9,12,15-TETRAOXA-3,18-DIAZAHENICOSANOYL)-4- HYDROXY-N-(4-(4-METHYLTHIAZOL-5-YL)BENZYL)PYRROLIDINE-2- CARBOXAMIDE (5, BODIPY FL VH032) [00261] Under room temperature, VH032-PEG4-amine hydrochloride (10, 50 mg, 0.075 mmol) was added to a solution of BODIPY FL propionic acid (13, 24.20 mg, 0.083 mmol) and DIPEA (29.2 mg, 0.226 mmol) in DMSO (1 mL). EDCI (21.66 mg, 0.113 mmol) and HOBt (12.21 mg, 0.090 mmol) were then added. The reaction was stirred for 3h and then the reaction mixture was directly purified by a Biotage Isolera four flash chromatography system (Biotage, LLC, Charlotte, NC) with a Sfär C18 flash column and gradient mobile phase (H2O + 0.1% formic acid → acetonitrile + 0.1% formic acid) to yield the product BODIPY FL VH032 (5, 45.4 mg, 64.3% yield and 95.9% purity) as a brown-red solid. 1 H NMR (500 MHz, Chloroform-d) δ 8.70 (s, 1H), 7.36 (dd, J = 7.6, 4.2 Hz, 5H), 7.07 (s, 1H), 6.87 (d, J = 4.0 Hz, 1H), 6.45 (s, 1H), 6.29 (d, J = 4.0 Hz, 1H), 6.10 (s, 1H), 4.73 (t, J = 8.0 Hz, 1H), 4.59 – 4.47 (m, 3H), 4.35 (dd, J = 15.0, 5.4 Hz, 1H), 4.06 (d, J = 11.0 Hz, 1H), 4.01 (d, J = 2.7 Hz, 2H), 3.71 – 3.54 (m, 13H), 3.50 (t, J = 5.2 Hz, 2H), 3.39 (dq, J = 8.3, 5.2, 4.6 Hz, 2H), 3.28 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 7.6 Hz, 2H), 2.56 – 2.47 (m, 7H), 2.24 (s, 3H), 2.18 – 2.10 (m, 1H), 0.94 (s, 9H). 13 C NMR (126 MHz, DMSO-d6) δ 170.11, 169.24, 167.47, 166.93, 157.44, 156.20, 149.80, 146.09, 142.39, 137.79, 132.76, 131.31, 129.48, 128.03, 127.26, 127.22, 127.03, 126.48, 125.80, 123.68, 118.59, 114.95, 68.78, 68.18, 68.13, 68.06, 67.94, 67.92, 67.89, 67.45, 67.22, 57.08, 54.92, 54.03, 40.01, 36.95, 36.26, 34.06, 31.97, 24.51, 22.31, 14.26, 12.84, 9.34. ESI-TOF HRMS m/z: [M + H] + Calcd for C 46 H 63 BF 2 N 7 O 9 S + 938.4464; Found 938.4484. 2. BIOLOGY METHODS [00262] Tb-anti-GST antibody, 1,4-dithiothreitol (DTT, 1 M), Tris (1 M, pH 7.5) and DMSO were purchased from Fisher Scientific (Pittsburgh, PA). HEPES (1 M, pH 7.4) was purchased from Teknova Inc (Hollister, CA). Triton X-100, Tween-20 and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). VH-298, VH032- amine, Me-VH032-amine and VH032-PEG4-NH 2 were purchased from MedChemExpress LLC (Monmouth Junction, NJ). BOC-VH032 was purchased from LabNetwork (Cambridge, MA). VH032 phenol and thalidomide-4’-O-acetamido-alkylC4-amine were purchased from Bio-Techne Corporation (Minneapolis, MN). dBET1, (+)-JQ1, and MZ1 were purchased from Cayman Chemical (Ann Arbor, Michigan). Echo 384-well low dead volume (LDV) compound plates were purchased from Labcyte Inc. (San Jose, CA).384-well, black low- volume assay plates were purchased from Corning Incorporated Life Sciences (Tewksbury, MA). c. GST-VCB PROTEIN PREPARATION [00263] The pGEX-4T-1-GST-VHL (54-213 aa) plasmid, pCDFDuet-1-flag-Elongin- C (17-112 aa)-strep II-Elongin-B (1-118 aa) plasmid and the GST-VCB protein complex were custom created, expressed and purified by GenScript USA, Inc. (Piscataway, NJ). Briefly, VHL (54-213 aa) was subcloned into the pGEX-4T-1-GST bacterial expression vector between the BamHI and XhoI restriction sites. Flag- Elongin-C (17-112 aa) and strep II-Elongin-B (1-118 aa) were respectively subcloned between the NcoI and HindIII restriction sites and between the NdeI and XhoI restriction sites into the pCDFDuet-1 bacterial expression vector. E.coli BL21(DE3) competent cells were transformed with the recombinant pGEX-4T-1-GST-VHL (54-213 aa) and pCDFDuet-1-flag-Elongin-C (17-112 aa)-strep II-Elongin-B (1-118 aa) plasmids. A single colony was inoculated into LB medium containing ampicillin and streptomycin and the culture was incubated in 37 °C at 200 rpm. Once the cell density reached to OD = 0.6-0.8 at 600 nm, 0.5 mM IPTG was introduced for induction at 25 °C for 16 h. Cells were then harvested and lysed. The supernatant of the cell lysate was subjected to a one-step purification by a GST column to provide the GST-VCB protein complex. Aliquots of the GST-VCB protein was stored under -80 °C in the buffer of 50 mM Tris (pH 8.0), 150 mM NaCl and 10% Glycerol. [00264] The protein sequence of the N-terminal GST-VHL (54–213 aa) protein is as follows: GST- Thrombin cleavage site-TEV cleavage site-VHL (54-213 aa, NCBI Reference Sequence: NP_000542.1). NO:1). [00265] The protein sequence of the N-terminal Flag-Elongin-C (17-112 aa) is as follows: Flag- Elongin-C (17-112 aa, NCBI Reference Sequence: XP_028374114.1). [00266] The protein sequence of the N-termianl Strep II-Elongin-B (1-118 aa) is as follows: Strep II-Elongin-B (1-118 aa, NCBI Reference Sequence: NP_009039.1). d. GENERAL TR-FRET AND FP BINDING ASSAY PROTOCOL [00267] The final DMSO concentration was 0.2% in all tests with 0.1% DMSO from the stock probe BODIPY FL VH032 (5) solution (1000x DMSO stock) and 0.1% DMSO from test compound stock solution (1000x DMSO stock). If there is no chemical tested under certain conditions, 0.1% DMSO was supplemented to make final 0.2% for these assay condition. The final assay volume was 20 µL/well and all assays were performed under room temperature (~ 25°C). All assays were performed three times independently with quadruplicate sample replicates. [00268] For the BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay, a reported TR-FRET assay protocol (Lin, et al. (2014) Development of BODIPY FL vindoline as a novel and high-affinity pregnane X receptor fluorescent probe. Bioconjug Chem 25, 1664-1677) was followed except that an Echo 555 Acoustic Liquid Handler (Labcyte Inc., San Jose, CA) was used to dispense chemicals and BODIPY FL VH032 (5), GST-VCB and Tb-anti-GST antibody were used. [00269] For the BODIPY FL VH032 (5)-mediated pVHL FP binding assay, the general TR-FRET assay protocol was followed except that the Tb-anti-anti-GST antibody was not added and the PHERAstar FS plate reader (BMG Labtech; Durham, NC) was equipped with a FP optic module (Excitation: 485 nm, Emission: 520 nm) to read FP assay signals. e. PVHL TR-FRET BINDING ASSAY BUFFER [00270] The pVHL TR-FRET binding assay buffer has a formula of 50 mM Tris pH 7.5, 0.1% Triton X-100, 0.01% bovine serum albumin, 1 mM DTT. It was freshly prepared every time before an experiment. f. PVHL FP ASSAY BUFFER [00271] The pVHL FP assay buffer has a formula of 25 mM HEPES pH 7.4, 0.01% Tween-20, 0.5 mM DTT, 0.01% BSA. It was freshly prepared every time before an experiment. g. CHEMICAL STOCK SOLUTION PREPARATION AND REAGENT DISPENSE [00272] Chemicals, including the fluorescent probe BODIPY FL VH032 (5), were solubilized in DMSO as 1,000× stock. Stock chemical DMSO solutions, positive control VH298 (2) and negative control DMSO were all plated in Echo LDV compound plates. For the probe or chemicals tested in dilutions, their stock dilutions were prepared in Echo LDV compound plates as 1,000× DMSO stocks for all concentration levels. During TR-FRET and FP assays, assay buffer (10 µL/well) was first dispensed. Fluorescent probe BODIPY FL VH032 (5) 1,000× time DMSO stock in dilutions or single concentration was dispensed (20 nL/well) with an Echo 555 Acoustic Liquid Handler, and then 1,000× time DMSO stock positive control VH298 (2), negative control DMSO, chemicals or dilutions of chemicals was transferred (20 nL/well) with the Echo 555 Acoustic Liquid Handler. Protein solution (2× stock, 10 µL/well) in corresponding assay buffer was finally dispensed to give a total of 20 µL/well assay volume. The fluorescent probe, each chemical, the positive control VH298 (2) or negative control DMSO (20 nL/well) was dispensed to a final total volume of 20 µL/well to achieve the 1-to-1,000× dilution. To avoid possible signal variation introduced by different DMSO concentrations (Lin, W., and Chen, T. (2018) Using TR-FRET to Investigate Protein- Protein Interactions: A Case Study of PXR-Coregulator Interaction. Adv Protein Chem Struct Biol 110, 31-63; Lin, W., Liu, J., Jeffries, C., Yang, L., Lu, Y., Lee, R. E., and Chen, T. (2014) Development of BODIPY FL vindoline as a novel and high-affinity pregnane X receptor fluorescent probe. Bioconjug Chem 25, 1664-1677; Lin, W., and Chen, T. (2013) A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay. Anal Biochem 443, 252-260), the 0.2% DMSO concentration was used for all assays in this article. h. DETERMINATION OF BODIPY FL VH032 (5) BINDING K D TO GST- VCB PROTEIN COMPLEX IN A TR-FRET BINDING ASSAY [00273] Dilutions of BODIPY FL VH032 (5, 1-to-2 dilutions, a concentration range of 0.06 nM to 500 nM) was incubated with 2 nM Tb-anti-GST along with, group 1: 2 nM GST- VCB + DMSO; group 2: 2 nM GST-VCB + VH298 (2, 30 µM); or group 3: without GST- VCB + DMSO. The TR-FRET signals were monitored every 30 min from 30 min to 300 min with a PHERAstar FS plate reader equipped with a TR-FRET optic module (excitation: 340 nm; emission 1: 520 nm; emission 2: 490 nm). The TR-FRET signals were fitted into GraphPad Prism 8.4.3 software (GraphPad Software; San Diego, CA) using a one-site total binding equation to derive curves for each group. The binding affinity K d values were derived from the group 1 with 2 nM GST-VCB. i. SIGNAL STABILITY TEST OF THE BODIPY FL VH032 (5)-MEDIATED PVHL TR-FRET BINDING ASSAY [00274] BODIPY FL VH032 (5, 4 nM) and 2 nM Tb-anti-GST was incubated with, group 1 (negative control group): 2 nM GST-VCB + DMSO; group 2 (positive control group): 2 nM GST-VCB + VH298 (2, 30 µM); group 3 (background control group): without GST-VCB + DMSO; or group 4 (positive control dose response group): 2 nM GST-VCB + dilutions of VH298 (2, 1-to-3 dilutions, a concentration range of 2.1 pM to 30 µM). The TR- FRET signals were monitored every 30 min from 30 min to 300 min. The TR-FRET signal of group 1, 2 or 3 was divided by that of group 2 to derive the TR-FRET signal fold change of each group. The TR-FRET signals or the signal fold changes of the group 1, 2 and 3 were plotted. The dose dependent TR-FRET signals of the positive control VH298 (2, group 4) were fitted into the GraphPad Prism software using a Sigmoidal dose-response equation to derive IC 50 values. [00275] Binding inhibitory activity test of selected pVHL ligands or non-ligands with the BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay. BODIPY FL VH032 (5, 4 nM) was incubated with the positive control VH298 (2, 30 µM), negative control DMSO, or dilutions of selected pVHL ligands or non-ligands (1-to-3 dilutions, a concentration range of 2.1 pM to 30 µM), along with 2 nM GST-VCB and 2 nM Tb-anti- GST. The TR-FRET signals were determined at the 90-min incubation time. The %Inhibition of each tested ligand at its individual concentration was calculated by normalized to that of the positive control VH298 (2, 30 µM) and negative control DMSO using Equation 1. (Signal Ligand ^ Signal 30μM VH298 ) %inhibition =100% ^ 100% ^ ( Signal DMSO ^ Signal 30μMVH298 ) Equation 1 [00276] When applicable, the normalized percent inhibition values for each ligand at various concentrations were fitted into a sigmoidal dose-response equation with the GraphPad Prism software to derive the IC 50 values. The TR-FRET K i values were then calculated with Equation 2 (the Cheng−Prusoff equation) (Cheng, Y., and Prusoff, W. H. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22, 3099-3108). Ki = IC 50 /(1 + [L]/KL) Equation 2 [00277] Where IC50 is the concentration of tested ligand that inhibits 50% of BODIPY FL VH032 (5) binding to GST-VCB, [L] is the BODIPY FL VH032 (5) concentration of 4 nM in the assay mixture, and KL is the Kd value of BODIPY FL VH032 (5) in the assay which is 3.01 nM. The TR-FRET K i values were used to compare the relative binding affinities of the test ligands to pVHL. j. BODIPY FL VH032 (5) CONCENTRATION OPTIMIZATION IN A PVHL FP ASSAY [00278] BODIPY FL VH032 (5, 70, 60, 50, 40, 30, 20, 10, 5, 2, or 1 nM) was incubated with dilutions of GST-VCB (1-to-2 dilutions, a concentration range of 0.03 nM to 1 µM). The FP signals were monitored with the PHERAstar FS plate reader equipped with a FP optic module (Excitation: 485 nm, Emission: 520 nm). The representative data collected at 90-min incubation time was plotted with the GraphPad Prism software. k. DETERMINATION OF BODIPY FL VH032 (5) BINDING KD TO GST- VCB PROTEIN COMPLEX IN A FP BINDING ASSAY [00279] BODIPY FL VH032 (5, 10 nM) was incubated with dilutions of GST-VCB (1-to-2 dilutions, a concentration range of 0.03 nM to 1 µM) along with the DMSO group or VH298 (2, 30 µM) group. In addition, BODIPY FL VH032 (5, 10 nM) was incubated with DMSO only, but without GST-VCB. The FP signals were determined with the PHERAstar FS plate reader. The representative data collected at 90-min incubation time was plotted with the GraphPad Prism software using a one-site total binding equation to derive curves for each group. The binding affinity K d values were derived from the DMSO with GST-VCB. l. BINDING ACTIVITY TEST OF SELECTED PVHL LIGANDS OR NON- LIGANDS WITH THE BODIPY FL VH032 (5)-MEDIATED PVHL FP BINDING ASSAY [00280] BODIPY FL VH032 (5, 10 nM) was incubated with the positive control VH298 (2, 30 µM), negative control DMSO, or dilutions of selected pVHL ligands or non- ligands (1-to-3 dilutions, a concentration range of 2.1 pM to 30 µM), along with 100 nM GST-VCB. The FP signals were determined at the 90-min incubation time. The FP signal fold change of the negative control DMSO group or the positive control VH298 (2, 30 µM) group was divided by that of the positive control VH298 (2, 30 µM) group to derive FP signal fold changes. The FP signals or the FP signal fold changes were plotted with the GraphPad Prism software. The %Inhibition of each tested ligand at its individual concentration was calculated by normalized to that of the positive control VH298 (2, 30 µM) and negative control DMSO using Equation 1. The FP K i values were calculated by the method developed by Nikolovska-Coleska et al. ((2004) Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal Biochem 332, 261-273) with the Ki calculator available at: http://www.umich.edu/~shaomengwanglab/software/calc_ki/index .html. The FP K i values were used to compare the relative binding affinities of the test ligands to pVHL. 3. RESULTS AND DISCUSSION a. SYNTHESIS OF VH032 (1) [00281] VH032 (1) is a potent VHL inhibitor (Frost, et al. (2016) Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition. Nat Commun 7, 13312; Soares, et al. (2018) Group-Based Optimization of Potent and Cell-Active Inhibitors of the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Structure- Activity Relationships Leading to the Chemical Probe (2S,4R)-1-((S)-2-(1- Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydrox y-N-(4-(4-methylthiazol- 5-yl)benzyl)pyrrolidine-2-carboxamide (VH298). J Med Chem 61, 599-618) and suitable for fluorescent probe development as a positive ligand control. However, it is not commercially available. VH032 (1) was prepared by acetylating the VH032 amine (6) with acetic anhydride (Ac2O) in the presence of N,N-diisopropylethylamine (DIPEA) with dichloromethane (DCM) as the solvent. The yield is 60.6% after purification with a Prep- HPLC system (Scheme 1). SCHEME 1. SYNTHETIC SCHEME OF VH032 (1) b. DESIGN OF BODIPY FL VH032 (5) [00282] The BODIPY FL VH032 (5) was designed based on the MZ1 (3). MZ1 (3) is a bivalent BRD-VHL PROTAC molecule with the BRD ligand (+)-JQ1 (4) joined to the VHL ligand VH032 (1) by a PEG linker (Gadd, et al. (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514-521). It maintains binding affinities to both BRD and pVHL proteins via its corresponding (+)-JQ1 (4) moiety and its VH032 (1) moiety. It was rationalized that a high affinity pVHL fluorescent probe will be obtained if the (+)-JQ1 (4) moiety in MZ1 (3) is replaced with a fluorescent moiety, such as a BODIPY fluorophore, while the PEG linker and VH032 portions remain intact (FIG.1). The BODIPY FL VH032 (5) was, thus, designed accordingly (FIG.3). c. SYNTHESIS OF BODIPY FL VH032 (5) [00283] The BODIPY FL VH032 (5) was prepared with an EDCI- and HOBt-mediated coupling (Chan, L. C., and Cox, B. G. (2007) Kinetics of Amide Formation through Carbodiimide/N-Hydroxybenzotriazole (HOBt) Couplings. The Journal of Organic Chemistry 72, 8863-8869) between VH032-PEG4-amine (10) and BODIPY FL propionic acid (13) in the presence of DIPEA under room temperature with a yield of 64.3% after flash column chromatography purification (Scheme 2). SCHEME 2. SYNTHETIC SCHEME OF BODIPY FL VH032 (5) d. BODIPY FL VH032 (5) DISPLAYED A HIGH BINDING AFFINITY TO PVHL IN A TR-FRET ASSAY [00284] To measure the binding affinity of BODIPY FL VH032 (5) to pVHL, dilutions of BODIPY FL VH032 (5, 1-to-2 dilutions, an optimized concentration range of 0.06 nM to 500 nM) were incubated with 2 nM Tb-anti-GST antibody in the presence of 2 nM GST- VCB. By comparison, groups of samples without GST-VCB or with additional VH298 (2, 30 µM) in the presence of 2 nM GST-VCB were also included to investigate the background interactions between the BODIPY FL VH032 (5) and the Tb-anti-GST antibody or between the BODIPY FL VH032 (5) and the complex of Tb-anti-GST antibody and GST-VCB protein in the presence of VH298 (2). The TR-FRET signals were measured at every 30 min from 30 min to 300 min. [00285] The TR-FRET signals of the groups with 2 nM GST-VCB and without GST- VCB were first analyzed by fitting in a one-site total binding equation with the GraphPad PRISM software (GraphPad Software; San Diego, CA) (FIG.4A). In the presence of Tb- anti-GST, the interaction between BODIPY FL VH032 (5) and GST-VCB increased exponentially in the BODIPY FL VH032 (5) concentration range of 0.06 nM to 15.6 nM, and then the interaction increased in a linear manner in the BODIPY FL VH032 (5) concentration range of 15.6 nM to 500 nM (top panel curves in FIG.4A). The binding dissociation constant (Kd) values were derived from the 2 nM GST-VCB group and the respective Kd values were 3.61, 3.22, 3.01, 3.04, 3.01, 2.98, 2.96, 2.98, 2.99 and 3.04 nM for the incubation times of 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 min. The binding dissociation constant (Kd) values were basically very stable at ca 3.0 nM from the 90 min to 300 min incubation time. In addition, the Kd value of ca 3.0 nM demonstrated it is a high affinity interaction between BODIPY FL VH032 (5) and GST-VCB. Foremost, BODIPY FL VH032 (5) is so far reported the first small molecule-based pVHL fluorescent probe. In the absence of GST- VCB, a linear and very low TR-FRET interaction was observed between BODIPY FL VH032 (5) and Tb-anti-GST in the entire BODIPY FL VH032 (5) concentration range of 0.06 nM to 500 nM which represents a low non-specific background interaction nature (bottom panel curves in FIG.4A) (Lin, W., and Chen, T. (2018) Using TR-FRET to Investigate Protein-Protein Interactions: A Case Study of PXR-Coregulator Interaction. Adv Protein Chem Struct Biol 110, 31-63; Lin, W., Liu, J., Jeffries, C., Yang, L., Lu, Y., Lee, R. E., and Chen, T. (2014) Development of BODIPY FL vindoline as a novel and high-affinity pregnane X receptor fluorescent probe. Bioconjug Chem 25, 1664-1677). [00286] Because the 90 min incubation time was the earliest signal stable time point, the 90 min time point was chosen for further examination with an additional test group, the group with 2 nM GST-VCB + VH298 (2, 30 µM), included. The three groups of data, 2 nM GST-VCB + DMSO, 2 nM GST-VCB + VH298 (2, 30 µM) and without GST-VCB + DMSO, at the 90 min incubation time point were graphed by fitting into the one-site total binding equation in GraphPad PRISM (FIG.4B). The curve derived from the group of data with 2 nM GST-VCB + DMSO represented the total interaction between BODIPY FL VH032 (5) and GST-VCB in the presence of Tb-anti-GST with a K d value of 3.01 nM (the top blue curve in FIG.4B). In the presence of VH298 (2, 30 µM) which is a potent pVHL inhibitor (Frost, J, et al. (2016) Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition. Nat Commun 7, 13312; Soares, P., et al. (2018) Group-Based Optimization of Potent and Cell-Active Inhibitors of the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Structure-Activity Relationships Leading to the Chemical Probe (2S,4R)-1-((S)-2-(1-Cyanocyclopropanecarboxamido)-3,3- dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzy l)pyrrolidine-2-carboxamide (VH298). J Med Chem 61, 599-618), as the red curve in the FIG.4B, the TR-FRET signal between BODIPY FL VH032 (5) and GST-VCB in the presence of Tb-anti-GST is very close to the TR-FRET signal observed in the group without GST-VCB (the background interaction curve or the green curve in FIG.4B). The overlap of VH298 (2, 30 µM) inhibited curve with the background curve demonstrated there was minimal non-specific interaction between BODIPY FL VH032 (5) and GST-VCB. [00287] In order to develop a TR-FRET assay to characterize pVHL ligands for their inhibitory binding activities, the concentration of the fluorescent probe BODIPY FL VH032 (5) is very important. To gain the insight of the most sensitive BODIPY FL VH032 (5) concentration for a TR-FRET assay, the fold changes of TR-FRET signal were plotted at various BODIPY FL VH032 (5) concentrations between two different group comparisons: with 2 nM GST-VCB + DMSO/with 2 nM GST-VCB + VH-298 (2, 30 μM) (blue curve in FIG.4C) and with 2 nM GST-VCB + DMSO/without GST-VCB + DMSO (red curve in FIG.4C). The curves of signal fold change overlapped well with only very small difference. The highest signal fold changes were observed at 3.9 or 7.8 nM of BODIPY FL VH032 (5): with 2 nM GST-VCB + DMSO/with 2 nM GST-VCB + VH-298 (2, 30 μM) curve at 7.8 nM (15.1-fold) and 2 nM GST-VCB + DMSO/without GST-VCB + DMSO curve at 3.9 nM (16.2-fold). Because using a probe concentration closer to its Kd concentration in an assay will lead to less deviation of K i calculation of tested ligands (Cheng, Y., and Prusoff, W. H. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22, 3099-3108), the BODIPY FL VH032 (5) at 4 nM which is close to its Kd concentration of 3.01 nM was selected for the further TR-FRET assay development. [00288] Referring to FIG.4A, the binding interaction of BODIPY FL VH032 (5) to 2 nM Tb-anti-GST in the presence of 2 nM GST-VCB or in the absence of GST-VCB at the designated incubation time is shown. The TR-FRET signals were expressed as relative TR- FRET units (RTU) which were calculated by 10,000 × 520nm/490 nm. [00289] Referring to FIG.4B, the binding interaction of BODIPY FL VH032 (5) to 2 nM Tb-anti-GST, with 2 nM GST-VCB, 2 nM GST-VCB + VH298 (2, 30 µM), or without GST-VCB + DMSO at the 90-min incubation time is shown. [00290] Referring to FIG.4C, fold changes of TR-FRET signal of BODIPY FL VH032 (5) with 2 nM GST-VCB + DMSO to 2 nM GST-VCB + VH298 (2, 30 µM) (blue curve), or to without GST-VCB + DMSO (red curve) are shown. e. THE BODIPY FL VH032 (5)-BASED PVHL TR-FRET BINDING ASSAY HAS STABLE SIGNAL [00291] An assay with stable signal is very important for obtaining consistent activities of tested compounds. The BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay has been established using the following assay conditions: 4 nM BODIPY FL VH032 (5), 2 nM GST-VCB, 2 nM Tb-anti-GST and a tentative 90-min incubation time based on the signal stability observed without competing compound [00292] To further evaluate the assay signal stability in the presence of competing compound, a potent pVHL inhibitor VH298 (2, 30 µM) was used as the positive control, and DMSO was used as the negative control (DMSO is a solvent used to prepare compound stock solutions). In addition, the group of samples with 4 nM BODIPY FL VH032 (5), 2 nM Tb- anti-GST and DMSO, but without GST-VCB was included as the background control. The performance of the controls at different incubation time points was summarized in the form of relative TR-FRET unit (FIG.5A) and signal fold change to 2 nM GST-VCB + VH298 (2, 30 µM) (FIG.5B). Consistent to the observation made without competing compound, the positive control group had TR-FRET signals (278.7 ± 4.5) or the signal fold change to 2 nM GST-VCB + VH298 (2, 30 µM) (1.00 ± 0.03) that were very similar to those (276.6 ± 5.4 for TR-FRET signals and 0.99 ± 0.03 for the fold signal change) of the background control group at all incubation times from 30 min to 300 min (FIG.5B). For the negative control group, the overall TR-FRET interaction signals was slightly low at 30 min and 60 min incubation time points with respective RTUs at 3451 count and 3679 count, but very stable with RTU at 3858 ± 46 from incubation times of 90 min to 300 min (FIG.5A). In terms of signal fold change to 2 nM GST-VCB + VH298 (2, 30 µM) (FIG.5B), the negative control group had 12.05 and 12.94-fold at corresponding 30- and 60-min incubation times, then stable at 13.93 ± 0.09 with a CV of 0.67% from the incubation time of 90 min to 300 min, which is consistent to the observation made without competing compound. Thus, the BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay maintains stable signal from incubation time of 90 min to 300 min. [00293] The signal stability of the assay was further evaluated with the positive control VH298 (2) in a dose dependent manner (1-to-3 dilutions, an optimized concentration range of 2.09 pM to 30 µM) (FIG.5C). IC 50 values of 42.17, 43.27, 44.46, 42.93, 43.20, 44.04, 43.86, 45.74, 45.13, and 48.27 nM were observed at respective time point from 30 min to 300 min, with the average IC 50 value being 44.31 nM with a standard deviation of 1.75 nM and a low CV of 3.95%. These results further demonstrated that the BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay has stable signal over a wide range of incubation times.90- min was chosen as the incubation time for the assay. [00294] Referring to FIG.5A, TR-FRET interaction of 4 nM BODIPY FL VH032 (5) and 2 nM Tb-anti-GST with 2 nM GST-VCB + DMSO (negative control), 2 nM GST-VCB + VH298 (2, 30 µM) (positive control), or without GST-VCB + DMSO (background control) at specified incubation time points is shown. [00295] Referring to FIG.5B, TR-FRET signal fold change to 2 nM GST-VCB + VH298 (2, 30 µM) of 2 nM GST-VCB + DMSO (negative control), 2 nM GST-VCB + VH298 (2, 30 µM) (positive control) or without GST-VCB + DMSO (background control) in the presence of 4 nM BODIPY FL VH032 (5) and 2 nM Tb-anti-GST at specified incubation time points is shown. [00296] Referring to FIG.5C, dose response inhibition curves of VH298 (2, 1-to-3 dilutions, a concentration range of 2.1 pM to 30 µM) at specified incubation time points in the presence of 4 nM BODIPY FL VH032 (5), 2 nM GST-VCB, and 2 nM Tb-anti-GST is shown. f. THE BODIPY FL VH032 (5)-MEDIATED PVHL TR-FRET BINDING ASSAY IS SENSITIVE AND SELECTIVE [00297] A panel of reported pVHL ligands including VH032 (1), VH298 (2), VH032 amine (6), Me-VH032 amine (7), BOC-VH032 (8), VH032 phenol (9), VH032-PEG4-amine (10), dual pVHL and BRD PROTAC ligand MZ1 (3), and non-pVHL ligands including (+)- JQ1 (4), Thalidomide-4’-oxyacetamido-alkylC4-amine (11) and dBET1 (12) were tested for their pVHL inhibitory activities in the new established BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay with an optimized assay condition of 4 nM BODIPY FL VH032 (5), 2 nM GST-VCB, 2 nM Tb-anti-GST and a 90-min incubation time, along with negative control DMSO and positive control VH298 (2, 30 µM). The dose response curves of the pVHL ligands VH032 (1), VH298 (2), VH032 amine (6), Me-VH032 amine (7), BOC- VH032 (8), and VH032 phenol (9) are summarized in FIG.6A. The dose response curves of PROTACs MZ1 (3) and dBET1 (12), together with the PROTAC components (+)-JQ1 (4), VH032-PEG4-amine (10) and Thalidomide-4’-oxyacetamido-alkylC4-amine (11), as well as the positive control VH298 (2) are listed in FIG.6B. The pVHL ligands VH032 (1), VH298 (2), VH032 amine (6), Me-VH032 amine (7), BOC-VH032 (8), VH032 phenol (9), VH032- PEG4-amine (10), dual pVHL and BRD PROTAC ligand MZ1 (3) had respective IC50 values of 77.8 nM, 44.0 nM, 13.3 µM, 7.9 µM, 4.9 µM, 34.0 nM, 5.9 nM, 14.7 nM and respective K i values of 33.4 nM, 18.9 nM, 5.7 µM, 3.4 µM, 2.1 µM, 14.6 nM, 6.8 nM, and 6.3 nM. Among the pVHL ligands tested, the most potent ligand was MZ1 (3) with a K i value of 6.3 nM and the least potent ligand was VH032 amine (6) with a Ki value of 5.7 µM. There was over 904- fold activity difference between the most and least potent ligands, indicating that the BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay is sensitive to characterize pVHL ligands with high, medium or low inhibitory activities. As expected, the non-pVHL ligands (+)-JQ1 (4, a BRD ligand), Thalidomide-4’-oxyacetamido-alkylC4-amine (11, a cereblon E3 ligase ligand) and dBET1 (12, a BRD-CRBN PROTAC) were inactive (FIG. 6B), demonstrating that the assay only selectively detects pVHL ligands. [00298] Referring to FIG.6A, dose response curves of pVHL ligands VH032 (1), VH298 (2), VH032 amine (6), Me-VH032 amine (7), BOC-VH032 (8), and VH032 phenol (9) are shown. [00299] Referring to FIG.6B, dose response curves of pVHL ligands of VH298 (2), MZ1 (3), VH032-PEG4-amine (10) and non-pVHL ligands (+)-JQ1 (4, a BRD ligand), Thalidomide-4’-oxyacetamido-alkylC4-amine (11, a cereblon E3 ligase ligand), and dBET1 (12, a BRD-CRBN PROTAC) are shown. g. BODIPY FL VH032 (5) CONCENTRATION OPTIMIZATION FOR A P VHL FP ASSAY [00300] To compare the TR-FRET assay format to an FP assay format, a pVHL FP assay was also developed with BODIPY FL VH032 (5) as the fluorescent probe. [00301] To establish an FP assay that is sensitive and robust, an optimal probe concentration is critical, because too much probe will decrease assay sensitivity while insufficient probe will reduce assay robustness. It was chosen to optimize the probe concentration by incubating dilutions of GST-VCB (1-to-2 dilutions, an optimal concentration range of 0.03 nM to 1000 nM) with BDOIPY FL VH032 (5) at 70, 60, 50, 40, 30, 20, 10, 5, 2, and 1 nM (FIG.7A and FIG.7B). BODIPY FL VH032 (5) at concentrations ranged from 70 nM to 10 nM (FIG.7A) did not affect the GST-VCB concentration curves, except slight FP signal increases at certain GST-VCB concentrations (125, 250, and 500 nM) with 10 nM BODIPY FL VH032 (5). However, lower concentrations of BODIPY FL VH032 (5) (5, 2, or 1 nM) (FIG.7B) caused a substantial background increase at lower GST-VCB concentrations (FIG.7B), with respective background interactions increased from 21 to 33, 52 and 71 mP for BODIPY FL VH032 (5) concentrations of 10, 5, 2, and 1 nM. In addition, significant FP signal variations were observed at lower BODIPY FL VH032 (5) concentrations, especially at 2 and 1 nM (FIG.7B).10 nM BODIPY FL VH032 (5) was chosen for the FP assay. [00302] Referring to FIG.7A, pVHL FP assay performance with BODIPY FL VH032 (5) concentrations at 70, 60, 50, 40, 30, 20, and 10 nM is shown. [00303] Referring to FIG.7B, pVHL FP assay performance with BODIPY FL VH032 (5) concentrations at 10, 5, 2, and 1 nM is shown. h. BODIPY FL VH032 (5) DISPLAYS HIGH PVHL AFFINITY IN AN FP ASSAY [00304] To determine the optimal GST-VCB concentration for a BODIPY FL VH032 (5)-mediated pVHL FP assay, 10 nM BODIPY FL VH032 (5) was incubated with dilutions of GST-VCB (1-to-2 dilutions, an optimized concentration range of 0.03 nM to 1000 nM) plus DMSO (total interaction) or VH298 (2, 30 µM) (GST-VCB-mediated non-specific interaction). In addition, 10 nM BODIPY FL VH032 (5) with DMSO only, but without GST- VCB was used to determine the background interaction. The FP signals from the 3 groups were fitted into the one-site total binding equation in GraphPad PRISM (FIG.8). The curve derived from the total interaction group (DMSO group) represented the total FP interaction between BODIPY FL VH032 (5) and GST-VCB with a Kd value of 100.8 nM (the blue curve in FIG.8). The FP signals from the GST-VCB-mediated non-specific interaction [in the presence of VH298 (2, 30 µM), the red curve in FIG.8] were very similar to those of the background interaction (without GST-VCB, the green curve in FIG.8), except that the FP signal (43.0 mP) at the highest GST-VCB concentration (1000 nM) in the presence of VH298 (2, 30 µM) was slightly higher than the background FP signal (24.8 mP).100 nM of GST- VCB was chosen, which is 10-time lower than the 1000 nM to minimize the the GST-VCB- mediated non-specific interaction (overlaps with the background signal). At 100 nM, the target protein GST-VCB has a concentration close to the Kd value of the fluorescent probe BODIPY FL VH032 (5) (100.8 nM), which helps maintain a balance between sensitivity and signal window for the FP assay (Nikolovska-Coleska, et al. (2004) Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal Biochem 332, 261-273). i. THE BODIPY FL VH032 (5)-MEDIATED PVHL FP ASSAY IS S ENSITIVE AND SELECTIVE IN DETECTING LIGANDS OF PVHL [00305] Next, the FP assay was applied with the established condition of 10 nM BODIPY FL VH032 (5), 100 nM GST-VCB, and a 90-min incubation time to characterize pVHL ligands for their binding affinities. The negative control (DMSO), positive control [VH298 (2, 30 µM)] and dilutions (1-to-3 dilutions, a concentration range of 2.1 pM to 30 µM) of the same panel of pVHL ligands and non-pVHL ligands as tested in the TR-FRET assay were tested in the FP assay. The negative control (DMSO) and the positive control [VH298 (2, 30 µM)] had corresponding FP signal of 143.75 and 14.5 mP (FIG.9A) and respective FP signal fold change to the positive control VH298 (2, 30 µM) of 9.91 and 1.00- fold (FIG.9B). The FP signal fold change of 9.91-fold between the negative control (DMSO) and the positive control [VH298 (2, 30 µM)] was only slightly less than that of the TR-FRET assay (13.88-fold). [00306] The dose response curves of the pVHL ligands VH032 (1), VH298 (2), VH032 amine (6), Me-VH032 amine (7), BOC-VH032 (8), and VH032 phenol (9) in the FP assay are summarized in FIG.9C, and those for the BRD-VHL PROTAC ligands MZ1 (3) and dBET1 (12), together with their PROTAC components (+)-JQ1 (4), VH032-PEG4-amine (10) and Thalidomide-4’-oxyacetamido-alkylC4-amine (11), as well as the positive control VH298 (2) in FIG.9D. pVHL ligands VH032 (1), VH298 (2), MZ1 (3), BOC-VH032 (8), VH032 phenol (9), and VH032-PEG4-amine (10) had respective pVHL inhibitory IC 50 values of 352.2 nM, 288.2 nM, 226.2 nM, 16.3 µM, 212.5 nM, and 430.8 nM and Ki values of 142.1 nM, 110.4 nM, 79.7 nM, 8.0 µM, 77.9 nM, and 181.0 nM. VH032 amine (6) had a maximal FP %Inhibition of only 36.6% at the maximal tested concentration of 30 µM; therefore, its IC 50 or K i value could not be determined with the FP assay. However, it had corresponding TR-FRET IC50 and Ki values of 13.3 µM and 5.7 µM (FIG.6A), thus, the TR-FRET assay is more sensitive than the FP assay in detecting pVHL ligands with lower binding affinity. Furthermore, the TR-FRET assay is more robust than the FP assay in testing ligands which may interfere with the assays. For example, in the FP assay, Me-VH032 amine (7) (the purple curve with purple solid inversed triangle in FIG.9C) disturbed the assay detection at concentration at or higher than 370 nM; however, such assay interference was not observed in the TR-FRET assay (FIG.6A). A respective IC50 and Ki values of 7.9 µM and 3.4 µM for Me-VH032 amine (7) was determined in the TR-FRET assay without any interference observed. However, the FP assay is as selective as the TR-FRET assay, because the non- pVHL ligands (+)-JQ1 (4), Thalidomide-4’-oxyacetamido-alkylC4-amine (11) and dBET1 (12) did not show pVHL inhibitory activity in the FP assay. [00307] Referring to FIG.9A, the FP assay performance of DMSO (the negative control group), VH298 (2, 30 µM) (the positive control group), and DMSO + without GST- VCB (the background control group) is shown. [00308] Referring to FIG.9B, the FP signal fold change [to VH298 (2, 30 µM, the positive control group] of DMSO (the negative control group), VH298 (2, 30 µM) (the positive control group) and DMSO + without GST-VCB (the background control group) is shown. [00309] Referring to FIG.9C, the FP dose response curves of pVHL ligands VH032 (1), VH298 (2), VH032 amine (6), Me-VH032 amine (7), BOC-VH032 (8), and VH032 phenol (9) are shown. [00310] Referring to FIG.9D, the FP dose response curves of pVHL ligands VH298 (2), MZ1 (3), VH032-PEG4-amine (10), and non-pVHL ligands of (+)-JQ1 (4, a BRD ligand), Thalidomide-4’-oxyacetamido-alkylC4-amine (11, a cereblon E3 ligase ligand), and dBET1 (12, a BRD-CRBN PROTAC) are shown. j. COMPARISON OF THE NEWLY DEVELOPED PVHL TR-FRT AND FP A SSAYS TO THE REPORTED PVHL FP ASSAY [00311] Fluorescently labeled peptides derived from HIF-1α protein have been used to characterize pVHL ligands in FP assays (Buckley, et al. (2012) Targeting the von Hippel- Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction. J Am Chem Soc 134, 4465-4468; Frost, J, et al. (2016) Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition. Nat Commun 7, 13312). Two versions of FAM- HIF-1α peptides, FAM-DEALAHyp-YIPD (10-mer, MW: 1477.48), and FAM-DEALAHyp-YIPMDDDFQLRSF (19-mer, M + H: 2617.167) have been reported. In one report (Buckley, et al. (2012) Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction. J Am Chem Soc 134, 4465-4468), the FAM-DEALAHyp-YIPD (10-mer) and FAM-DEALAHyp- YIPMDDDFQLRSF (19-mer) had respective K d of 560 nM and 36 nM with FP assays. The FAM-DEALAHyp-YIPMDDDFQLRSF (19-mer) has also been reported with a Kd of 3 nM under another FP assay condition (Frost, J, et al. (2016) Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition. Nat Commun 7, 13312). The small molecule probe BODIPY FL VH032 (5, MW: 937.91 Da) that was developed had a Kd value of 3.01 nM in the TR-FRET assay, which is similar to the reported Kd value of 3 nM of the FAM-DEALAHyp-YIPMDDDFQLRSF (19-mer, M + H: 2617.167) in one of the reported FP assay. However, BODIPY FL VH032 (5) is smaller in size based on its molecular weight, so it’s impressive for its Kd value to be similar to that of a big peptide. The BODIPY FL VH032 (5) had a FP Kd value of 100.8 nM, which is better than the FP Kd value of 560 nM for the 10-mer FAM-DEALAHyp-YIPD HIF-1α peptide (10-mer, MW: 1477.48), although the latter one was derived from protein with a bigger molecular size. [00312] Three pVHL ligands VH032 (1), VH298 (2) and BOC-VH032 (8) were tested in the FAM-HIF-1α peptide (19-mer)-based FP assay (the FP assay that reported the highest affinity peptide probe) and in the new BODIPY FL VH032 (5)-based TR-FRET and FP assays, and their activities summarized in Table 1. Although the affinity rank order [VH298 (2), VH032 (1), and BOC-VH032 (8) from high to low] is the same among the three assays, the BODIPY FL VH032 (5)-based TR-FRET assay is the most sensitive. The most potent inhibitor VH298 (2) had respective pVHL inhibitory activity values of 80 nM (Kd), 18.9 nM (K i ) and 110.4 nM (K i ) for the FAM-HIF-1α peptide (19-mer)-mediated FP assay, the BODIPY FL VH032 (5)-mediated TR-FRET and FP assay. For measuring the activity of VH298 (2), the BODIPY FL VH032 (5)-based TR-FRET assay was more sensitive (4.23- fold) than the FAM-HIF-1α peptide (19-mer)-based FP assay. Similar sensitivity improvements of the TR-FRET assay were also observed for VH032 (1) and BOC-VH032 (8) (4.49-fold and 3.09-fold, respectively). In addition, less VCB protein (2 nM) was consumed with the TR-FRET assay, compared to the best FP assay reported that consumed 15 nM VCB protein. However, the BODIPY FL VH032 (5)-based FP assay just had comparable sensitivity to the reported FP assay using FAM-HIF-1α peptide (19-mer) as the probe and reported the highest affinity for that peptide probe. [00313] The activities of the pVHL ligands active in both BODIPY FL VH032 (5)- mediated TR-FRET and FP assays are also summarized (Table 1). The TR-FRET assay is more sensitive (with lower Ki values) than the FP assay for all the active ligands including VH032 (1), VH298 (2), MZ1 (3), BOC-VH032 (8), VH032 phenol (9), and VH032-PEG4- amine (10). The most potent ligand tested with the TR-FRET assay was MZ1 (3) with a Ki value of 6.3 nM. The weakest pVHL ligand tested with the TR-FRET assay was VH032 amine (6) with a Ki value of 5.7 µM. Thus, the TR-FRET assay was able to detect compounds with various affinity, at least ranging from 6.3 nM to 5.7 µM in K i values based on the tested ligands. The activity rank orders are generally similar for those pVHL ligands tested with the two assays, although with slight differences observed. It has been reported that TR-FRET assay and FP assay may give slightly different activity rank orders when a common set of ligands are tested (Newman, M., and Josiah, S. (2004) Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system. J Biomol Screen 9, 525-532; Cashman, J. R., et al. (2010) Inhibition of Bfl-1 with N-aryl maleimides. Bioorg Med Chem Lett 20, 6560-6564; Klink, T. A., et al. (2008) Evaluating PI3 kinase isoforms using Transcreener ADP assays. J Biomol Screen 13, 476-485), but TR-FRET assay was believed to be superior than FP assay because of its lower assay variability, lower nonspecific interference, and better correlation to cell-based assay (Raucy, J. L., and Lasker, J. M. (2010) Current in vitro high throughput screening approaches to assess nuclear receptor activation. Curr Drug Metab 11, 806-814). TABLE 1. COMPARISON OF THE BEST REPORTED PVHL FP ASSAY AND THE PVHL TR- F RET AND FP ASSAYS DEVELOPED IN THIS REPORT
a : assay developed in this report; b : NA, not available. [00314] In conclusion, the BODIPY FL VH032 (5) has been developed as the first small molecule fluorescent probe with high affinity to pVHL (a K d value of 3.01 nM in a TR- FRET assay). The BODIPY FL VH032 (5)-mediated pVHL TR-FRET binding assay is more sensitive than reported FP assays and the FP assay developed based on the same BODIPY FL VH032 (5) as the fluorescent probe, and it is less susceptible to interference of certain as observed in an FP assay. Both BODIPY FL VH032 (5)-mediated TR-FRET and FP assays only selectively detect pVHL ligands. It has been reported that assays based on a high- affinity probe are more sensitive in detecting the binding of ligands with a wide range of inhibitory potency (Huang, X. (2003) Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand. J Biomol Screen 8, 34-38). The high-affinity VHL fluorescent probe BODIPY FL VH032 (5)-based TR-FRET assay is sensitive, selective, resistant to assay interference, and capable of detecting ligands with a wide range of activity, and is therefore suitable for identification and characterization of VHL ligands in large scale screenings. H. REFERENCES [00315] Kibel, A., Iliopoulos, O., DeCaprio, J. A., and Kaelin, W. G., Jr. (1995) Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C. Science 269, 1444-1446. [00316] Stebbins, C. E., Kaelin, W. G., Jr., and Pavletich, N. P. (1999) Structure of the VHL-ElonginC-ElonginB complex: implications for VHL tumor suppressor function. Science 284, 455-461. [00317] Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R., and Ratcliffe, P. J. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271-275. [00318] Buckley, D. L., Van Molle, I., Gareiss, P. C., Tae, H. S., Michel, J., Noblin, D. J., Jorgensen, W. L., Ciulli, A., and Crews, C. M. (2012) Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction. J Am Chem Soc 134, 4465-4468. [00319] Buckley, D. L., Gustafson, J. L., Van Molle, I., Roth, A. G., Tae, H. S., Gareiss, P. C., Jorgensen, W. L., Ciulli, A., and Crews, C. M. (2012) Small-molecule inhibitors of the interaction between the E3 ligase VHL and HIF1α. Angew Chem Int Ed Engl 51, 11463-11467. [00320] Van Molle, I., Thomann, A., Buckley, D. L., So, E. C., Lang, S., Crews, C. M., and Ciulli, A. (2012) Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface. Chem Biol 19, 1300-1312. [00321] Galdeano, C., Gadd, M. S., Soares, P., Scaffidi, S., Van Molle, I., Birced, I., Hewitt, S., Dias, D. M., and Ciulli, A. (2014) Structure-guided design and optimization of small molecules targeting the protein-protein interaction between the von Hippel-Lindau (VHL) E3 ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit with in vitro nanomolar affinities. J Med Chem 57, 8657-8663. [00322] Frost, J., Galdeano, C., Soares, P., Gadd, M. S., Grzes, K. M., Ellis, L., Epemolu, O., Shimamura, S., Bantscheff, M., Grandi, P., Read, K. D., Cantrell, D. A., Rocha, S., and Ciulli, A. (2016) Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition. Nat Commun 7, 13312. [00323] Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G., Jr., and Pavletich, N. P. (2002) Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science 296, 1886-1889. [00324] Hon, W. C., Wilson, M. I., Harlos, K., Claridge, T. D., Schofield, C. J., Pugh, C. W., Maxwell, P. H., Ratcliffe, P. J., Stuart, D. I., and Jones, E. Y. (2002) Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature 417, 975-978. [00325] Wang, Y., Jiang, X., Feng, F., Liu, W., and Sun, H. (2020) Degradation of proteins by PROTACs and other strategies. Acta Pharm Sin B 10, 207-238. [00326] Gadd, M. S., Testa, A., Lucas, X., Chan, K. H., Chen, W., Lamont, D. J., Zengerle, M., and Ciulli, A. (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13, 514-521. [00327] Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., Morse, E. M., Keates, T., Hickman, T. T., Felletar, I., Philpott, M., Munro, S., McKeown, M. R., Wang, Y., Christie, A. L., West, N., Cameron, M. J., Schwartz, B., Heightman, T. D., La Thangue, N., French, C. A., Wiest, O., Kung, A. L., Knapp, S., and Bradner, J. E. (2010) Selective inhibition of BET bromodomains. Nature 468, 1067-1073. [00328] Soares, P., Gadd, M. S., Frost, J., Galdeano, C., Ellis, L., Epemolu, O., Rocha, S., Read, K. D., and Ciulli, A. (2018) Group-Based Optimization of Potent and Cell-Active Inhibitors of the von Hippel-Lindau (VHL) E3 Ubiquitin Ligase: Structure-Activity Relationships Leading to the Chemical Probe (2S,4R)-1-((S)-2-(1- Cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydrox y-N-(4-(4-methylthiazol- 5-yl)benzyl)pyrrolidine-2-carboxamide (VH298). J Med Chem 61, 599-618. [00329] Testa, A., Lucas, X., Castro, G. V., Chan, K. H., Wright, J. E., Runcie, A. C., Gadd, M. S., Harrison, W. T. A., Ko, E. J., Fletcher, D., and Ciulli, A. (2018) 3-Fluoro-4- hydroxyprolines: Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation. J Am Chem Soc 140, 9299-9313. [00330] Crew, A. P., Raina, K., Dong, H., Qian, Y., Wang, J., Vigil, D., Serebrenik, Y. V., Hamman, B. D., Morgan, A., Ferraro, C., Siu, K., Neklesa, T. K., Winkler, J. D., Coleman, K. G., and Crews, C. M. (2018) Identification and Characterization of Von Hippel- Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1. J Med Chem 61, 583-598. [00331] Zoppi, V., Hughes, S. J., Maniaci, C., Testa, A., Gmaschitz, T., Wieshofer, C., Koegl, M., Riching, K. M., Daniels, D. L., Spallarossa, A., and Ciulli, A. (2019) Iterative Design and Optimization of Initially Inactive Proteolysis Targeting Chimeras (PROTACs) Identify VZ185 as a Potent, Fast, and Selective von Hippel-Lindau (VHL) Based Dual Degrader Probe of BRD9 and BRD7. J Med Chem 62, 699-726. [00332] Lea, W. A., and Simeonov, A. (2011) Fluorescence polarization assays in small molecule screening. Expert Opin Drug Discov 6, 17-32. [00333] Parker, G. J., Law, T. L., Lenoch, F. J., and Bolger, R. E. (2000) Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand- binding and kinase/phosphatase assays. J Biomol Screen 5, 77-88. [00334] Du, Y., Nikolovska-Coleska, Z., Qui, M., Li, L., Lewis, I., Dingledine, R., Stuckey, J. A., Krajewski, K., Roller, P. P., Wang, S., and Fu, H. (2011) A dual-readout F2 assay that combines fluorescence resonance energy transfer and fluorescence polarization for monitoring bimolecular interactions. Assay Drug Dev Technol 9, 382-393. [00335] Moerke, N. J. (2009) Fluorescence Polarization (FP) Assays for Monitoring Peptide-Protein or Nucleic Acid-Protein Binding. Curr Protoc Chem Biol 1, 1-15. [00336] Lin, W., and Chen, T. (2018) Using TR-FRET to Investigate Protein-Protein Interactions: A Case Study of PXR-Coregulator Interaction. Adv Protein Chem Struct Biol 110, 31-63. [00337] Lin, W., Liu, J., Jeffries, C., Yang, L., Lu, Y., Lee, R. E., and Chen, T. (2014) Development of BODIPY FL vindoline as a novel and high-affinity pregnane X receptor fluorescent probe. Bioconjug Chem 25, 1664-1677. [00338] Lin, W., and Chen, T. (2013) A vinblastine fluorescent probe for pregnane X receptor in a time-resolved fluorescence resonance energy transfer assay. Anal Biochem 443, 252-260. [00339] Thorne, N., Auld, D. S., and Inglese, J. (2010) Apparent activity in high- throughput screening: origins of compound-dependent assay interference. Curr Opin Chem Biol 14, 315-324. [00340] Glickman, J. F., Wu, X., Mercuri, R., Illy, C., Bowen, B. R., He, Y., and Sills, M. (2002) A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. J Biomol Screen 7, 3-10. [00341] Lai, A. C., Toure, M., Hellerschmied, D., Salami, J., Jaime-Figueroa, S., Ko, E., Hines, J., and Crews, C. M. (2016) Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL. Angew Chem Int Ed Engl 55, 807-810. [00342] Raina, K., Lu, J., Qian, Y., Altieri, M., Gordon, D., Rossi, A. M., Wang, J., Chen, X., Dong, H., Siu, K., Winkler, J. D., Crew, A. P., Crews, C. M., and Coleman, K. G. (2016) PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc Natl Acad Sci U S A 113, 7124-7129. [00343] Maniaci, C., Hughes, S. J., Testa, A., Chen, W., Lamont, D. J., Rocha, S., Alessi, D. R., Romeo, R., and Ciulli, A. (2017) Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat Commun 8, 830. [00344] Winter, G. E., Buckley, D. L., Paulk, J., Roberts, J. M., Souza, A., Dhe- Paganon, S., and Bradner, J. E. (2015) DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376-1381. [00345] Chan, L. C., and Cox, B. G. (2007) Kinetics of Amide Formation through Carbodiimide/N-Hydroxybenzotriazole (HOBt) Couplings. The Journal of Organic Chemistry 72, 8863-8869. [00346] Cheng, Y., and Prusoff, W. H. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22, 3099-3108. [00347] Nikolovska-Coleska, Z., Wang, R., Fang, X., Pan, H., Tomita, Y., Li, P., Roller, P. P., Krajewski, K., Saito, N. G., Stuckey, J. A., and Wang, S. (2004) Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization. Anal Biochem 332, 261-273. [00348] Newman, M., and Josiah, S. (2004) Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system. J Biomol Screen 9, 525-532. [00349] Cashman, J. R., MacDonald, M., Ghirmai, S., Okolotowicz, K. J., Sergienko, E., Brown, B., Garcia, X., Zhai, D., Dahl, R., and Reed, J. C. (2010) Inhibition of Bfl-1 with N-aryl maleimides. Bioorg Med Chem Lett 20, 6560-6564. [00350] Klink, T. A., Kleman-Leyer, K. M., Kopp, A., Westermeyer, T. A., and Lowery, R. G. (2008) Evaluating PI3 kinase isoforms using Transcreener ADP assays. J Biomol Screen 13, 476-485. [00351] Raucy, J. L., and Lasker, J. M. (2010) Current in vitro high throughput screening approaches to assess nuclear receptor activation. Curr Drug Metab 11, 806-814. [00352] Huang, X. (2003) Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand. J Biomol Screen 8, 34-38. [00353] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.