Identification of novel small-molecule inhibitors of α-methylacyl-CoA racemase (AMACR; P504S) and structure-activity relationships

Abstract

α-Methylacyl-CoA racemase (AMACR; P504S; EC 5.1.99.4) catalyses epimerization of 2-methylacyl-CoAs and is important for the degradation of branched-chain fatty acids and the pharmacological activation of ibuprofen and related drugs. It is also a novel drug target for prostate and other cancers. However, development of AMACR as a drug target has been hampered by the difficulties in assaying enzyme activity. Consequently, reported inhibitors have been rationally designed acyl-CoA esters, which are delivered as their carboxylate prodrugs. The novel colorimetric assay for AMACR based on the elimination of 2,4-dinitrophenolate was developed for high-throughput screening and 20,387 'drug-like compounds' were screened, with a throughput of 768 compounds assayed per day. Pyrazoloquinolines and pyrazolopyrimidines were identified as novel scaffolds and investigated as AMACR inhibitors. The most potent inhibitors have IC50 values of ~2 µM. The pyrazoloquinoline inhibitor 10a displayed uncompetitive inhibition, whilst 10j displayed mixed competitive inhibition. The pyrazolopyr-imidine inhibitor 11k displayed uncompetitive inhibition. This is the first report of the identification of specific drug-like small-molecule AMACR inhibitors by high-throughput screening. Pyrazoloquinolines and pyr-azolopyrimidines may also be useful as inhibitors of other CoA-utilizing enzymes.

Current Knowledge on the Function of α-Methyl Acyl-CoA Racemase in Human Diseases

“…One of the inhibitors found to date, N-Dodecyl-N-methylcarbamoyl-CoA 1 is a representative inhibitor ( Yevglevskis et al, 2017 ). In other prostate cells expressing AMACR (LAPC4, LNCaP, and PC3), ebselen and ebselen are selective covalent inhibitors, and 2-trifluoromethyl tetradecanoyl-CoA and E-13-iodo-2 methylenetridec-12-enoyl-CoA as competitive inhibitors Was reported ( Carnell et al, 2007 , 2013 ; Morgenroth et al, 2011 ; Festuccia et al, 2014 ; Yevglevskis et al, 2018 , 2019 ; Petrova et al, 2019 ). These results suggest that ibuprofen and other nonsteroidal anti-inflammatory drugs are effective in preventing prostate and colon cancer, and this effect may be caused by AMACR inhibition based on previous studies ( He et al, 2018 ).…”

“…Based on these results, we hypothesize that this diet can be used for the prevention of breast cancer, ovarian cancer, and other major cancers. According to the results reported so far, target drugs that inhibit the expression of AMACR in prostate cancer are typically ibuprofenoyl-CoA derivatives ( Petrova et al, 2019 ), and 2-(phenylthio)propanoyl-CoA derivatives ( Yevglevskis et al, 2018 ). All of these have been reported to be involved in the growth and survival of cancer cells by inhibiting the expression of AMACR in cancer cells.…”

Phase‐Regulated Sensing Mechanism of MoS2 Based Nanohybrids toward Point‐of‐Care Prostate Cancer Diagnosis

“…AMACR functions to metabolize certain fatty acids in human system, such as pristanic acid. [ 26,27 ] We herein utilize this specific metabolism pathway for AMACR detection. The functions of AMACR is to invert the 2R‐isomer of pristanic acid (as the coenzyme A ester) to its 2S form so and further processed by β‐oxidation.…”

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition

“…120,121,161 Attempts to use fluoride sensors to measure enzymatic activity with substrates eliminating fluorine has met with limited success. 161 A notable example of this approach is the elimination reaction of an unnatural acyl-CoA substrate 37 by AMACR to give 2,4dinitrophenoxide 38 and acyl-CoA 39 (Scheme 9); 127 this assay was used in a high-throughput screening campaign of 20 387 compounds which identified novel pyrazoloquinolines and pyrazolopyrimidines as inhibitors 163 and also in the first extensive inhibitor structure-activity relationship studies on any racemase/epimerase (vide infra). 164,165…”