High-Throughput Screening to identify novel inhibitors of human α-methylacyl-CoA racemase 1A (AMACR; P504S)

α-Methylacyl-CoA racemase (AMACR; P504S) catalyzes a key step in the degradation of branched-chain fatty acids and is important for the pharmacological activation of Ibuprofen and related drugs. Both the concentration and activity of AMACR are increased in prostate and other cancer cells, and the enzyme is a recognized drug target. However, all of the reported inhibitors are acyl-CoA esters (which do not comply with Lipinski guidelines) or non-specific protein modifying agents.
Libraries of ~20,000 drug-like compounds were screened using a novel colorimetric assay; Incubation of R,S-2-3-(2,4-dinitrophenoxy)-2-methylpropanoyl-CoA with active AMACR resulted in the elimination of the strongly yellow 2,4-dinitrophenoxide and allows continuous measurement of activity in a microtitre plate format. Inhibitors were identified by a reduction in the rate of reaction in the presence of the library compound vs. the control. A number of novel reversible inhibitors were identified and their potency determined using dose-response curves. The results demonstrate the utility of the assay for the discovery and characterization of AMACR inhibitors as anti-cancer agents.

This work was funded by Prostate Cancer UK (PG14-009), a Biochemical Society Summer Vacation Studentship Award, The Nuffield Foundation and MRC technology.