A versatile continuous colorimetric assay for AMACR is reported.

α-Methylacyl-CoA racemase (AMACR; P504S) catalyses 'racemization' of 2-methylacyl-CoAs, the activation of R-ibuprofen and is a promising cancer drug target. Human recombinant AMACR 1A catalyses elimination of 3-fluoro-2-methyldecanoyl-CoAs to give E-2-methyldec-2-enoyl-CoA and fluoride anion, a previously unknown reaction. 'Racemization' of 2-methyldec-3-enoyl-CoAs was also catalysed, without double bond migration.

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the 'racemisation' reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure-activity relationship study has been performed. This paper describes the first structure-activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC = 400-750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure-activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.

The elimination of fluoride from 3-fluoro-2-methylacyl-CoA substrates by α-methylacyl-CoA racemase (AMACR 1A; P504S) was investigated as a method for determining enzyme activity and inhibitor potency.

Branched-chain fatty acid metabolism Castrate-resistant prostate cancer (CRPC) Drug lipophilicity Enzyme inhibitors Ibuprofen Mixed competitive inhibition Rational drug design Structure-activity relationships ABSTRACT α-Methylacyl-CoA racemase (AMACR; P504S) catalyses an essential step in the degradation of branched-chain fatty acids and the activation of ibuprofen and related drugs. AMACR has gained much attention as a drug target and biomarker, since it is found at elevated levels in prostate cancer and several other cancers. Herein, we report the synthesis of 2-(phenylthio)propanoyl-CoA derivatives which provided potent AMACR inhibitory activity (IC50 = 22-100 nM), as measured by the AMACR colorimetric activity assay. Inhibitor potency positively correlates with calculated logP, although 2-(3-benzyloxyphenylthio)propanoyl-CoA and 2-(4-(2-methylpropoxy) phenylthio)propanoyl-CoA were more potent than predicted by this parameter. Subsequently, carboxylic acid precursors were evaluated against androgen-dependent LnCaP prostate cancer cells and androgen-independent Du145 and PC3 prostate cancer cells using the MTS assay. All tested precursor acids showed inhibitory activity against LnCaP, Du145 and PC3 cells at 500 µM, but lacked activity at 100 µM. This is the first extensive structureactivity relationship study on the influence of side-chain interactions on the potency of novel rationally designed AMACR inhibitors.