A convenient colorimetric assay for α-methylacyl-CoA racemase (AMACR; P504S) and testing of inhibitors

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. Diminution of AMACR levels using siRNA reduces proliferation of cancer cells. Therapeutic development of AMACR inhibitors has been hampered by the lack of a convenient assay. A novel substrate, 3-(2,4-dinitrophenoxy)-2-methylpropanoyl-CoA, was designed and synthesized. Incubation with active AMACR results in the elimination of the strongly yellow 2,4-dinitrophenoxide. A series of acyl-CoA substrates, previously reported AMACR inhibitors and non-specific protein modifying agents were tested as inhibitors using this assay. The fully-developed assay was used to determine IC50 values and other inhibitor characteristics. Medium-length straight-chain acyl-CoA esters (C6 – C10) were modest competitive inhibitors of AMACR, with IC50 values of 3 - 16 µM. R- and S-2-methyldecanoyl-CoA were ~ 3-fold more potent inhibitors than decanoyl-CoA. Ibuprofenoyl-CoA and similar compounds were reversible inhibitors with an IC50 value of ~0.5 µM. N-dodecyl-N-methylcarbamoyl-CoA (IC50 = 400 pM) was the most potent reversible inhibitor. Ebselen Oxide was a non-specific inactivator of AMACR, whilst Rose Bengal degraded the enzyme in a time and light-dependent manner. This assay will facilitate exploration of the biology of AMACR and the development of inhibitors as anti-cancer agents.

This work was funded by Prostate Cancer UK (S10-03 and PG14-009), a University of Bath Overseas University Research Studentship, and a Biochemical Society Summer Vacation Studentship Award.