Abstract
α-Methylacyl-CoA racemase (AMACR; P504S) catalyses a key step in the degradation of branched-chain fatty acids and is important for the pharmacological activation of Ibuprofen and related drugs. Levels of AMACR are increased in prostate and other cancers, and it is a drug target. Development of AMACR as a drug target is hampered by lack of a convenient assay. AMACR irreversibly catalyses the elimination of HF from 3-fluoro-2-methylacyl-CoA substrates, and this reaction was investigated for use as an assay. Several known inhibitors and alternative substrates reduced conversion of 3-fluoro-2-methyldecanoyl-CoA by AMACR, as determined by (1)H NMR. The greatest reduction of activity was observed with known potent inhibitors. A series of novel acyl-CoA esters with aromatic side chains were synthesised for testing as chromophoric substrates. These acyl-CoA esters were converted to unsaturated products by AMACR, but their use was limited by non-enzymatic elimination. Fluoride sensors were also investigated as a method of quantifying released fluoride and thus AMACR activity. These sensors generally suffered from high background signal and lacked reproducibility under the assay conditions. In summary, the elimination reaction can be used to characterise inhibitors, but it was not possible to develop a convenient colorimetric or fluorescent assay using 3-fluoro-2-methylacyl-CoA substrates.
Highlights
Introduction αMethylacyl-CoA racemase (AMACR, P504S; E.C. 5.1.99.4) catalyses a key step in the degradation of branched-chain fatty acids.[1,2,3] The enzyme catalyses the conversion of either epimer of a 2-methylacyl-CoA ester into a ca. 1 : 1 mixture of 2R- and 2S-epimers.[4,5] β-Oxidation of 2-methylacyl-CoAs requires 2Sconfiguration,[6,7] but both R- and S-2-methylacyl-CoAs are produced in vivo and are derived from dietary fatty acids.[3]
AMACR irreversibly catalyses the elimination of HF from 3-fluoro-2-methylacyl-CoA substrates, and this reaction was investigated for use as an assay
Use of the elimination assay for inhibitor characterisation was initially performed by incubation of recombinant human AMACR 1A4 with a series of known ‘inhibitors’ (Table 1) and substrate 1
Summary
AMACR irreversibly catalyses the elimination of HF from 3-fluoro-2-methylacyl-CoA substrates, and this reaction was investigated for use as an assay. The elimination reaction can be used to characterise inhibitors, but it was not possible to develop a convenient colorimetric or fluorescent assay using 3-fluoro-2-methylacyl-CoA substrates. Assays using fluoride-specific electrodes to measure enzyme activity have been reported,[36,37,38] but these are generally low-throughput, require relatively large volumes and are not easy to adapt to a microtitre plate format.[39] A number of highly sensitive molecular fluoride sensors have been reported in the literature, which give an increase in absorbance or fluorescence upon reaction with fluoride. The use of 3-fluoro-2-methylacyl-CoA substrates with aromatic side-chains and fluoride sensors in order to translate this reaction into a colorimetric or fluorescent assay format is investigated
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