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 (IC50â¯=â¯22-100â¯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⯵M, but lacked activity at 100⯵M. This is the first extensive structure-activity relationship study on the influence of side-chain interactions on the potency of novel rationally designed AMACR inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Prostatic Neoplasms/drug therapy , Racemases and Epimerases/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Male , Molecular Structure , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Racemases and Epimerases/metabolism , Structure-Activity RelationshipABSTRACT
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 (IC50â¯=â¯400-750â¯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.
Subject(s)
Acyl Coenzyme A/chemistry , Enzyme Inhibitors/chemistry , Racemases and Epimerases/antagonists & inhibitors , Acyl Coenzyme A/chemical synthesis , Drug Design , Enzyme Assays , Enzyme Inhibitors/chemical synthesis , Humans , Hydrophobic and Hydrophilic Interactions , Isoenzymes/antagonists & inhibitors , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
α-Methylacyl-CoA racemase (AMACR; P504S) regulates branched-chain fatty acid degradation, activates Ibuprofen and is a recognised cancer drug target. A novel, facile colorimetric assay was developed based on elimination of 2,4-dinitrophenolate. The assay was used to test 5 known inhibitors, determining IC50 and Ki values, reversibility and characterizing irreversible inhibition.
Subject(s)
Colorimetry/methods , Hydroxybenzoates/chemistry , Nitro Compounds/chemistry , Racemases and Epimerases/analysis , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Molecular Structure , Racemases and Epimerases/antagonists & inhibitors , Racemases and Epimerases/metabolism , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Acyl Coenzyme A/pharmacology , Drug Evaluation, Preclinical , Enzyme Assays , Enzyme Inhibitors/pharmacology , Esters/pharmacology , Racemases and Epimerases/antagonists & inhibitors , Racemases and Epimerases/metabolism , Acyl Coenzyme A/chemical synthesis , Acyl Coenzyme A/chemistry , Biocatalysis , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Esters/chemical synthesis , Esters/chemistry , Humans , Molecular Structure , Racemases and Epimerases/chemistry , Structure-Activity RelationshipABSTRACT
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.
