Acetyl coenzyme A analogues as rationally designed inhibitors of citrate synthase.

In this study we probe inhibition of pig heart citrate synthase (E. C. 4.1.3.7) by synthesis of seven analogues designed to mimic either the proposed enolate intermediate in this enzyme reaction or develop from historical inhibitors. The most potent inhibitor was fluorovinyl thioether 9 (Ki = 4.3 μM), where a fluorine replaces the oxygen atom of the enolate. A comparison of the potency of 9 versus its non-fluorinated vinyl thioether analogue 10 (Ki = 68.3 μM), revealed a clear 'fluorine effect' favouring 9 by an order of magnitude. Inhibitors 11 and 12 were dethia analogues of 9 and 10; these proved to be poor inhibitors. Methyl sulfoxide 13 was a moderate inhibitor (Ki = 11.1 μM) suggesting hydrogen bonding interactions in the enoate site. Finally two propenoate thioether isomers 14(E) and 15(Z) were explored as conformationally constrained carboxylates, but these were not inhibitors. All compounds were prepared by the synthesis of the appropriate pantetheinyl diol and then assembly of the coenzyme A structure using a three enzyme biotransformation protocol. A computational QM study, modelling both inhibitors 9 and 10 into the active site indicated short CF…H contacts ~2.0Å, consistent with fluorine making two stabilising hydrogen bonds, and mimicking an enolate rather than an enol intermediate. Computation also indicated that when 9 binds to citrate synthase this increases the basicity of a key Asp carboxylate which becomes protonated.