Mechanism and Inhibition of Bacterial Enoyl-ACP Reductases: Towards Novel Antibacterial Drugs
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Resistance to existing antimicrobial agents is a global threat to human health care, and new drugs with novel mechanisms of action are required in order to keep pace with the emergence of drug resistant pathogens. The bacterial fatty acid biosynthesis (FAS-II) pathway represents a validated yet relatively unexploited target for new antibiotic development and consequently there is significant interest in developing potent inhibitors of FAS-II enzymes. In this work we have focused on the FAS-II enoyl-ACP reductase enzyme which is known to be essential for bacterial survival. We cloned, expressed and purified the enoyl-ACP reductase from Francisella tularensis (ftuFabI). The enzyme was shown to catalyze substrate reduction through a sequential bi bi mechanism and to prefer substrate with long (C12) acyl chains. In addition, a series of diaryl ether inhibitors were designed and synthesized. These compounds are all subnanomolar slow binding inhibitors of ftuFabI with MIC values as low as 0.00018 æg/ml. A linear correlation between the Ki values for enzyme inhibition and the MIC values for inhibiting bacterial growth strongly suggests that ftuFabI is the primary cellular target of these compounds. Finally, we found that the in vivo efficacy of the inhibitors correlates best with their residence time on the enzyme but not with their thermodynamic affinity (Ki) for the enzyme, nor with their MIC values for inhibiting bacterial growth in vitro. We conclude therefore that residence time is the best predictor of the drug's in vivo efficacy. Studies on enoyl-ACP reductases have also been extended to the pathogen Burkholderia mallei which contains both FabI (bmFabI) and FabV (bmFabV) homologues of this enzyme. FabV is a newly discovered member of the enoyl-ACP reductase family, and so steady-state kinetics together with site directed mutagenesis have been used to study the reaction mechanism of bmFabV and to explore the role of proposed catalytic residues in substrate reduction. Rational drug design together with structure activity relationship (SAR) studies has lead to the identification of several novel bmFabV inhibitors that have submicromolar affinity for the enzyme. Finally, since there are two enoyl-ACP reductases (bmFabI and bmFabV) in B. mallei, the in vivo function of both enzymes has been investigated. Cell complementation experiments and kinetic studies suggest that bmFabV might function in unsaturated fatty acid biosynthesis while bmFabI only reduces the intermediates for saturated fatty acid biosynthesis.