Investigating Bacterial Malonyl-COA:Acyl Transferase as a Potential Secondary Target of 3-Hydroxy-3-Methyl-Glutaryl-COA Reductase Inhibitors

Abstract

Antibiotic-resistant gram positive bacteria are a serious global health hazard, causing severe infections as well as significant healthcare associated costs. Among these pathogens, Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis rely on the conserved mevalonate pathway to produce the peptidoglycan precursor isopentenyl diphosphate (IPP). Inhibitors developed against bacterial HMG-CoA reductase (HMGR), which converts HMG-CoA to mevalonate, appear to have a secondary bactericidal effect that is not yet understood. Evidence exists that HMG-CoA causes toxicity at accumulated levels, possibly due to inhibition of Malonyl-CoA:acyl transferase (FabD). FabD is therefore being investigated for inhibition both by HMG-CoA and our bacterial HMGR inhibitors. FabD is an initiating enzyme of the type II fatty acid cycle (FAS II) that transfers the malonyl moiety of malonyl-CoA to Acyl Carrier Protein(ACP), producing malonyl-ACP. Crosslinking and computational studies show that the interaction of ACP with each Fab enzyme in the elongation cycle, while apparently highly conserved, is transient and likely influenced by the size of the growing acyl chain. This study seeks to examine FabD-ACP binding in gram positive pathogens and identify HMGR inhibitors with secondary activity against FabD using structural and biophysical techniques. The native structure of E. faecalis FabD has been solved to1.74Å using Xray crystallography. Ligand co-crystallization and kinetics studies are ongoing with HMGR inhibitors that show thermal shift activity against FabD. The FabD-ACP interface will be examined by ITC using interface residue mutants, which have been identified through computational FabD-ACP docking simulations performed with LZERD (Local 3D Zernike descriptor-based Docking algorithm) and comparison to existing structures. Information gained will determine whether established mevalonate pathway inhibitors have dual activity against the fatty acid cycle, and will contribute a better understanding of FabD-ACP binding in gram positive pathogens.