Mechanism and inhibition of saFabI, the enoyl reductase from Staphylococcus aureus

Abstract

Approximately one-third of the world's population carries Staphylococcus aureus. The recent emergence of extreme drug resistant strains that are resistant to the "antibiotic of last resort", vancomycin, has caused a further increase in the pressing need to discover new drugs against this organism. The S. aureus enoyl reductase, saFabI, is a validated target for drug discovery. To drive the development of potent and selective saFabI inhibitors, we have studied the mechanism of the enzyme and analyzed the interaction of saFabI with triclosan and two related diphenyl ether inhibitors. Results from kinetic assays reveal that saFabI is NADPH-dependent, and prefers acyl carrier protein substrates carrying fatty acids with long acyl chains. On the basis of product inhibition studies, we propose that the reaction proceeds via an ordered sequential ternary complex, with the ACP substrate binding first, followed by NADPH. The interaction of NADPH with the enzyme has been further explored by site-directed mutagenesis, and residues R40 and K41 have been shown to be involved in determining the specificity of the enzyme for NADPH compared to NADH. Finally, in preliminary inhibition studies, we have shown that triclosan, 5-ethyl-2-phenoxyphenol (EPP), and 5-chloro-2-phenoxyphenol (CPP) are all nanomolar slow-onset inhibitors of saFabI. These compounds inhibit the growth of S. aureus with MIC values of 0.03-0.06 microg/mL. Upon selection for resistance, three novel safabI mutations, A95V, I193S, and F204S, were identified. Strains containing these mutations had MIC values approximately 100-fold larger than that of the wild-type strain, whereas the purified mutant enzymes had K i values 5-3000-fold larger than that of wild-type saFabI. The increase in both MIC and K i values caused by the mutations supports the proposal that saFabI is the intracellular target for the diphenyl ether-based inhibitors.

Figure 1. Structure of the ecFabI:NAD⁺ Complex

Structure of ecFabI complexed with NAD⁺ (pdb code 1DFI) showing interactions between the protein and the NAD⁺ ribose. ecFabI is colored yellow and polar interactions between the protein residues (green) and NAD⁺ (cyan) are indicated with red dashed lines. Q40 interacts with the 2’-hydroxyl group in the adenosine moity of NAD⁺. The Figure was made using pymol ().

Figure 2. FabI Sequence Alignment

Sequence alignment of FabIs from E. coli, S. aureus, H. influenzae, M. tuberculosis and B. subtilis performed using ClustalW. The red-colored amino acids are proposed to interact with the cofactor, according to the X-ray structure of ecFabI:NAD⁺ complex.

Figure 3. Structure of ecFabI Complexed with NAD⁺ and Triclosan

Structure of triclosan bound to ecFabI (pdb code 1D8A) showing the proximity of residues G93, I192 and F203 to the inhibitor binding site. The corresponding residues in saFabI were found to be mutated in the diphenyl ether resistant S. aureus strains. ecFabI is colored yellow, while the polar interactions between the residues (green) in ecFabI and NAD/triclosan (cyan), are indicated with red dashed lines. The Figure was made using pymol ().

Figure 4. Inhibition of Wild Type saFabI by the Diphenyl Ether Inhibitors

(A) Dependence of K_i,app on [NADP⁺] for inhibition by triclosan. (B) Dependence of K_i,app on [NADPH] for inhibition by triclosan. (C) Dependence of K_i,app on [NADP⁺] for inhibition by EPP. (D) Dependence of K_i,app on [NADP⁺] for inhibition by CPP.

Scheme 1

The Type II Fatty Acid Biosynthesis Pathway.

Scheme 2

The Diphenyl Ether saFabI Inhibitors