Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)

Charles O Rock,Jiangwei Yao,John B Maxwell

doi:10.1074/jbc.m113.512905

Charles O Rock, Jiangwei Yao + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m113.512905

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Abstract

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Ki(app) increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.

Highlights

AFN-1252 is a FabI inhibitor developed to treat Staphylococcus aureus
Selection of AFN-1252-resistant Strains—AFN-1252 was a potent antibiotic against S. aureus strain RN4220 with a MIC of 3.9 ng/ml in LB broth (Fig. 1B)
Methionine is the only residue found at position 99 in S. aureus FabIs, and the gene expression data indicated that FabI(M99T) was catalytically compromised in vivo

Summary

Background

AFN-1252 is a FabI inhibitor developed to treat Staphylococcus aureus. Results: AFN-1252 resistance arises from a single missense mutation that produces a FabI(M99T) protein. AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). The discovery that the antibiotic activity of triclosan (Fig. 1A) against E. coli was due to the selective inhibition of FabI [3, 4] validated FabI as a bona fide drug target and stimulated the biochemical and structural characterization of FabI [5,6,7,8,9,10,11,12] as well as the development of multiple small molecule FabI inhibitors as potential antibacterial agents [11,12,13,14,15]. Two other FabI-related proteins carry out enoyl-ACP reduction, FabL [5] and FabV [18] These enzymes are related to FabI, bacteria that express these proteins are resistant to triclosan [5, 16, 18]. Strains expressing FabI mutants had increased resistance to AFN-1252 inhibition, they still had submicromolar MICs for AFN-1252 due to on-target inhibition of FabI

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