AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei--Crystal structure, mode of action, and biological activity

Abstract

Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC50 of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors.

Keywords: AFN-1252; Burkholderia pseudomallei; FabI; Melioidosis.

Figure 1

Chemical structure of (a) AFN-1252 and (b) Triclosan.

Figure 2

Dose-response curve for the inhibition of BpmFabI by AFN-1252.

Figure 3

Mechanism of inhibition of BpmFabI by AFN-1252 (a) at 300 µM crotonyl-CoA and different concentrations of NADH and (b) at 375 µM NADH and different concentrations of crotonyl- CoA. The concentrations of AFN-1252 used were: 0 nM (•), 2.5 nM (▪), 5 nM (▴), 10 nM (▾), 20 nM (♦), and 40 nM () [Fig. 3(a)]; and 0 nM (•), 5 nM (▪), 20 nM (▴), 40 nM (▾), 80 nM (♦), and 160 nM () [Fig. 3(b)].

Figure 4

Thermal melting curves of BpmFabI alone (▪) and in presence of AFN-1252 (▴) and Triclosan (○).

Figure 5

Ribbon representation of the monomeric structure of BpmFabI (cyan) with the bound ligand AFN-1252 (yellow). The monomer is composed of seven α helices and seven β strands.

Figure 6

Ribbon diagram of BpmFabI tetrameric assembly viewed down the twofold noncrystallographic axis. Each monomer along with the bound AFN-1252 is shown in a different color.

Figure 7

Active site structure of AFN-1252 bound to BpmFabI. The interacting residues seen within 3.6 Å distance from AFN-1252 are labeled. Hydrogen bonds are represented as dotted lines and water molecules as red spheres.

Figure 8

Cartoon diagram showing the superposed structures of Bpm (magenta), Sa (yellow), and EcFabI (green). The bound NAD/NADP and AFN-1252 ligands are also shown. The flexible loop (residues T194-K199) in BpmFabI was found protruding away from the active site.