A Structural and Energetic Model for the Slow-Onset Inhibition of the Mycobacterium tuberculosis Enoyl-ACP Reductase InhA

Abstract

Slow-onset enzyme inhibitors are of great interest for drug discovery programs since the slow dissociation of the inhibitor from the drug–target complex results in sustained target occupancy leading to improved pharmacodynamics. However, the structural basis for slow-onset inhibition is often not fully understood, hindering the development of structure-kinetic relationships and the rational optimization of drug-target residence time. Previously we demonstrated that slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA correlated with motions of a substrate-binding loop (SBL) near the active site. In the present work, X-ray crystallography and molecular dynamics simulations have been used to map the structural and energetic changes of the SBL that occur upon enzyme inhibition. Helix-6 within the SBL adopts an open conformation when the inhibitor structure or binding kinetics is substrate-like. In contrast, slow-onset inhibition results in large-scale local refolding in which helix-6 adopts a closed conformation not normally populated during substrate turnover. The open and closed conformations of helix-6 are hypothesized to represent the EI and EI* states on the two-step induced-fit reaction coordinate for enzyme inhibition. These two states were used as the end points for nudged elastic band molecular dynamics simulations resulting in two-dimensional potential energy profiles that reveal the barrier between EI and EI*, thus rationalizing the binding kinetics observed with different inhibitors. Our findings indicate that the structural basis for slow-onset kinetics can be understood once the structures of both EI and EI* have been identified, thus providing a starting point for the rational control of enzyme–inhibitor binding kinetics.

Rational Design of Broad Spectrum Antibacterial Activity Based on a Clinically Relevant Enoyl-Acyl Carrier Protein (ACP) Reductase Inhibitor

“…6). Furthermore, the structure of InhA in complex with the 4-pyridone PT155 shares a very similar open SBL conformation with the substrate-bound form of this enzyme (74,76). As expected based on these considerations, the SAR and predicted binding mode for the 4-pyridones is highly reminiscent of the 2-pyridone series, but 4-pyridones possess superior potency at the enzymatic and cellular levels (Tables 1 and 2).…”

“…Similar to the ecFabI-NADH-CG400549 structure, the substrate-binding loop was found to be disordered or in a very open conformation in these ecFabI and bpFabI structures, respectively. Interestingly, an open SBL conformation was very recently also observed in the structure of PT155 bound to InhA (74).…”

Biological Evaluation of Potent Triclosan‐Derived Inhibitors of the Enoyl–Acyl Carrier Protein Reductase InhA in Drug‐Sensitive and Drug‐Resistant Strains of Mycobacterium tuberculosis

“…In lieu of X-ray crystal structures, we offer the docking calculations provide an understanding of reasonable inhibitor binding modes to InhA, given the assumptions made by the software. Since InhA is a flexible enzyme which contains a substrate-binding loop that samples multiple distinct conformations when it forms a lid on the active site, [49] several conformations of InhA from multiple chains of four different ligand-bound crystal structures of InhA were utilized as targets for these docking studies. 2X23 [50] and 3FNH [32] are both bound to triclosan derivatives, while 4COD [37] and 4BQP [38] are new crystal structures of InhA bound to structurally distinct lead compounds from GlaxoSmithKline and AstraZeneca, respectively.…”

Drug–Target Kinetics in Drug Discovery

“…5 The figure shows a simple one-step mechanism, although in many cases slow-binding inhibitors operate through a two-step induced-fit mechanism. 13,15,28−31 …”