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.

Figure 1

Time scale of slow-onset inhibition. The rates of many common protein motions are shown, ranging from bond vibrations and enzyme turnover to slow-onset inhibition.^,,− Also shown is the time scale for drug pharmacokinetics and the time scales for enzyme assays and MD simulations.⁻

Figure 2

InhA reaction mechanism and induced-fit inhibition. (a) The reaction catalyzed by the FabI enoyl-ACP reductase InhA. (b) The kinetic mechanism for two-step, induced-fit inhibition of InhA by the diphenyl ether inhibitors.

Figure 3

Open and closed conformations of InhA. (a) Structure of the closed state is represented by the PT70 ternary complex (PDB code 2X23, chain A) in which helix-6 is close to strand-4. (b) Structure of the open state is represented by the C16-NAC ternary complex (PDB code 1BVR, chain A) in which helix-6 has moved away from strand-4 to accommodate the substrate. (c) Structure of the PT155 ternary complex observed in chain B in the I2₁2₁2₁ crystal.

Figure 4

Kinetics, structural and energetic changes in InhA inhibition and catalysis. (a) Comparison of the conformational changes resulting from slow inhibition and substrate binding. Helix-6/helix-7 from the cofactor-bound binary complex is shown in gray in both overlays. PT70 (PDB code 2X23, chain B) and substrate analogue (PDB code 1BVR, chain A) ternary complexes are shown in blue on the left and in red on the right, respectively. Cα of residue 203 and 211 are highlighted, showing relative motions of helix-6 with respect to helix-7. (b) Difference in the kinetic rates involved in the inhibition and the catalytic cycle. The slow rates in the inhibition cycle relative to those in the catalytic cycle result in time-dependent inhibition observed in the InhA kinetic assay. The inhibition cycle is shown for diphenyl ethers, which form a ternary complex with the cofactor through the two-step, induced-fit mechanism. In the absence of the inhibitor, the equilibrium with the Michaelis complex, E·NADH·S, is reached rapidly on the time scale of the enzyme assay. (c) Proposed 1D energy profiles before (right) and after (left) binding of a slow inhibitor (solid line). The right and left energy landscapes depict the initial and final cross sections on the binding reaction coordinate for slow-onset inhibition as shown in Figure 2. Black solid line, violet dashed line, and brown dotted lines in the right panel depict the change in landscape that occurs during catalysis. E, ES, and EP denote the cofactor binary complex, substrate ternary complex, and product ternary complex, respectively; EI and EI* denote the inhibitor ternary complexes in Figure 2

Figure 5

Open to closed transition determined by the computational approach. (a) The colors (red to blue) represent structures along the reaction coordinate. Helix6 has larger scale of motion compared to helix7. (b) Definition of step and shear torsions that reduce the dimensionality of the structural change.

Figure 6

Free energy profiles and summary of X-ray structural data. Free energy profiles are shown for (a) the cofactor-bound binary complex, (b) the PT155 ternary complex, (c) the PT70 ternary complex, (d) the PT92 ternary complex, and (e) the PT03 ternary complex. (f) Locations of crystal structures on the same conformational coordinates. Area occupied by open structures is shaded red; area occupied by diphenyl ether ternary complex structures is shaded blue: (i) E-NADH binary complex 2AQ8; (ii) C16-NAC ternary complex 1BVR; (iii) PT70 ternary complex 2X23, chain A; (iv_a) PT10 ternary complex, chain A; (iv_b) PT10 ternary complex, chain B; (v) PT91 ternary complex; (vi_a) PT92 ternary complex, chain A; (vi_b) PT92 ternary complex, chain B; (vii_a) PT155 ternary complex, chain A, P2₁2₁2₁ crystal; (vii_d) PT155 ternary complex, chain D, P2₁2₁2₁ crystal.

Figure 7

Helix-6 and -7 conformations along the open to closed reaction coordinate. Structures are taken from the binary complex (PDB code 2AQ8, red), PT155 ternary complex (P2₁2₁2₁ chain C/D, green), PT92 ternary complex (blue), PT10 ternary complex (yellow), and PT70 ternary complex (purple). Individual structures are shown in Supplementary Figure S7.