A Slow, Tight Binding Inhibitor of InhA, the Enoyl-Acyl Carrier Protein Reductase from Mycobacterium tuberculosis

Sylvia R Luckner,Nina Liu,Christopher W Am Ende,Peter J Tonge,Caroline Kisker

doi:10.1074/jbc.m109.090373

Abstract

InhA, the enoyl-ACP reductase in Mycobacterium tuberculosis is an attractive target for the development of novel drugs against tuberculosis, a disease that kills more than two million people each year. InhA is the target of the current first line drug isoniazid for the treatment of tuberculosis infections. Compounds that directly target InhA and do not require activation by the mycobacterial catalase-peroxidase KatG are promising candidates for treating infections caused by isoniazid-resistant strains. Previously we reported the synthesis of several diphenyl ethers with nanomolar affinity for InhA. However, these compounds are rapid reversible inhibitors of the enzyme, and based on the knowledge that long drug target residence times are an important factor for in vivo drug activity, we set out to generate a slow onset inhibitor of InhA using structure-based drug design. 2-(o-Tolyloxy)-5-hexylphenol (PT70) is a slow, tight binding inhibitor of InhA with a K(1) value of 22 pm. PT70 binds preferentially to the InhA x NAD(+) complex and has a residence time of 24 min on the target, which is 14,000 times longer than that of the rapid reversible inhibitor from which it is derived. The 1.8 A crystal structure of the ternary complex between InhA, NAD(+), and PT70 reveals the molecular details of enzyme-inhibitor recognition and supports the hypothesis that slow onset inhibition is coupled to ordering of an active site loop, which leads to the closure of the substrate-binding pocket.

Highlights

Months to nearly 2 years and thereby increasing the cost for therapy 20-fold
Introduction of a methyl group ortho to the diphenyl ether linkage resulted in a compound, PT70 (Fig. 1), that is a slow onset inhibitor of InhA with a K1 value of 22 pM
Progress curve analysis was used to determine whether PT70 is a slow onset inhibitor of InhA

Summary

EXPERIMENTAL PROCEDURES

Synthesis of PT70—PT70 was synthesized using the procedure described previously for the synthesis of alkyl-substituted diphenyl ethers [16]. Progress Curve Analysis—The slow onset inhibition of InhA by PT70 was monitored by adding the enzyme (5 nM) to assay mixtures containing glycerol (8%), bovine serum albumin (0.1 mg/ml), DMSO (2% v/v), DD-CoA (300 ␮M), NADH (250 ␮M), NADϩ (200 ␮M), and inhibitor (0 – 480 nM). The values of kobs, vi, and vs obtained from Equation 2 were fitted to Equations 3 and 4, which describe a two-step inhibition mechanism in which rapid binding of the inhibitor to the enzyme is followed by a second slow step that results in the final complex In these equations, KϪ ap1p and Kiapp are the apparent dissociation constants for the initial enzyme1⁄7inhibitor complex (E-I) and the final enzyme1⁄7inhibitor complex (E-I*), respectively. Direct Determination of koff—Having shown that PT70 bound preferentially to the E-NADϩ product complex, the rate of dissociation of PT70 from InhA was monitored using 32PNADϩ to provide a direct estimate for koff These experiments followed a similar protocol to that described previously [5]. In the initial refinement rounds, the density improved significantly and allowed the

RESULTS

62 Ϯ 2 62 Ϯ 2 100 Ϯ 75

DISCUSSION