Rational Modulation of the Induced-Fit Conformational Change for Slow-Onset Inhibition in Mycobacterium tuberculosis InhA.

Slow-onset enzyme inhibitors are the subject of considerable interest as an approach to increasing the potency of pharmaceutical compounds by extending the residence time of the inhibitor on the target (the lifetime of the drug-receptor complex). However, rational modulation of residence time presents significant challenges because it requires additional mechanistic insight, such as the nature of the transition state for postbinding isomerization. Our previous work, based on X-ray crystallography, enzyme kinetics, and molecular dynamics simulation, suggested that the slow step in inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA involves a change in the conformation of the substrate binding loop from an open state in the initial enzyme-inhibitor complex to a closed state in the final enzyme-inhibitor complex. Here, we use multidimensional free energy landscapes for loop isomerization to obtain a computational model for the transition state. The results suggest that slow-onset inhibitors crowd key side chains on helices that slide past each other during isomerization, resulting in a steric clash. The landscapes become significantly flatter when residues involved in the steric clash are replaced with alanine. Importantly, this lower barrier can be increased by rational inhibitor redesign to restore the steric clash. Crystallographic studies and enzyme kinetics confirm the predicted effects on loop structure and flexibility, as well as inhibitor residence time. These loss and regain of function studies validate our mechanistic hypothesis for interactions controlling substrate binding loop isomerization, providing a platform for the future design of inhibitors with longer residence times and better in vivo potency. Similar opportunities for slow-onset inhibition via the same mechanism are identified in other pathogens.

Time-dependent diaryl ether inhibitors of InhA: structure-activity relationship studies of enzyme inhibition, antibacterial activity, and in vivo efficacy.

The diaryl ethers are a novel class of antituberculosis drug candidates that inhibit InhA, the enoyl-ACP reductase involved in the fatty acid biosynthesis (FASII) pathway, and have antibacterial activity against both drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis. In the present work, we demonstrate that two time-dependent B-ring modified diaryl ether InhA inhibitors have antibacterial activity in a mouse model of TB infection when delivered by intraperitoneal injection. We propose that the efficacy of these compounds is related to their residence time on the enzyme, and to identify structural features that modulate drug-target residence time in this system, we have explored the inhibition of InhA by a series of B-ring modified analogues. Seven ortho-substituted compounds were found to be time-dependent inhibitors of InhA, where the slow step leading to the final enzyme-inhibitor complex (EI*) is thought to correlate with closure and ordering of the InhA substrate binding loop. A detailed mechanistic understanding of the molecular basis for residence time in this system will facilitate the development of InhA inhibitors with improved in vivo activity.

A structural and energetic model for the slow-onset inhibition of the Mycobacterium tuberculosis enoyl-ACP reductase InhA.

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.

Mechanism of the intramolecular Claisen condensation reaction catalyzed by MenB, a crotonase superfamily member.

MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 Å structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable, and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate α-proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase (enoyl-CoA hydratase), is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in α-proton acidity.

CoA Adducts of 4-Oxo-4-Phenylbut-2-enoates: Inhibitors of MenB from the M. tuberculosis Menaquinone Biosynthesis Pathway.

A high-throughput screen led to the discovery of 2-amino-4-oxo-4-phenylbutanoate inhibitors of the 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) from the menaquinone biosynthesis pathway in Mycobacterium tuberculosis. However, these compounds are unstable in solution and eliminate to form the corresponding 4-oxo-4-phenylbut-2-enoates that then react with CoA in situ to form nanomolar inhibitors of MenB. The potency of these compounds results from interaction of the CoA adduct carboxylate with the MenB oxyanion hole, a conserved structural motif in the crotonase superfamily. 4-Oxo-4-chlorophenylbutenoyl methyl ester has MICs of 0.6 and 1.5 µg/ml against replicating and nonreplicating M. tuberculosis, respectively, and it is proposed that the methyl ester penetrates the cell where it is hydrolyzed and reacts with CoA to generate the active antibacterial. The CoA adducts thus represent an important foundation for the development of novel MenB inhibitors, and suggest a general approach to the development of potent inhibitors of acyl-CoA binding enzymes.

High affinity InhA inhibitors with activity against drug-resistant strains of Mycobacterium tuberculosis.

Novel chemotherapeutics for treating multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (MTB) are required to combat the spread of tuberculosis, a disease that kills more than 2 million people annually. Using structure-based drug design, we have developed a series of alkyl diphenyl ethers that are uncompetitive inhibitors of InhA, the enoyl reductase enzyme in the MTB fatty acid biosynthesis pathway. The most potent compound has a Ki' value of 1 nM for InhA and MIC99 values of 2-3 microg mL(-1) (6-10 microM) for both drug-sensitive and drug-resistant strains of MTB. Overexpression of InhA in MTB results in a 9-12-fold increase in MIC99, consistent with the belief that these compounds target InhA within the cell. In addition, transcriptional response studies reveal that the alkyl diphenyl ethers fail to upregulate a putative efflux pump and aromatic dioxygenase, detoxification mechanisms that are triggered by the lead compound triclosan. These diphenyl ether-based InhA inhibitors do not require activation by the mycobacterial KatG enzyme, thereby circumventing the normal mechanism of resistance to the front line drug isoniazid (INH) and thus accounting for their activity against INH-resistant strains of MTB.