Gopal R. Bommineni

Rational Optimization of Drug-Target Residence Time: Insights from Inhibitor Binding to the Staphylococcus aureus FabI Enzyme–Product Complex

Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for a series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI)--an important target for the development of new anti-staphylococcal drugs--as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient, and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Because of its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 h. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme.

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.

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.

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

Background: The FabI inhibitor CG400549 is a promising new anti-staphylococcal drug candidate with recently validated human efficacy. Results: We revealed the molecular determinants conferring S. aureus FabI selectivity to rationally design a compound with an improved antibacterial activity spectrum. Conclusion: The 4-pyridone PT166 represents a critical step toward Gram-negative and mycobacterial coverage. Significance: We provide an approach to expand the spectrum of antimicrobial activity. Determining the molecular basis for target selectivity is of particular importance in drug discovery. The ideal antibiotic should be active against a broad spectrum of pathogenic organisms with a minimal effect on human targets. CG400549, a Staphylococcus-specific 2-pyridone compound that inhibits the enoyl-acyl carrier protein reductase (FabI), has recently been shown to possess human efficacy for the treatment of methicillin-resistant Staphylococcus aureus infections, which constitute a serious threat to human health. In this study, we solved the structures of three different FabI homologues in complex with several pyridone inhibitors, including CG400549. Based on these structures, we rationalize the 65-fold reduced affinity of CG400549 toward Escherichia coli versus S. aureus FabI and implement concepts to improve the spectrum of antibacterial activity. The identification of different conformational states along the reaction coordinate of the enzymatic hydride transfer provides an elegant visual depiction of the relationship between catalysis and inhibition, which facilitates rational inhibitor design. Ultimately, we developed the novel 4-pyridone-based FabI inhibitor PT166 that retained favorable pharmacokinetics and efficacy in a mouse model of S. aureus infection with extended activity against Gram-negative and mycobacterial organisms.

Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin

Thiolactomycin (TLM), a natural product thiolactone antibiotic produced by species of Nocardia and Streptomyces, is an inhibitor of the β-ketoacyl-acyl carrier protein synthase (KAS) enzymes in the bacterial fatty acid synthase pathway. Using enzyme kinetics and direct binding studies, TLM has been shown to bind preferentially to the acyl-enzyme intermediates of the KASI and KASII enzymes from Mycobacterium tuberculosis and Escherichia coli. These studies, which utilized acyl-enzyme mimics in which the active site cysteine was replaced by a glutamine, also revealed that TLM is a slow onset inhibitor of the KASI enzymes KasA and ecFabB but not of the KASII enzymes KasB and ecFabF. The differential affinity of TLM for the acyl-KAS enzymes is proposed to result from structural change involving the movement of helices α5 and α6 that prepare the enzyme to bind malonyl-AcpM or TLM and that is initiated by formation of hydrogen bonds between the acyl-enzyme thioester and the oxyanion hole. The finding that TLM is a slow onset inhibitor of ecFabB supports the proposal that the long residence time of TLM on the ecFabB homologues in Serratia marcescens and Klebsiella pneumonia is an important factor for the in vivo antibacterial activity of TLM against these two organisms despite the fact that the in vitro MIC values are only 100–200 μg/ml. The mechanistic data on the interaction of TLM with KasA will provide an important foundation for the rational development of high affinity KasA inhibitors based on the thiolactone skeleton.

Thiolactomycin-based β-Ketoacyl-AcpM Synthase A (KasA) Inhibitors FRAGMENT-BASED INHIBITOR DISCOVERY USING TRANSIENT ONE-DIMENSIONAL NUCLEAR OVERHAUSER EFFECT NMR SPECTROSCOPY

Background: Potent inhibitors of the tuberculosis drug target KasA are needed. Results: Three-position analogs of the natural product thiolactomycin (TLM) were designed based on transient one-dimensional NOEs that reveal the relative orientation of TLM and a pantetheine analog bound simultaneously to KasA. Conclusion: Three-position analogs of TLM bind to KasA with increased potency. Significance: Optimization of TLM will lead to improved inhibitors of KasA. Thiolactomycin (TLM) is a natural product inhibitor of KasA, the β-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy.

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.

The Francisella tularensis FabI enoyl-acyl carrier protein reductase gene is essential to bacterial viability and is expressed during infection.

Francisella tularensis is classified as a category A priority pathogen and causes fatal disseminated disease in humans upon inhalation of less than 50 bacteria. Although drugs are available for treatment, they are not ideal because of toxicity and route of delivery, and in some cases patients relapse upon withdrawal. We have an ongoing program to develop novel FAS-II FabI enoyl-ACP reductase enzyme inhibitors for Francisella and other select agents. To establish F. tularensis FabI (FtFabI) as a clinically relevant drug target, we demonstrated that fatty acid biosynthesis and FabI activity are essential for growth even in the presence of exogenous long-chain lipids and that FtfabI is not transcriptionally altered in the presence of exogenous long-chain lipids. Inhibition of FtFabI or fatty acid synthesis results in loss of viability that is not rescued by exogenous long-chain lipid supplementation. Importantly, whole-genome transcriptional profiling of F. tularensis with DNA microarrays from infected tissues revealed that FtfabI and de novo fatty acid biosynthetic genes are transcriptionally active during infection. This is the first demonstration that the FabI enoyl-ACP-reductase enzyme encoded by F. tularensis is essential and not bypassed by exogenous fatty acids and that de novo fatty acid biosynthetic components encoded in F. tularensis are transcriptionally active during infection in the mouse model of tularemia.

Thiolactomycin-Based Inhibitors of Bacterial β-Ketoacyl-ACP Synthases with in Vivo Activity

β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5R-thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection.

Substituted diphenyl ethers as a novel chemotherapeutic platform against Burkholderia pseudomallei

ABSTRACT Identification of a novel class of anti-Burkholderia compounds is key in addressing antimicrobial resistance to current therapies as well as naturally occurring resistance. The FabI enoyl-ACP reductase in Burkholderia is an underexploited target that presents an opportunity for development of a new class of inhibitors. A library of substituted diphenyl ethers was used to identify FabI1-specific inhibitors for assessment in Burkholderia pseudomallei ex vivo and murine efficacy models. Active FabI1 inhibitors were identified in a two-stage format consisting of percent inhibition screening and MIC determination by the broth microdilution method. Each compound was evaluated against the B. pseudomallei 1026b (efflux-proficient) and Bp400 (efflux-compromised) strains. In vitro screening identified candidate substituted diphenyl ethers that exhibited MICs of less than 1 μg/ml, and enzyme kinetic assays were used to assess potency and specificity against the FabI1 enzyme. These compounds demonstrated activity in a Burkholderia ex vivo efficacy model, and two demonstrated efficacy in an acute B. pseudomallei mouse infection model. This work establishes substituted diphenyl ethers as a suitable platform for development of novel anti-Burkholderia compounds that can be used for treatment of melioidosis.