Kirk E. Hevener

Hit Identification and Optimization in Virtual Screening: Practical Recommendations Based on a Critical Literature Analysis

A critical analysis of virtual screening results published between 2007 and 2011 was performed. The activity of reported hit compounds from over 400 studies was compared to their hit identification criteria. Hit rates and ligand efficiencies were calculated to assist in these analyses, and the results were compared with factors such as the size of the virtual library and the number of compounds tested. A series of promiscuity, druglike, and ADMET filters were applied to the reported hits to assess the quality of compounds reported, and a careful analysis of a subset of the studies that presented hit optimization was performed. These data allowed us to make several practical recommendations with respect to selection of compounds for experimental testing, definition of hit identification criteria, and general virtual screening hit criteria to allow for realistic hit optimization. A key recommendation is the use of size-targeted ligand efficiency values as hit identification criteria.

Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching

Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with submicromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented.

Comparison of radii sets, entropy, QM methods, and sampling on MM-PBSA, MM-GBSA, and QM/MM-GBSA ligand binding energies of F. tularensis enoyl-ACP reductase (FabI)

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM‐PBSA, MM‐GBSA, and QM/MM‐GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM‐GBSA using 6 ns MD simulation trajectories together with GBneck2, PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r2 = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called “multiple independent sampling”), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GBHCT and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r2 = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazoles physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive “multiple independent sampling method” may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM‐P(G)BSA were limited to inhibitors’ relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program.

Fragment-based drug discovery using a multidomain, parallel MD-MM/PBSA screening protocol

We have developed a rigorous computational screening protocol to identify novel fragment-like inhibitors of N(5)-CAIR mutase (PurE), a key enzyme involved in de novo purine synthesis that represents a novel target for the design of antibacterial agents. This computational screening protocol utilizes molecular docking, graphics processing unit (GPU)-accelerated molecular dynamics, and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) free energy estimations to investigate the binding modes and energies of fragments in the active sites of PurE. PurE is a functional octamer comprised of identical subunits. The octameric structure, with its eight active sites, provided a distinct advantage in these studies because, for a given simulation length, we were able to place eight separate fragment compounds in the active sites to increase the throughput of the MM/PBSA analysis. To validate this protocol, we have screened an in-house fragment library consisting of 352 compounds. The theoretical results were then compared with the results of two experimental fragment screens, Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) binding analyses. In these validation studies, the protocol was able to effectively identify the competitive binders that had been independently identified by experimental testing, suggesting the potential utility of this method for the identification of novel fragments for future development as PurE inhibitors.

Structural and enzymatic analyses reveal the binding mode of a novel series of Francisella tularensis enoyl reductase (FabI) inhibitors

Because of structural and mechanistic differences between eukaryotic and prokaryotic fatty acid synthesis enzymes, the bacterial pathway, FAS-II, is an attractive target for the design of antimicrobial agents. We have previously reported the identification of a novel series of benzimidazole compounds with particularly good antibacterial effect against Francisella tularensis, a Category A biowarfare pathogen. Herein we report the crystal structure of the F. tularensis FabI enzyme in complex with our most active benzimidazole compound bound with NADH. The structure reveals that the benzimidazole compounds bind to the substrate site in a unique conformation that is distinct from the binding motif of other known FabI inhibitors. Detailed inhibition kinetics have confirmed that the compounds possess a novel inhibitory mechanism that is unique among known FabI inhibitors. These studies could have a strong impact on future antimicrobial design efforts and may reveal new avenues for the design of FAS-II active antibacterial compounds.

High-throughput screening (HTS) and hit validation to identify small molecule inhibitors with activity against NS3/4A proteases from multiple hepatitis C virus genotypes.

Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC50 value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. The bacterial FASII pathway is a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. These compounds display an improved low nanomolar enzymatic activity as well as promising low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). The improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.

Synergistic inhibitor binding to the papain-like protease of human SARS coronavirus: mechanistic and inhibitor design implications.

We previously developed two potent chemical classes that inhibit the essential papain‐like protease (PLpro) of severe acute respiratory syndrome coronavirus. In this study, we applied a novel approach to identify small fragments that act synergistically with these inhibitors. A fragment library was screened in combination with four previously developed lead inhibitors by fluorescence‐based enzymatic assays. Several fragment compounds synergistically enhanced the inhibitory activity of the lead inhibitors by approximately an order of magnitude. Surface plasmon resonance measurements showed that three fragments bind specifically to the PLpro enzyme. Mode of inhibition, computational solvent mapping, and molecular docking studies suggest that these fragments bind adjacent to the binding site of the lead inhibitors and further stabilize the inhibitor‐bound state. We propose potential next‐generation compounds based on a computational fragment‐merging approach. This approach provides an alternative strategy for lead optimization for cases in which direct co‐crystallization is difficult.

High-level expression, purification, and characterization of Staphylococcus aureus dihydroorotase (PyrC) as a cleavable His-SUMO fusion

Staphylococcus aureus is a pathogenic bacterium that causes a variety of mild to lethal human diseases. The rapid spread of multidrug-resistant strains makes the discovery of new antimicrobial agents critical. Dihydroorotase (PyrC), the third enzyme in the bacterial pyrimidine biosynthesis pathway, is structurally and mechanistically distinct from its mammalian counterpart. It has been confirmed to be essential in S. aureus making it an attractive antibacterial drug target. No protocol to express and purify S. aureus PyrC (SaPyrC) has been reported. To obtain the SaPyrC enzyme and overcome anticipated solubility problems, the SaPyrC gene was cloned into the pET-SUMO vector. The N-terminal His-SUMO fused SaPyrC was expressed in Escherichia coli BL21 (DE3) with an HRV 3C protease recognition site inserted between the SUMO tag and SaPyrC to allow for improved cleavage by HRV protease. Purification of cleaved protein using HisTrap affinity and gel filtration columns resulted in native SaPyrC with estimated 95% purity and 40% yield. Both His-SUMO tagged and native SaPyrC form dimers, and enzyme characterization studies have shown that the His-SUMO tag affects enzyme activity slightly. Forward and reverse kinetic rate constants for both tagged and native SaPyrC were determined, and pH profiling studies revealed the optimal pH values for forward and reverse reactions.

Special Challenges to the Rational Design of Antibacterial Agents

Abstract The rational design of pharmaceutical agents with activity against bacterial targets presents several unique challenges due to the significant differences in the target bacterial cells and the eukaryotic cells of their mammalian hosts. The structural features and cellular components commonly targeted in drug design programs are often unique to bacteria. While this provides an excellent opportunity in terms of selectivity and decreased toxicities, there are also special factors that must be considered, including distribution to the target, bacterial cell penetration, efflux, metabolism and elimination, and the rapid emergence of bacterial resistance. These factors can play a key role in the design of compounds intended for use against bacterial targets and the application of traditional and nontraditional screening strategies aimed at identifying such compounds. This report discusses these special issues pertaining to antibacterial drug discovery, presents practical approaches to overcoming these challenges, and highlights some recent examples of their application.