Fine tuning for success in structure-based virtual screening.

Structure-based virtual screening plays a significant role in drug-discovery. The method virtually docks millions of compounds from corporate or public libraries into a binding site of a disease-related protein structure, allowing for the selection of a small list of potential ligands for experimental testing. Many algorithms are available for docking and assessing the affinity of compounds for a targeted protein site. The performance of affinity estimation calculations is highly dependent on the size and nature of the site, therefore a rationale for selecting the best protocol is required. To address this issue, we have developed an automated calibration process, implemented in a Knime workflow. It consists of four steps: preparation of a protein test set with structures and models of the target, preparation of a compound test set with target-related ligands and decoys, automatic test of 24 scoring/rescoring protocols for each target structure and model, and graphical display of results. The automation of the process combined with execution on high performance computing resources greatly reduces the duration of the calibration phase, and the test of many combinations of algorithms on various target conformations results in a rational and optimal choice of the best protocol. Here, we present this tool and exemplify its application in setting-up an optimal protocol for SBVS against Retinoid X Receptor alpha.

User-Friendly Quantum Mechanics: Applications for Drug Discovery.

Quantum mechanics (QM) methods provide a fine description of receptor-ligand interactions and of chemical reactions. Their use in drug design and drug discovery is increasing, especially for complex systems including metal ions in the binding sites, for the design of highly selective inhibitors, for the optimization of bi-specific compounds, to understand enzymatic reactions, and for the study of covalent ligands and prodrugs. They are also used for generating molecular descriptors for predictive QSAR/QSPR models and for the parameterization of force fields. Thanks to the continuous increase of computational power offered by GPUs and to the development of sophisticated algorithms, QM methods are becoming part of the standard tools used in computer-aided drug design (CADD). We present the most used QM methods and software packages, and we discuss recent representative applications in drug design and drug discovery.

Bactericidal Disruption of Magnesium Metallostasis in Mycobacterium tuberculosis Is Counteracted by Mutations in the Metal Ion Transporter CorA.

A defining characteristic of treating tuberculosis is the need for prolonged administration of multiple drugs. This may be due in part to subpopulations of slowly replicating or nonreplicating Mycobacterium tuberculosis bacilli exhibiting phenotypic tolerance to most antibiotics in the standard treatment regimen. Confounding this problem is the increasing incidence of heritable multidrug-resistant M. tuberculosis A search for new antimycobacterial chemical scaffolds that can kill phenotypically drug-tolerant mycobacteria uncovered tricyclic 4-hydroxyquinolines and a barbituric acid derivative with mycobactericidal activity against both replicating and nonreplicating M. tuberculosis Both families of compounds depleted M. tuberculosis of intrabacterial magnesium. Complete or partial resistance to both chemotypes arose from mutations in the putative mycobacterial Mg2+/Co2+ ion channel, CorA. Excess extracellular Mg2+, but not other divalent cations, diminished the compounds' cidality against replicating M. tuberculosis These findings establish depletion of intrabacterial magnesium as an antimicrobial mechanism of action and show that M. tuberculosis magnesium homeostasis is vulnerable to disruption by structurally diverse, nonchelating, drug-like compounds.IMPORTANCE Antimycobacterial agents might shorten the course of treatment by reducing the number of phenotypically tolerant bacteria if they could kill M. tuberculosis in diverse metabolic states. Here we report two chemically disparate classes of agents that kill M. tuberculosis both when it is replicating and when it is not. Under replicating conditions, the tricyclic 4-hydroxyquinolines and a barbituric acid analogue deplete intrabacterial magnesium as a mechanism of action, and for both compounds, mutations in CorA, a putative Mg2+/Co2+ transporter, conferred resistance to the compounds when M. tuberculosis was under replicating conditions but not under nonreplicating conditions, illustrating that a given compound can kill M. tuberculosis in different metabolic states by disparate mechanisms. Targeting magnesium metallostasis represents a previously undescribed antimycobacterial mode of action that might cripple M. tuberculosis in a Mg2+-deficient intraphagosomal environment of macrophages.

Pushing the Limits of Computational Structure-Based Drug Design with a Cryo-EM Structure: The Ca2+ Channel α2δ-1 Subunit as a Test Case.

Cryo-electron microscopy (cryo-EM) is emerging as a real alternative for structural elucidation. In spite of this, very few cryo-EM structures have been described so far as successful platforms for in silico drug design. Gabapentin and pregabalin are some of the most successful drugs in the treatment of epilepsy and neuropathic pain. Although both are in clinical use and are known to exert their effects by binding to the regulatory α2δ subunit of voltage gated calcium channels, their binding modes have never been characterized. We describe here the successful use of an exhaustive protein-ligand sampling algorithm on the α2δ-1 subunit of the recently published cryo-EM structure, with the goal of characterizing the ligand entry path and binding mode for gabapentin, pregabalin, and several other amino acidic α2δ-1 ligands. Our studies indicate that (i) all simulated drugs explore the same path for accessing the occluded binding site on the interior of the α2δ-1 subunit; (ii) they all roughly occupy the same pocket; (iii) the plasticity of the binding site allows the accommodation of a variety of amino acidic modulators, driven by the flexible "capping loop" delineated by residues Tyr426-Val435 and the floppy nature of Arg217; (iv) the predicted binding modes are in line with previously available mutagenesis data, confirming Arg217 as key for binding, with Asp428 and Asp467 highlighted as additional anchoring points for all amino acidic drugs. The study is one of the first proofs that latest-generation cryo-EM structures combined with exhaustive computational methods can be exploited in early drug discovery.

Inhibition of Human Enhancer of Zeste Homolog 2 with Tambjamine Analogs.

Combining computational modeling, de novo compound synthesis, and in vitro and cellular assays, we have performed an inhibition study against the enhancer of zeste homolog 2 (EZH2) histone-lysine N-methyltransferase. This enzyme is an important catalytic component of the PRC2 complex whose alterations have been associated with different cancers. We introduce here several tambjamine-inspired derivatives with low micromolar in vitro activity that produce a significant decrease in histone 3 trimethylation levels in cancer cells. We demonstrate binding at the methyl transfer active site, showing, in addition, that the EZH2 isolated crystal structure is capable of being used in molecular screening studies. Altogether, this work provides a successful molecular model that will help in the identification of new specific EZH2 inhibitors and identify a novel class of tambjamine-derived EZH2 inhibitors with promising activities for their use in cancer treatment.

Combating virulence of Gram-negative bacilli by OmpA inhibition.

Preventing the adhesion of pathogens to host cells provides an innovative approach to tackling multidrug-resistant bacteria. In this regard, the identification of outer membrane protein A (OmpA) as a key bacterial virulence factor has been a major breakthrough. The use of virtual screening helped us to identify a cyclic hexapeptide AOA-2 that inhibits the adhesion of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli to host cells and the formation of biofilm, thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. Inhibition of OmpA offers a strategy as monotherapy to address the urgent need for treatments for infections caused by Gram-negative bacilli.

Challenges of docking in large, flexible and promiscuous binding sites.

After decades of work, the correct determination of the binding mode of a small molecule into a target protein is still a challenging problem, whose difficulty depends on: (i) the sizes of the binding site and the ligand; (ii) the flexibility of both interacting partners, and (iii) the differential solvation of bound and unbound partners. We have evaluated the performance of standard rigid(receptor)/flexible(ligand) docking approaches with respect to last-generation fully flexible docking methods to obtain reasonable poses in a very challenging case: soluble Epoxide Hydrolase (sEH), a flexible protein showing different binding sites. We found that full description of the flexibility of both protein and ligand and accurate description of solvation leads to significant improvement in the ability of docking to reproduce well known binding modes, and at the same time capture the intrinsic binding promiscuity of the protein.

Active-Site-Directed Inhibitors of Prolyl Oligopeptidase Abolish Its Conformational Dynamics.

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond-millisecond motions opens the opportunity for regulating protein-protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation.

Combined Use of Oligopeptides, Fragment Libraries, and Natural Compounds: A Comprehensive Approach To Sample the Druggability of Vascular Endothelial Growth Factor.

The modulation of protein-protein interactions (PPIs) is emerging as a highly promising tool to fight diseases. However, whereas an increasing number of compounds are able to disrupt peptide-mediated PPIs efficiently, the inhibition of domain-domain PPIs appears to be much more challenging. Herein, we report our results related to the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR). The VEGF-VEGFR interaction is a typical domain-domain PPI that is highly relevant for the treatment of cancer and some retinopathies. Our final goal was to identify ligands able to bind VEGF at the region used by the growth factor to interact with its receptor. We undertook an extensive study, combining a variety of experimental approaches, including NMR-spectroscopy-based screening of small organic fragments, peptide libraries, and medicinal plant extracts. The key feature of the successful ligands that emerged from this study was their capacity to expose hydrophobic functional groups able to interact with the hydrophobic hot spots at the interacting VEGF surface patch.

Unveiling prolyl oligopeptidase ligand migration by comprehensive computational techniques.

Prolyl oligopeptidase (POP) is a large 80 kDa protease, which cleaves oligopeptides at the C-terminal side of proline residues and constitutes an important pharmaceutical target. Despite the existence of several crystallographic structures, there is an open debate about migration (entrance and exit) pathways for ligands, and their coupling with protein dynamics. Recent studies have shown the capabilities of molecular dynamics and classical force fields in describing spontaneous binding events and nonbiased ligand migration pathways. Due to POP's size and to the buried nature of its active site, an exhaustive sampling by means of conventional long enough molecular dynamics trajectories is still a nearly impossible task. Such a level of sampling, however, is possible with the breakthrough protein energy landscape exploration technique. Here, we present an exhaustive sampling of POP with a known inhibitor, Z-pro-prolinal. In >3000 trajectories Z-pro-prolinal explores all the accessible surface area, showing multiple entrance events into the large internal cavity through the pore in the ß-propeller domain. Moreover, we modeled a natural substrate binding and product release by predicting the entrance of an undecapeptide substrate, followed by manual active site cleavage and nonbiased exit of one of the products (a dipeptide). The product exit shows preference from a flexible 18-amino acid residues loop, pointing to an overall mechanism where entrance and exit occur in different sites.

Molecular mechanics (MM3(pi)) conformational analysis of molecules containing conjugated pi-electron fragments: Leucomycin-V.

The conformations of the 16-membered macrolide antibiotic leucomycin-V (1) were studied with molecular mechanics. Leucomycin-V contains a conjugated pi-electron fragment and necessitates special treatment with the MM3(pi) modeling protocol. Comparison was made with results from the standard MM3 scheme. The CONFLEX conformational search procedure was used for finding low-energy conformations. The computed data are indicative for the existence of mainly one conformation of the macro-ring of 1 and minor participation of several others. Intramolecular hydrogen bonds play important roles for the preferred geometry of the macro-ring and the conformations of the side chains. The most probable macro-ring conformation of 1 is very similar to the preferred conformation of another 16-ring macrolide antibiotic, tylosin. The same order of conformational preference for 1 was estimated with the MM3 and the MM3(pi) methods. Surprisingly, when changing the chirality of the C(9) macro-ring atom of 1, the two methods produced different order of conformational preferences for the 9-epi form (2), as well as enhanced population of several clusters of conformations.