Discovery of an Allosteric Ligand Binding Site in SMYD3 Lysine Methyltransferase.

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug-like compounds was screened in a biosensor-based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (KD =42 and 84 µM, respectively). Co-crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C-terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3-HSP90 binding was confirmed (KD =13 µM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action.

Cosolvent-Based Protein Pharmacophore for Ligand Enrichment in Virtual Screening.

Virtual screening of large compound databases, looking for potential ligands of a target protein, is a major tool in computer-aided drug discovery. Throughout the years, different techniques such as similarity searching, pharmacophore matching, or molecular docking have been applied with the aim of finding hit compounds showing appreciable affinity. Molecular dynamics simulations in mixed solvents have been shown to identify hot spots relevant for protein-drug interaction, and implementations based on this knowledge were developed to improve pharmacophore matching of small molecules, binding free-energy estimations, and docking performance in terms of pose prediction. Here, we proved in a retrospective manner that cosolvent-derived pharmacophores from molecular dynamics (solvent sites) improve the performance of docking-based virtual screening campaigns. We applied a biased docking scheme based on solvent sites to nine relevant target proteins that have a set of known ligands or actives and compounds that are, presumably, nonbinders (decoys). Our results show improvement in virtual screening performance compared to traditional docking programs both at a global level, with up to 35% increase in areas under the receiver operating characteristic curve, and in early stages, with up to a 7-fold increase in enrichment factors at 1%. However, the improvement in pose prediction of actives was less profound. The presented application makes use of the AutoDock Bias method and is the only cosolvent-derived pharmacophore technique that employs its knowledge both in the ligand conformational search algorithm and the final affinity scoring for virtual screening purposes.

LigQ: A Webserver to Select and Prepare Ligands for Virtual Screening.

Virtual screening is a powerful methodology to search for new small molecule inhibitors against a desired molecular target. Usually, it involves evaluating thousands of compounds (derived from large databases) in order to select a set of potential binders that will be tested in the wet-lab. The number of tested compounds is directly proportional to the cost, and thus, the best possible set of ligands is the one with the highest number of true binders, for the smallest possible compound set size. Therefore, methods that are able to trim down large universal data sets enriching them in potential binders are highly appreciated. Here we present LigQ, a free webserver that is able to (i) determine best structure and ligand binding pocket for a desired protein, (ii) find known binders, as well as potential ligands known to bind to similar protein domains, (iii) most importantly, select a small set of commercial compounds enriched in potential binders, and (iv) prepare them for virtual screening. LigQ was tested with several proteins, showing an impressive capacity to retrieve true ligands from large data sets, achieving enrichment factors of over 10%. LigQ is available at http://ligq.qb.fcen.uba.ar/ .

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.

Virtual screening: an in silico tool for interlacing the chemical universe with the proteome.

In silico screening both in the forward (traditional virtual screening) and reverse sense (inverse virtual screening (IVS)) are helpful techniques for interlacing the chemical universe of small molecules with the proteome. The former, which is using a protein structure and a large chemical database, is well-known by the scientific community. We have chosen here to provide an overview on the latter, focusing on validation and target prioritization strategies. By comparing it to complementary or alternative wet-lab approaches, we put IVS in the broader context of chemical genomics, target discovery and drug design. By giving examples from the literature and an own example on how to validate the approach, we provide guidance on the issues related to IVS.

Structure-activity relationships in purine-based inhibitor binding to HSP90 isoforms.

Inhibition of the ATPase activity of the chaperone protein HSP90 is a potential strategy for treatment of cancers. We have determined structures of the HSP90alpha N-terminal domain complexed with the purine-based inhibitor, PU3, and analogs with enhanced potency both in enzyme and cell-based assays. The compounds induce upregulation of HSP70 and downregulation of the known HSP90 client proteins Raf-1, CDK4, and ErbB2, confirming that the molecules inhibit cell growth by a mechanism dependent on HSP90 inhibition. We have also determined the first structure of the N-terminal domain of HSP90beta, complexed with PU3. The structures allow a detailed rationale to be developed for the observed affinity of the PU3 class of compounds for HSP90 and also provide a structural framework for design of compounds with improved binding affinity and drug-like properties.