Target identification of Mycobacterium tuberculosis phenotypic\textit{Mycobacterium tuberculosis phenotypic} hits using a concerted chemogenomic, biophysical and structural approach

Mycobacterium phenotypic hits are a good reservoir for new chemotypes for the treatment of tuberculosis. However, the absence of defined molecular targets and modes of action could lead to failure in drug development. Therefore, a combination of ligand-based and structure-based chemogenomic approaches followed by biophysical and biochemical validation have been used to identify targets for Mycobacterium tuberculosis phenotypic hits. Our approach identified EthR and InhA as targets for several hits, with some showing dual activity against these proteins. From the 35 predicted EthR inhibitors, eight exhibited an IC50 below 50 μM against M. tuberculosis EthR and three were confirmed to be also simultaneously active against InhA. Further hit validation was performed using X-ray crystallography yielding eight new crystal structures of EthR inhibitors. Although the EthR inhibitors attain their activity against M. tuberculosis by hitting yet undefined targets, these results provide new lead compounds that could be further developed to be used to potentiate the effect of EthA activated pro-drugs, such as ethionamide, thus enhancing their bactericidal effect.GM is grateful to the European Molecular Biology Laboratory and Marie Sklodowska-Curie Actions for funding this work. VM and MB acknowledge Bill & Melinda Gates Foundation [subcontract by the Foundation for the National Institutes of Health (NIH)] (OPP1024021). VM and MS acknowledge the European Community’s Seventh Framework Programme [grant number 260872]. GP would like to acknowledge the Wellcome Trust and the European Molecular Biology Laboratory for funding. JPO was funded by the member nation states of the European Molecular Biology Laboratory. TLB acknowledges The Wellcome Trust for funding and support (grant number 200814/Z/16/Z)

Flipping the odds of drug development success through human genomics

Drug development depends on accurately identifying molecular targets that both play a causal role in a disease and are amenable to pharmacological action by small molecule drugs or bio-therapeutics, such as monoclonal antibodies. Errors in drug target specification contribute to the extremely high rates of drug development failure. Integrating knowledge of genes that encode druggable targets with those that influence susceptibility to common disease has the potential to radically improve the probability of drug development success

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β2-adrenergic receptor (β2AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β2AR. We show that the select ligands bind preferentially to different predicted conformers of β2AR, and identify a role of β2AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as "computational probes" to systematically identify protein conformers with likely biological significance. © 2012 Isin et al

diXa: a data infrastructure for chemical safety assessment

Motivation: The field of toxicogenomics (the application of ‘-omics' technologies to risk assessment of compound toxicities) has expanded in the last decade, partly driven by new legislation, aimed at reducing animal testing in chemical risk assessment but mainly as a result of a paradigm change in toxicology towards the use and integration of genome wide data. Many research groups worldwide have generated large amounts of such toxicogenomics data. However, there is no centralized repository for archiving and making these data and associated tools for their analysis easily available. Results: The Data Infrastructure for Chemical Safety Assessment (diXa) is a robust and sustainable infrastructure storing toxicogenomics data. A central data warehouse is connected to a portal with links to chemical information and molecular and phenotype data. diXa is publicly available through a user-friendly web interface. New data can be readily deposited into diXa using guidelines and templates available online. Analysis descriptions and tools for interrogating the data are available via the diXa portal. Availability and implementation: http://www.dixa-fp7.eu Contact: [email protected]; [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Examining the effects of sodium ions on the binding of antagonists to dopamine D2 and D3 receptors

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo

IUPHAR-DB: An Open-Access, Expert-Curated Resource for Receptor and Ion Channel Research

[Image: see text] This contribution highlights efforts by the International Union of Basic and Clinical Pharmacology (IUPHAR) Nomenclature Committee (NC-IUPHAR) to classify human receptors and ion channels, to document their properties, and to recommend ligands that are useful for characterization. This effort has inspired the creation of an online database (IUPHAR-DB), which is intended to provide free information to all scientists, summarized from primary literature by experts

Controlling transmembrane protein concentration and orientation in supported lipid bilayers

The trans-membrane protein – proteorhodopsin (pR) has been incorporated into supported lipid bilayers (SLB). In-plane electric fields have been used to manipulate the orientation and concentration of these proteins, within the SLB, through electrophoresis leading to a 25-fold increase concentration of pR

The role of kinetic context in apparent biased agonism at GPCRs

Biased agonism describes the ability of ligands to stabilize different conformations of a GPCR linked to distinct functional outcomes and offers the prospect of designing pathway-specific drugs that avoid on-target side effects. This mechanism is usually inferred from pharmacological data with the assumption that the confounding influences of observational (that is, assay dependent) and system (that is, cell background dependent) bias are excluded by experimental design and analysis. Here we reveal that ‘kinetic context’, as determined by ligand-binding kinetics and the temporal pattern of receptor-signalling processes, can have a profound influence on the apparent bias of a series of agonists for the dopamine D2 receptor and can even lead to reversals in the direction of bias. We propose that kinetic context must be acknowledged in the design and interpretation of studies of biased agonism

Small Molecule Inhibited Parathyroid Hormone Mediated cAMP Response by N–Terminal Peptide Binding

Ligand binding to certain classes of G protein coupled receptors (GPCRs) stimulates the rapid synthesis of cAMP through G protein. Human parathyroid hormone (PTH), a member of class B GPCRs, binds to its receptor via its N–terminal domain, thereby activating the pathway to this secondary messenger inside cells. Presently, GPCRs are the target of many pharmaceuticals however, these drugs target only a small fraction of structurally known GPCRs (about 10%). Coordination complexes are gaining interest due to their wide applications in the medicinal field. In the present studies we explored the potential of a coordination complex of Zn(II) and anthracenyl–terpyridine as a modulator of the parathyroid hormone response. Preferential interactions at the N–terminal domain of the peptide hormone were manifested by suppressed cAMP generation inside the cells. These observations contribute a regulatory component to the current GPCR–cAMP paradigm, where not the receptor itself, but the activating hormone is a target. To our knowledge, this is the first report about a coordination complex modulating GPCR activity at the level of deactivating its agonist. Developing such molecules might help in the control of pathogenic PTH function such as hyperparathyroidism, where control of excess hormonal activity is essentially required

Therapeutic opportunities within the DNA damage response

The DNA damage response (DDR) is essential for maintaining the genomic integrity of the cell, and its disruption is one of the hallmarks of cancer. Classically, defects in the DDR have been exploited therapeutically in the treatment of cancer with radiation therapies or genotoxic chemotherapies. More recently, protein components of the DDR systems have been identified as promising avenues for targeted cancer therapeutics. Here, we present an in-depth analysis of the function, role in cancer and therapeutic potential of 450 expert-curated human DDR genes. We discuss the DDR drugs that have been approved by the US Food and Drug Administration (FDA) or that are under clinical investigation. We examine large-scale genomic and expression data for 15 cancers to identify deregulated components of the DDR, and we apply systematic computational analysis to identify DDR proteins that are amenable to modulation by small molecules, highlighting potential novel therapeutic targets