ABSTRACT
In silico drug discovery is a complex process requiring flexibility and ingenuity in method selection and a careful validation of work protocols. GPCR in silico drug discovery poses additional challenges due to the paucity of crystallographic data. This paper starts by reviewing selected GPCR in silico screening programs reported in the literature, including both structure-based and ligand-based approaches. Particular emphasis is given to library design, binding mode selection, process validation and compound selection for biological testing. Following literature review, we provide insights into in silico methodologies and process workflows used at EPIX to drive over 20 highly successful screening and lead optimization programs performed since 2001. Applications of the various methodologies discussed are demonstrated by examples from recent programs that have not yet been published.
Subject(s)
Drug Discovery , Receptors, G-Protein-Coupled/antagonists & inhibitors , Drug Design , Ligands , Models, Molecular , Structure-Activity RelationshipABSTRACT
The diverse selection of targets in the CAPRI experiments provides grounds for determining the limits of our rigid-body docking program MolFit, and for extending it. We find that the sensitivity of MolFit is high, enabling it to produce reasonably accurate docking solutions when the structures undergo moderate local conformation changes upon complex formation or when the docked molecules are modeled. Yet the ranks of these solutions are sometimes too low to meet the requirements of CAPRI assessment. This indicates that the selectivity of MolFit, which was optimized for docking of unbound X-ray structures, and which relies on the availability of external data from biochemical and bioinformatic sources, needs readjustment in order to meet the challenges presented by NMR or modeled structures. A different challenge is presented by large global conformation changes such as movements of domains. We show that such changes can be accommodated within the rigid-body approximation by employing rigid multibody multistage docking procedures. We also address the difficulty of ranking results from 2-body and multibody docking scans in cases in which there are no external data favoring one option over the other.
Subject(s)
Computational Biology/methods , Protein Interaction Mapping/methods , Proteomics/methods , Algorithms , Computer Simulation , Crystallography, X-Ray , Databases, Protein , Dimerization , Internet , Macromolecular Substances , Models, Molecular , Models, Statistical , Molecular Conformation , Mutation , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Reproducibility of Results , Software , Static Electricity , Structural Homology, Protein , ThermodynamicsABSTRACT
Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vitro evolution of these biologically important molecules. However, the general usefulness of this technology is still limited by some technical difficulties. One of the most debilitating obstacles to the widespread application of the technology is the accumulation of "insert loss" clones in the libraries; phagemid clones from which the DNA encoding part or all of the cloned antibody fragment had been deleted. Another difficulty arises when phage technology is applied for cloning hybridoma-derived antibody genes, where myeloma derived light chains, irrelevant to the hybridoma's antibody specificity may be fortuitously cloned. Here, we report the construction of a novel phage-display system designed to address these problems. In our system a single-chain Fv (scFv) is expressed as an in-frame fusion protein with a cellulose-binding domain (CBD) derived from the Clostridium thermocellum cellulosome. The CBD domain serves as an affinity tag allowing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produced thereof. We demonstrate the utility of our system in solving the technical difficulties described above, and in speeding up the process of scFv isolation from combinatorial antibody repertoires.
