ALFA: Automatic Ligand Flexibility Assignment

ALFA is a fast computational tool for the conformational analysis of small molecules that uses a custom-made iterative algorithm to provide a set of representative conformers in an attempt to reproduce the diversity of states in which small molecules can exist, either isolated in solution or bound to a target. The results shown in this work prove that ALFA is fast enough to be integrated into massive high-throughput virtual screening protocols with the aim of incorporating ligand flexibility and also that ALFA reproduces crystallographic X-ray structures of bound ligands with great accuracy. Furthermore, the application includes a graphical user interface that allows its use through the popular molecular graphics program PyMOL to make it accessible to nonexpert users. ALFA is distributed free of charge upon request from the authors

CRDOCK: An Ultrafast Multipurpose Protein–Ligand Docking Tool

An ultrafast docking and virtual screening program, CRDOCK, is presented that contains (1) a search engine that can use a variety of sampling methods and an initial energy evaluation function, (2) several energy minimization algorithms for fine tuning the binding poses, and (3) different scoring functions. This modularity ensures the easy configuration of custom-made protocols that can be optimized depending on the problem in hand. CRDOCK employs a precomputed library of ligand conformations that are initially generated from one-dimensional SMILES strings. Testing CRDOCK on two widely used benchmarks, the ASTEX diverse set and the Directory of Useful Decoys, yielded a success rate of ∼75% in pose prediction and an average AUC of 0.66. A typical ligand can be docked, on average, in just ∼13 s. Extension to a representative group of pharmacologically relevant G protein-coupled receptors that have been recently cocrystallized with some selective ligands allowed us to demonstrate the utility of this tool and also highlight some current limitations. CRDOCK is now included within VSDMIP, our integrated platform for drug discovery

Interactions of Bacterial Cell Division Protein FtsZ with C8-Substituted Guanine Nucleotide Inhibitors. A Combined NMR, Biochemical and Molecular Modeling Perspective

FtsZ is the key protein of bacterial cell-division and target for new antibiotics. Selective inhibition of FtsZ polymerization without impairing the assembly of the eukaryotic homologue tubulin was demonstrated with C8-substituted guanine nucleotides. By combining NMR techniques with biochemical and molecular modeling procedures, we have investigated the molecular recognition of C8-substituted-nucleotides by FtsZ from <i>Methanococcus jannaschii</i> (Mj-FtsZ) and <i>Bacillus subtilis</i> (Bs-FtsZ). STD epitope mapping and trNOESY bioactive conformation analysis of each nucleotide were employed to deduce differences in their recognition mode by each FtsZ species. GMP binds in the same anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn conformation. However, the anti conformation of 8-morpholino-GMP is selected by Bs-FtsZ, while Mj-FtsZ binds both anti- and syn-geometries. The inhibitory potencies of the C8-modified-nucleotides on the assembly of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities. Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces formation of Mj-FtsZ curved filaments, resembling polymers formed by the inactive forms of this protein. NMR data, combined with molecular modeling protocols, permit explanation of the mechanism of FtsZ assembly impairment by C8-substituted GTP analogs. The presence of the C8-substituent induces electrostatic remodeling and small structural displacements at the association interface between FtsZ monomers to form filaments, leading to complete assembly inhibition or to formation of abnormal FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be simply explained by steric clashes of the C8-GTP-analogs with the incoming FtsZ monomer. This information may facilitate the design of antibacterial FtsZ inhibitors replacing GTP

Engineering Erg10 Thiolase from <i>Saccharomyces cerevisiae</i> as a Synthetic Toolkit for the Production of Branched-Chain Alcohols

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from <i>Saccharomyces cerevisiae</i> showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)­chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered <i>Clostridium</i>’s 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound