Frog: a FRee Online druG 3D conformation generator

In silico screening methods based on the 3D structures of the ligands or of the proteins have become an essential tool to facilitate the drug discovery process. To achieve such process, the 3D structures of the small chemical compounds have to be generated. In addition, for ligand-based screening computations or hierarchical structure-based screening projects involving a rigid-body docking step, it is necessary to generate multi-conformer 3D models for each input ligand to increase the efficiency of the search. However, most academic or commercial compound collections are delivered in 1D SMILES (simplified molecular input line entry system) format or in 2D SDF (structure data file), highlighting the need for free 1D/2D to 3D structure generators. Frog is an on-line service aimed at generating 3D conformations for drug-like compounds starting from their 1D or 2D descriptions. Given the atomic constitution of the molecules and connectivity information, Frog can identify the different unambiguous isomers corresponding to each compound, and generate single or multiple low-to-medium energy 3D conformations, using an assembly process that does not presently consider ring flexibility. Tests show that Frog is able to generate bioactive conformations close to those observed in crystallographic complexes. Frog can be accessed at http://bioserv.rpbs.jussieu.fr/Frog.html

Molecular basis of coagulation factor V deficiency caused by the R1698W inter-domain mutation.

Coagulation factor V (FV) deficiency is characterised by variable bleeding phenotypes and heterogeneous mutations. To add new insights into the FV genotype-phenotype relationship, we characterised the R1698W change in the A3 domain, at the poorly investigated interface with the A2 domain. The FV R1698W mutation was responsible for a markedly reduced expression level (10% of FV-WT) and specific activity in thrombin generation (0.39). Interestingly, the FVa1698W showed rapid activity decay upon activation due to increased dissociation rate between the heavy and light chains. The importance of the size and charge of the residue at position 1698 was investigated by three additional recombinant mutants, FVR1698A, FVR1698Q, and FVR1698E. FVR1698A and FVR1698Q expression (30 and 45% of FV-WT), specific activity (both 0.57) and stability were all reduced. Noticeably, FVR1698E showed normal activity and stability despite poor expression (10% of FV-WT). These data indicate the essential role of R1698 for normal biosynthetic process and support local flexibility for positively or negatively charged residues to produce stable and functional A3-A2 domain interactions. Their experimental alteration produces a gradient of FV defects, which help to interpret the wide spectrum of phenotypes in FV-deficient patients

Mutations in complement factor I as found in atypical hemolytic uremic syndrome lead to either altered secretion or altered function of factor I

The complement system is regulated by inhibitors such as factor I (FI), a serine protease that degrades activated complement factors C4b and C3b in the presence of specific cofactors. Mutations and polymorphisms in FI and its cofactors are associated with atypical hemolytic uremic syndrome (aHUS). All 14 complement factor I mutations associated with aHUS analyzed in this study were heterozygous and generated premature stop codons (six) or amino acid substitutions (eight). Almost all of the mutants were expressed by human embryonic kidney 293 cells but only six mutants were secreted into the medium, three of which were at lower levels than WT. The remaining eight mutants were not secreted but sensitive to deglycosylation with endoglycosidase H, indicating that they were retained early in the secretory pathway. Six secreted mutants were purified and five of them were functionally altered in degradation of C4b/C3b in the fluid-phase in the presence of various cofactors and on endothelial cells. Three mutants cleaved surface-bound C3b less efficiently than WT. The D501N mutant was severely impaired both in solution and on surface irrespective of the cofactor used. in conclusion, mutations in complement factor I affect both secretion and function of FI, which leads to impaired regulation of the complement system in aHUS.Pathophysiology and treatment of rheumatic disease

A critical role for Gly25 in the B chain of human thrombin

We have recently identified (Akhavan S et al., Thromb Haemost 2000; 84: 989-97) a patient with a mild bleeding diathesis associated to an homozygous mutation in the thrombin B chain (Gly25Ser, chymotrypsinogen numbering, i.e. position 330 in human prothrombin numbering). Transient transfection of wild-type prothrombin (FII-WT) and mutant prothrombin (designated FII-G25((330))S) cDNA in COS-7 cells showed a mild reduction (50%) in FII-G25((330))S production. Recombinant proteins, stably expressed in Chinese hamster ovary cells, were isolated and activated by TaYpan snake or Echis carinatus venoms. We show that the G25((330))S mutation results in a decrease in the rate of prothrombin proteolytic activation. The mutation also significantly decreases (i) the catalytic activity of thrombin with a 9-fold reduction in catalytic efficiency of the mutant toward S-2238; (ii) the interaction with benzamidine; (iii) the rate of inhibition by TLCK and antithrombin; and (iv) the rate of hydrolysis of macromolecular substrates (fibrinogen, protein C). In contrast, exosite I does not appear to be affected by the molecular defect. These results, together with molecular modeling and dynamics, indicate that Gly25((330)) is important for proper expression and probably proper folding of prothrombin, and also plays a critical role in both the alignment of the catalytic triad and the flexibility of one of the activation segments of prothrombi

1,2,4-Oxadiazoles Identified by Virtual Screening and their Non-Covalent Inhibition of the Human 20S Proteasome

International audienceAlthough several constitutive proteasome inhibitors have been reported these recent years, potent organic, noncovalent and readily available inhibitors are still poorly documented. Here we used a structure- and ligand-based in silico approach to identify commercially available 1,2,4-oxadiazole derivatives as non-covalent human 20S proteasome inhibitors. Their optimization led to the newly synthesized compound 4h that is a mixed proteasomal inhibitor of the chymotrypsin- like activity (K(i) of 26,1 nM and K'(i) of 7.5 nM) which is in addition selective versus the challenging cathepsin B and calpain proteases. Molecular modelling studies corroborated the mechanism of inhibition and suggest an unusual binding of the inhibitor within the S5 binding pocket (β6 subunit). The cellular effects of our compounds validate their utility as potential pharmacological agents for anti-cancer pre-clinical studies