ABCG2/BCRP transport mechanism revealed through kinetically excited targeted molecular dynamics simulations.

ABCG2/BCRP is an ABC transporter that plays an important role in tissue protection by exporting endogenous substrates and xenobiotics. ABCG2 is of major interest due to its involvement in multidrug resistance (MDR), and understanding its complex efflux mechanism is essential to preventing MDR and drug-drug interactions (DDI). ABCG2 export is characterized by two major conformational transitions between inward- and outward-facing states, the structures of which have been resolved. Yet, the entire transport cycle has not been characterized to date. Our study bridges the gap between the two extreme conformations by studying connecting pathways. We developed an innovative approach to enhance molecular dynamics simulations, 'kinetically excited targeted molecular dynamics', and successfully simulated the transitions between inward- and outward-facing states in both directions and the transport of the endogenous substrate estrone 3-sulfate. We discovered an additional pocket between the two substrate-binding cavities and found that the presence of the substrate in the first cavity is essential to couple the movements between the nucleotide-binding and transmembrane domains. Our study shed new light on the complex efflux mechanism, and we provided transition pathways that can help to identify novel substrates and inhibitors of ABCG2 and probe new drug candidates for MDR and DDI.

1,2,4-Oxadiazoles identified by virtual screening and their non-covalent inhibition of the human 20S proteasome.

Although 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 (ß6 subunit). The cellular effects of our compounds validate their utility as potential pharmacological agents for anti-cancer pre-clinical studies.

AMMOS software: method and application.

Recent advances in computational sciences enabled extensive use of in silico methods in projects at the interface between chemistry and biology. Among them virtual ligand screening, a modern set of approaches, facilitates hit identification and lead optimization in drug discovery programs. Most of these approaches require the preparation of the libraries containing small organic molecules to be screened or a refinement of the virtual screening results. Here we present an overview of the open source AMMOS software, which is a platform performing an automatic procedure that allows for a structural generation and optimization of drug-like molecules in compound collections, as well as a structural refinement of protein-ligand complexes to assist in silico screening exercises.

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 models of the procoagulant factor VIIIa-factor IXa complex.

BACKGROUND: Formation of the intrinsic tenase complex is an essential event in the procoagulant reactions that lead to clot formation. The tenase complex is formed when the activated serine protease, Factor IXa (FIXa), and its cofactor Factor VIIIa (FVIIIa) assemble on a phospholipid surface to proteolytically convert the zymogen Factor X (FX) into its active form FXa. The physiological relevance of the tenase complex is evident in hemophilia A or B patients who present with bleeding disorders. OBJECTIVES: The purpose of this study was to establish three-dimensional (3D) models of the FVIIIa-FIXa complex. METHODS: First, we built two new theoretical models of FVIIIa via homology modeling, inter-domain docking and loop simulation algorithms as well as a model for FIXa. This was followed by pseudo-Brownian protein-protein docking in internal coordinates with the ICM (Internal Coordinates Mechanics) program between the two FVIIIa and the FIXa structures. RESULTS: Ten representative models of this complex are presented based on agreements with known experimental data and according to structural criteria. CONCLUSIONS: These novel 3D models will help guide future site directed mutagenesis aimed at improving the functionality of FVIIIa and/or FIXa and will contribute to a better understanding of the role of this macromolecular complex in the blood coagulation cascade.

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 Taipan 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 prothrombin.

Theoretical and experimental study of the D2194G mutation in the C2 domain of coagulation factor V.

Coagulation factor V (FV) is a large plasma glycoprotein with functions in both the pro- and anticoagulant pathways. In carriers of the so-called R2-FV haplotype, the FV D2194G mutation, in the C2 membrane-binding domain, is associated with low expression levels, suggesting a potential folding/stability problem. To analyze the molecular mechanisms potentially responsible for this in vitro phenotype, we used molecular dynamics (MD) and continuum electrostatic calculations. Implicit solvent simulations were performed on the x-ray structure of the wild-type C2 domain and on a model of the D2194G mutant. Because D2194 is located next to a disulfide bond (S-S bond), MD calculations were also performed on S-S bond depleted structures. D2194 is part of a salt-bridge network and investigations of the stabilizing/destabilizing role of these ionic interactions were carried out. Five mutant FV molecules were created and the expression levels measured with the aim of assessing the tolerance to amino acid changes in this region of molecule. Analysis of the MD trajectories indicated increased flexibility in some areas and energetic comparisons suggested overall destabilization of the structure due to the D2194G mutation. This substitution causes electrostatic destabilization of the domain by approximately 3 kcal/mol. Together these effects likely explain the lowered expression levels in R2-FV carriers.

Local electrostatic potentials in pyridoxal phosphate labelled horse heart cytochrome c.

The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt.c). PLP has also been used to calculate the pKa values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental pKa values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluorescence measurements, as follows: in PLP-Lys-79-cyt.c for pyridine nitrogen 4.5 (absorbance) and 5.1 (fluorescence), for phenolic hydroxyl 8.6 (absorbance) and 8.3 (fluorescence); in PLP-Lys-86-cyt.c for pyridine nitrogen 4.7 (absorbance) and 5.8 (fluorescence), for phenolic hydroxyl 8.3 (absorbance) and 8.5 (fluorescence). The differences between absorbance and fluorescence data are related to differences in the behaviour of the bound label in the ground and excited electronic states and to intermolecular charge-charge interactions. Molecular modelling was used to generate the atomic co-ordinates of the PLP-modified horse heart cytochrome c necessary for the theoretical calculations of the pKa values and electrostatic potentials.