NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease

Dengue and West Nile virus infections put an estimated 2.5 billion people at risk. Neither drugs nor vaccines are currently available against these diseases. The non-structural protein NS3 is a protease that, together with the cofactor NS2B, is essential for viral maturation. The NS2B-NS3 proteases of dengue and West Nile viruses are highly homologous and present promising drug targets. Crystal structures of the West Nile virus protease with and without bound inhibitor revealed large structural differences in NS2B, while no crystal structure of the dengue virus protease could be determined with a bound inhibitor. We investigated the structural change in solution and found that the C-terminal segment (CTS) of the NS2B cofactor is prone to dissociation from NS3. In the case of the West Nile virus protease, the CTS of NS2B is mostly associated with NS3, especially in the presence of inhibitors. In the case of the dengue virus protease and in the absence of inhibitors, the CTS of NS2B is mostly dissociated from NS3. Finding drug candidates to inhibit the association of the NS2B cofactor may thus be easier for the dengue virus protease

The Free Energy Landscape of Small Molecule Unbinding

The spontaneous dissociation of six small ligands from the active site of FKBP (the FK506 binding protein) is investigated by explicit water molecular dynamics simulations and network analysis. The ligands have between four (dimethylsulphoxide) and eleven (5-diethylamino-2-pentanone) non-hydrogen atoms, and an affinity for FKBP ranging from 20 to 0.2 mM. The conformations of the FKBP/ligand complex saved along multiple trajectories (50 runs at 310 K for each ligand) are grouped according to a set of intermolecular distances into nodes of a network, and the direct transitions between them are the links. The network analysis reveals that the bound state consists of several subbasins, i.e., binding modes characterized by distinct intermolecular hydrogen bonds and hydrophobic contacts. The dissociation kinetics show a simple (i.e., single-exponential) time dependence because the unbinding barrier is much higher than the barriers between subbasins in the bound state. The unbinding transition state is made up of heterogeneous positions and orientations of the ligand in the FKBP active site, which correspond to multiple pathways of dissociation. For the six small ligands of FKBP, the weaker the binding affinity the closer to the bound state (along the intermolecular distance) are the transition state structures, which is a new manifestation of Hammond behavior. Experimental approaches to the study of fragment binding to proteins have limitations in temporal and spatial resolution. Our network analysis of the unbinding simulations of small inhibitors from an enzyme paints a clear picture of the free energy landscape (both thermodynamics and kinetics) of ligand unbinding

Discovery of a Non-Peptidic Inhibitor of West Nile Virus NS3 Protease by High-Throughput Docking

An estimated 2.5 billion people are at risk of diseases caused by dengue and West Nile virus. As of today, there are neither vaccines to prevent nor drugs to cure the severe infections caused by these viruses. The NS3 protease is one of the most promising targets for drug development against West Nile virus because it is an essential enzyme for viral replication and because success has been demonstrated with the closely related hepatitis C virus protease. We have discovered a small molecule that inhibits the NS3 protease of West Nile virus by computer-aided high-throughput docking, and validated it using three experimental techniques. The inhibitor has potential to be developed to a drug candidate to combat West Nile virus infections

Flaviviral protease inhibitors identified by fragment-based library docking into a structure generated by molecular dynamics

Fragment-based docking was used to select a conformation for virtual screening from a molecular dynamics trajectory of the West Nile virus nonstructural 3 protease. This conformation was chosen from an ensemble of 100 molecular dynamics snapshots because it optimally accommodates benzene, the most common ring in known drugs, and two positively charged fragments (methylguanidinium and 2-phenylimidazoline). The latter fragments were used as probes because of the large number of hydrogen bond acceptors in the substrate binding site of the protease. Upon high-throughput docking of a diversity set of 18,694 molecules and pose filtering, only five compounds were chosen for experimental validation, and two of them are active in the low micromolar range in an enzymatic assay and a tryptophan fluorescence quenching assay. Evidence for specific binding to the protease active site is provided by nuclear magnetic resonance spectroscopy. The two inhibitors have different scaffolds (diphenylurea and diphenyl ester) and are promising lead candidates because they have a molecular weight of about 300 Da

Transplantation of a hydrogen bonding network from West Nile virus protease onto Dengue-2 protease improves catalytic efficiency and sheds light on substrate specificity

10.1093/protein/gzs049Protein Engineering, Design and Selection2512843-850PEDS

Structure-guided fragment-based in silico drug design of dengue protease inhibitors

10.1007/s10822-011-9418-0Journal of Computer-Aided Molecular Design253263-274JCAD

Dynamics in the active site of β-Secretase: a network analysis of atomistic simulations

The aspartic protease β-secretase (BACE) catalyzes the hydrolysis of the amyloid precursor protein (APP) which leads to amyloid-β aggregation and, ultimately, the perilous Alzheimer's disease. The conformational dynamics and free energy surfaces of BACE at three steps of the catalytic cycle are studied here by explicit solvent molecular dynamics simulations (multiple runs for a total of 2.2 μs). The overall plasticity of BACE is essentially identical for the three states of the substrate: the octapeptide reactant, gem-diol intermediate, and cleavage products. In contrast, the network of hydrogen bonds in the active site is more stable in the complex of BACE with the gem-diol intermediate than the other two states of the substrate. The spontaneous release of the C-terminal (P1'-P4') fragment of the product follows a single-exponential time dependence with a time constant of 50 ns and does not require the opening of the flap. The fast dissociation of the C-terminal fragment is consistent with the transmembrane location and orientation of APP and its further processing by γ-secretase. On the other hand, the N-terminal (P4-P1) fragment of the product does not exit the BACE active site within the simulation time scale of 80 ns. A unified network analysis of the complexes of BACE with the three states of the substrate provides an estimation of the activation free energy associated with the structural rearrangements that involve only noncovalent interactions. The estimated rearrangement barriers are not negligible (up to 3 kcal/mol) but are significantly smaller than the barrier of the peptide bond hydrolysis reaction