11 research outputs found
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 flexibility analyzed by molecular dynamics and exploited to identify non-peptidic inhibitors by high-throughput docking
Protein modeling with reduced representation: statistical potentials and protein folding mechanism
A high resolution reduced model of proteins is used in Monte Carlo dynamics studies of the folding mechanism of a small globular protein, the B1 immunoglobulin-binding domain of streptococcal protein G. It is shown that in order to reproduce the physics of the folding transition, the united atom based model requires a set of knowledge-based potentials mimicking the short-range conformational propensities and protein-like chain stiffness, a model of directional and cooperative hydrogen bonds, and properly designed knowledge-based potentials of the long-range interactions between the side groups. The folding of the model protein is cooperative and very fast. In a single trajectory, a number of folding/unfolding cycles were observed. Typically, the folding process is initiated by assembly of a native-like structure of the C-terminal hairpin. In the next stage the rest of the four-ribbon β-sheet folds. The slowest step of this pathway is the assembly of the central helix on the scaffold of the β-sheet
Molecular Dynamics of Biomolecules through Direct Analysis of Dipolar Couplings
Residual dipolar
couplings (RDCs) are important probes in structural
biology, but their analysis is often complicated by the determination
of an alignment tensor or its associated assumptions. We here apply
the maximum entropy principle to derive a tensor-free formalism which
allows for direct, dynamic analysis of RDCs and holds the classic
tensor formalism as a special case. Specifically, the framework enables
us to robustly analyze data regardless of whether a clear separation
of internal and overall dynamics is possible. Such a separation is
often difficult in the core subjects of current structural biology,
which include multidomain and intrinsically disordered proteins as
well as nucleic acids. We demonstrate the method is tractable and
self-consistent and generalizes to data sets comprised of observations
from multiple different alignment conditions
Molecular Determinants for Unphosphorylated STAT3 Dimerization Determined by Integrative Modeling
Signal
transducer and activator of transcription factors (STATs)
are proteins that can translocate into the nucleus, bind DNA, and
activate gene transcription. STAT proteins play a crucial role in
cell proliferation, apoptosis, and differentiation. The prevalent
view is that STAT proteins are able to form dimers and bind DNA only
upon phosphorylation of specific tyrosine residues in the transactivation
domain. However, this paradigm has been questioned recently by the
observation of dimers of unphosphorylated STATs (USTATs) by X-ray,
Förster resonance energy transfer, and site-directed mutagenesis.
A more complex picture of the dimerization process and of the role
of the dimers is, thus, emerging. Here we present an integrated modeling
study of STAT3, a member of the STAT family of utmost importance in
cancer development and therapy, in which we combine available experimental
data with several computational methodologies such as homology modeling,
protein–protein docking, and molecular dynamics to build reliable
atomistic models of USTAT3 dimers. The models generated with the integrative
approach presented here were then validated by performing computational
alanine scanning for all the residues in the protein–protein
interface. These results confirmed the experimental observation of
the importance of some of these residues (in particular Leu78 and
Asp19) in the USTAT3 dimerization process. Given the growing importance
of USTAT3 dimers in several cellular pathways, our models provide
an important tool for studying the effects of pathological mutations
at the molecular and/or atomistic level, and in the rational design
of new inhibitors of dimerization
NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease
The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor