Daniela C Vaz
University of Coimbra, Portugal
Title: Use of molecular interaction fields to understand drug resistance in HIV 1 protease caused by single point mutations
Biography
Daniela C Vaz has completed her PhD in Biological Chemistry from the University of Coimbra. Her research focuses mainly on protein structure, folding and stability, in relation to function and disease. She is currently working as a Professor at the School of Health Sciences of Leiria and is also a Member of the Coimbra Chemistry Centre at the University of Coimbra, Portugal.
Abstract
Molecular Interaction Fields (MIF) is an archetypal computational chemistry technique that can be applied to capture a singular fingerprint of an ensemble of atoms on a protein and encode its physicochemical environment. Thus, MIFs have particular relevance in the context of binding hot spots and binding site analysis. Taking HIV 1 Protease (HIVPR) as case study, the present work focuses on a MIF-based in silico approach to achieve a qualitative interpretation and quantitative determination of mutation effects on HIVPR’s binding site, to help to understand translated changes in the enzyme’s structure and physicochemical environment. Assuming that binding sites with similar chemical environments have similar affinity for inhibitors, our method calculates and compares MIF similarities, visually assessing structural differences and quantifying their overlap through a Tanimoto coefficient. To assess the method’s ability to capture mutation induced chemical perturbations within HIVPR’s binding site, we collected 48 X-ray structures from the Protein Data Bank (PDB), from HIV strains either resistant or susceptible to protease inhibitors and quantified their binding site MIF similarities against a high quality, susceptible, reference structure. We observed and defined a threshold that discriminated most susceptible and resistant structures, confirming the MIF's suitability for our approach. Subsequently, we built homology models containing different reported single point resistance-conferring mutations using a single high-quality PDB structure as template. Root-Mean-Square Deviation (RMSD) values between template and model structures were calculated on residue by residue basis, confirming that the mutation was the only structural change. Then, the MIF similarities were determined, showing that this technique effectively captured subtle changes on HIVPR’s binding sites induced by the studied mutations. Along with the perspective of following an equivalent ligand based approach, we believe our results can be a promising starting point for developing an algorithm with drug resistance predictive power.
Premkumar Dinadayala
Sanofi Pasteur, France
Title: Structural and functional characterization of various forms of glycated heat labile toxin protein
Biography
Premkumar Dinadayala has completed his PhD from University of Toulouse and Postdoctoral studies from Colorado State University, Colorado. He is working as a Scientist since 12 years at Sanofi Pasteur. He has published more than 10 papers in journals.
Abstract
Enterotoxigenic Escherichia coli (ETEC) are responsible for a high diarrheal disease burden, especially in children living in endemic countries and travelers visiting those countries. After oro-fecal transmission, ETEC reaches the small intestine where adhesion occurs through colonization factors. Then heat Labile Toxin (LT), one of the two enterotoxins produced by ETEC, is secreted and causes aqueous diarrhea. LT consists of five B sub-units, which are able to bind the monosialoganglioside GM1 and a single, catalytically-active A subunit stimulating the intracellular synthesis of cyclic Adenosine Monophosphate (cAMP), leading ultimately to fluid and electrolyte secretions into the intestinal lumen. Herein we characterized various purified forms of LT: (1) recombinant B subunit of LT (rLTB) (2) native LT purified from ETEC strain (nLT) and (3) recombinant LT purified from E. coli expressing the protein (rLT). SDS-PAGE analysis showed a difference of migration between the different LT forms confirmed by liquid chromatography coupled to MS (mass shift of 162 Da). This modification was found to be due to the glycation of LT subunits by galactose, a reducing sugar that is used in the LT purification process and remains present during the LT Lyophilization process. This observation has to be taken into account for the purification and storage of LT. Experiments are ongoing to determine if LT glycation could have an impact on the functional activity of LT using an in vitro assay based on cAMP release by epithelial cell line.
