Our targets include HIV-1 integrase, the c-Kit and src-abl proteins , and the metalloproteinases associated with CXCl16 shedding. Student researchers trained in these techniques are also responsible for peer instruction in our general education science curriculum, leading discovery-based experimental modules in each of the areas outlined above.
The personnel include Dr. Eamonn F. Healy , who has published extensively on the development and application of quantum mechanical methods. Dr. Healy is responsible for the modeling components of this work. Dr. Peter King has more than a decade of experience in HIVand Mtb-related research, and is responsible for directing the molecular biologic aspects of the group, including molecular cloning of enzymatic targets, over-expression and purification of target proteins, and the development and performance of fluorescence-based enzymatic assays.Dr. Charles Hauser has examined the structure function relationships of the nuclease BAL 31, was a member of the research team that initiated the genome sequencing efforts for Chlamydomonas, and is an active participant in the NSF-funded Genome Consortium for Active Teaching (GCAT). Dr. Hauser directs the bioinformatics component of the research.
HIV-1 Integrase
Human Immunodeficiency Virus 1 Integrase (HIV-1 IN) is the enzyme responsible for integrating the viral DNA into the host genome, and is essential to the life cycle of the virus . L-chicoric acid (L-CA) is a bidentate catechol that has been identified as a potent inhibitor of HIV-1 IN. By combining high level ab initio calculations, a flexible docking scheme, and the new Autodock 4.0 free-energy function we have obtained a L-CA binding mode that explains its observed potency and is consistent with available experimental data. Because of the a,b-unsaturated ester functionality of the side arms of L-CA we first performed an extensive conformational analysis of L-CA using molecular mechanics, semiempirical calculations, and high level ab initio calculations. As a result we have identified two distinct L-CA binding modes, one for the s-cis /s-cis and another for the s-cis /s-trans isomers. The most stable conformer was found to be the structure with the a,b-unsaturated ester in the s-cis conformation for both arms of L-CA. This conformer also gave the top ranked docking solution. Analysis of the interactions with key IN residues, combined with results using a L-CA tetramethoxy derivative and a Q148A IN mutant, correlate well with the experimental data.
Cite: Eamonn F. Healy, Jonathan Sanders†, Peter J. King and W. Edward Robinson, Jr “A Docking Study of L-Chicoric Acid with HIV-1 Integrase” J Mol. Graph. Model. 2009, 27 , 14.
† student author
Tyrosine Kinases
The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamiliy often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.
Cite: Eamonn F. Healy , Skylar Johnson† , Charles Hauser, and Peter King “Tyrosine kinase inhibition: Ligand binding and conformational change in c-Kit and c-Abl.” FEBS Lett. 2009, 583, 2899-2906.
† student author
Mtb acr
A number of studies utilizing various strategies have identified Mtb proteins specifically expressed under conditions that mimic some of those thought to be present during the establishment of latency. One such protein Acr, expressed from the Mtb hspX gene is the predominantly expressed protein, in deference to most other Mtb genes, under micro-aerobic conditions such as those postulated to be present during establishment of latency. In similar studies, Acr has been shown to be released either through direct secretion or from lysed bacteria. Therefore, overexpression and extracellular release of Acr may represent a critical step in the establishment of latency by modulating intracellular bacterial conditions, modifying extracellular conditions such as host cells, or both. In support of this hypothesis, previous data from our group has demonstrated that phagocytosis of Acr-coated polystyrene beads by murine macrophages induces expression of a number of chemokines, cytokines and other factors when compared to phagocytosis of control beads. Of note, macrophage release of the chemokine CXCL16 was induced 3-fold in these experiments and correlated with a 5-fold increase in transcription of CXCL16 mRNA. In that CXCL16 is released from macrophages due to the activities of two transmembrane proteases ADAM-10 and ADAM-17, this suggests that Acr either directly or indirectly activates ADAM-10 or ADAM-17 leading to secretion of CXCL16. Importantly the role of CXCL16 in latency and pathogenesis of tuberculosis has yet to be addressed.
