PROJECT 1: HIV-1 REVERSE TRANSCRIPTASE
Research in the Peliska lab involves the discovery and characterization of novel inhibitors against the clinically important viral target HIV-1 Reverse Transcriptase. This project encompasses a broad range of study in protein chemistry, advanced enzyme kinetics, organic synthesis, biophysical chemistry, and bio-organic reaction mechanisms.
DNA strand transfer reactions (where the extending DNA primer transfers from one template strand to another) must occur twice during reverse transcription, and homologous recombination during reverse transcription is frequent (see Peliska & Benkovic, (1992) Science 258: 1112-1118 for an introduction to strand transfer reactions associated with reverse transcription). The two proteins known to be involved in these reactions, HIV-1 reverse transcriptase (RT) and nucleocapsid (NC) protein, are being studied using kinetic, biochemical and genetic approaches to understand the mechanism and inhibition of DNA strand transfer. It is known that strand transfer reactions require both the polymerase and RNase H activities of RT. We have identified novel inhibitors of these HIV-1 RT catalyzed reactions and we have begun to elucidate the mechanisms of inhibition by these compounds. In addition, we have begun to develop bacteria-based selection methods to investigate inhibitor-resistant mutants of HIV-1 RT. Our approaches should provide important information regarding possible new inhibitor designs targeting this important viral protein, as well as aid in our understanding of the mechanism of DNA polymerization, RNase H and strand transfer reactions catalyzed by HIV-1 RT.
We are currently conducting structure-activity relationship (SAR) studies on one of our novel inhibitors of reverse transcriptase. We are designing, synthesizing and kinetically characterizing these new inhibitors to 1) increase their potency against RT polymerase and/or RNaseH activities, and 2) elucidate their mechanism of action and identify their binding site on the enzyme. These studies will provide information regarding the mode of inhibitor binding relative to the template-primer and deoxynucleoside triphosphate substrates, as well as which step in the polymerization and DNA strand transfer reactions are impacted by these inhibitors. These insights will in turn allow us to strategically design and create better next-generation inhibitors.
Undergraduate Opportunities for Research Involvement
Our lab, comprised of undergraduate students in the Biochemistry major, has been investigating the kinetics and mechanism of reverse transcriptase for some time. The project presented here entails a wide range of strategies and methods that are attractive to the undergraduate student interested in independent research opportunities:
Synthetic organic chemistry: synthesis of inhibitors and their derivatives.
Protein biochemistry: analysis of protein modification, development of fluorescence-based enzyme assays.
Enzyme kinetics: analysis of RT mechanism and inhibitor function.
Molecular biology: creation of mutant RTs (site specific and libraries), development of genetic selections, creation and characterization of
selection strains using recombineering techniques.
Structural analysis: inhibitor-protein computer-aided docking (e.g. AutoDock Vina) experiments for the design of second generation inhibitors.
The structure of HIV-1 Reverse transcriptase bound to a DNA template-primer
PROJECT 2: QUANTITATIVE ANALYSIS OF METABOLITE BIOMARKERS OF HUMAN DISEASES
We are designing novel sensors for the quantification of established or suspected metabolite biomarkers for certain human medical disorders. Metabolites (small molecules associated with cellular metabolism) fluctuate depending on the cell type and growth conditions. Studies in the field of metabolomics are identifying metabolites that change in concentration as a result of aberrant cellular metabolic changes associated with disorders such as autism, cancer, infection or other disease states. The monitoring of these biomarkers will lead to important new information for the research scientist, and could potentially serve as the basis of useful clinical diagnostic tools for the early detection of a wide variety of disorders. Because of the complexities of biological samples (containing thousands of metabolites, proteins, etc.), current methods of metabolite identification and quantification involve the use of technically demanding, equipment-intensive and expensive techniques such as GC or LC-MS and magic angle spinning NMR spectroscopy. We are approaching the analysis of specific metabolites from a philosophy that Manu Prakash at Stanford coined frugal science. The idea is to take complex, critically important problems (such as disease detection, prevention and treatment) and find scientifically rigorous yet inexpensive and simple methods of analysis. Using protein-based receptors (easily and inexpensively obtained in large quantities by expression of recombinant proteins and purification by affinity chromatography), inexpensively fabricated microfluidic devices and economical fluorescence spectroscopy techniques, we are developing novel ways of quantitatively assaying several of the metabolic intermediates potentially useful as medical diagnostics. These methods promise to provide sensitive, yet inexpensive tools for the monitoring of these compounds in small (microliter quantities) biological samples.