Research Interests

    One of the greatest obstacles in the treatment of HIV-1 infection and AIDS is the rapid appearance of drug resistance due to the great variability that develops in the retroviral genome.  Significant evidence shows that much of this genetic variability is acquired during retroviral reverse transcription, a process catalyzed by the virally encoded enzyme reverse transcriptase (RT).  The two principle mechanisms responsible for the rapid evolution of the retroviral genome include nucleotide misincorporation by HIV-1 RT during proviral DNA synthesis, and recombination events occurring during reverse transcription.  HIV-1 RT is involved in both of these processes.  HIV-1 RT is a multifunctional enzyme that can catalyze RNA-dependent DNA polymerization and DNA-dependent DNA polymerization as well as the efficient hydrolysis of RNA from RNA·DNA hybrids.

This is the x-ray crystal structure of HIV-1 reverse transcriptase showing the locations of the polymerase and RNaseH active sites.  Our lab is studying inhibitors that target each activity separately and together.  Our research efforts focus on questions concerning 1) where do the inhibitors bind to the enzyme and 2) can we design and synthesize more potent, more selective inhibitors based on structure-activity studies.

   The discovery of novel RT inhibitors and a better understanding of the mechanisms of drug resistance are essential for the development of more effective treatments of retroviral infections, and for a more complete understanding of the complex process of reverse transcription.   More generally, such studies will lead to a more complete understanding of the origin of replicative errors during DNA synthesis that can lead to the generation of genetic mutations and disease states.  In an effort to explore these broad issues, my research efforts fall into two interrelated areas and span a range of interests in bio-organic chemistry, organic synthesis, biophysical chemistry, protein chemistry and advanced enzyme kinetics.  The goal of the first project is to identify, characterize and optimize the activity of novel inhibitors of HIV-1 reverse transcriptase (RT).  We have several such inhibitors in hand and we are currently characterizing their mode of action using structure-activity studies, enzyme kinetics and inhibitor-enzyme cross-linking experiments.   

    The second area of study involves the elucidation of the mechanism of nucleoside analog drug resistance and altered replication fidelity in clinically important mutants of the HIV-1 RT.  The ability of HIV to development drug resistance is the major problem in the therapeutic treatment of this infection.  One of the most common classes of drugs used in the treatment of HIV-1 infection is the so-called nucleoside analog (i.e. AZT, ddG, ddI, d4T).  These drugs (in their triphosphate form) serve as substrates for RT during reverse transcription.  Since they lack the 3'-hydroxyl required for subsequent primer extension, they serve as replication chain terminators.  Some 20 HIV-1 RT mutations have been clinically identified that individually or in combination lead to a pronounced resistance to the incorporation these nucleoside analogs.  The goal of this project is generate recombinant RT containing these mutations, and investigate the mechanism of resistance to the incorporation of these analogs. The mechanism by which RT mutants accomplish this resistance is not obvious since nucleoside triphosphates bind to RT via a minimal two-step mechanism:  
(E = RT; D = template-primer; N = dNTP/analogue)

    The mechanism of resistance to incorporation of nucleoside triphosphate analogs by mutant RT must be investigated within the context of this reaction mechanism.  A mutant RT might accomplish resistance to incorporation of the dNTP analog (i.e. AZT triphosphate) by altering one of the three steps shown in the figure above.  The mutation could reduce the affinity of RT for the dNTP at the step of initial binding and recognition.  Alternatively, the second binding step involving a protein conformational change might be less efficient in the resistant mutant.  Finally, dNTP binding may not be compromised in the mutant enzyme, but the bound substrates may not be properly aligned for catalysis.  We are using a number of advanced kinetic and biophysical techniques to understand which step or steps of the above kinetic mechanism are altered in mutant, resistant RTs.