The broad, long-term objective of this research is to use a combination of NMR, crystallographic and quantum chemical methods to investigate the structure, function and inhibition of metalloproteins of interest as drug targets. The first specific aim is to use NMR and computational chemistry to devise novel inhibitors of HIV-1 reverse transcriptase catalyzed excision of AZT (azidothymidine), responsible for many cases of drug-resistant HIV/AIDS in the United States. This work will follow up on the recent discovery of a class of compounds, bisphosphonates, which block the ATP-dependent excision of AZT in both enzyme and cell-based assays, restoring drug sensitivity. The hypothesis to be tested is that the bisphosphonates bind to the Mg2+ site in RT, as does ATP, inhibiting the ATP - catalyzed excision process. Since there are no x-ray structures of these bound inhibitors, NMR will be used to determine bound drug conformations, plus quantum chemistry will be used to determine structure/activity relationships aimed at developing improved inhibitors.
The second aim i s to investigate 3 Mg2+-enzymes involved in isoprenoid biosynthesis, of importance in e.g. cell signaling and sterol biosynthesis: farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR). We will use NMR, crystallography and quantum chemistry to guide the development of novel bisphosphonate and diphosphate inhibitors of these metalloenzymes. FPPS and DXR are of interest in the context of the therapy of infectious diseases while FPPS and IPPI are of interest in the immunotherapy of certain cancers. The third and final aim is to investigate the effects of these inhibitors on FPPS, IPPI, DXR and other isoprene biosynthesis pathway enzymes in cells, using microarray techniques, together with targeted gene silencing using siRNA techniques. The objective here is to develop a better understanding of drug synergisms and mechanisms of action, at the cellular level. This work is of interest in the context of the development of anti-bacterial, anti-fungal, anti-protozoal and immunomodulator drugs. Overall, this project is aimed at developing novel inhibitors of metalloenzyme using NMR, crystallography and computational chemistry methods and in investigating the pharmacological effects of these drug candidates at the cellular level, using modern gene expression techniques.
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