Dr. Arnon Lavie
- PhD, Brandeis University
- Postdoctoral: Max Planck Institute for Molecular Physiology, Dortmund, Germany.
Structure-based design of novel therapeutic agents by prodrug development and enzyme modification. X-ray crystallographic structure determination of enzymes involved in prodrug activation. A limiting factor of many drugs is the toxicity associated with their administration at the required dose. Toxicity arises when a drug elicits an additional undesired biological response due to insufficient specificity. Our laboratory is working to develop new drugs that possess enhanced specificity, and thus less toxicity, in comparison to current available treatment.
Due to problems of uptake or degradation many drugs are not given in their biologically active form and are therefore termed prodrugs. Notable prodrugs are AZT (azidothymidine) against AIDS, acyclovir against herpes simplex virus, and 5-FU against cancer. These drugs require activation to their active metabolite before they can induce their therapeutic effect. The activation of prodrugs to their active form is catalyzed by cellular and/or viral enzymes. In many cases it is this essential activation that determines both how effective and how toxic a drug is.
A specific problem that this laboratory is trying to solve is the low level of the active form of AZT, which is its triphosphate metabolite AZT-TP, found in patients taking this drug. AZT must be phosphorylated three times by cellular enzymes, and the second phosphorylation step, catalyzed by the enzyme thymidylate kinase (TmpK), has been shown to be the rate-limiting step of the AZT activation pathway. This bottleneck results in a high concentration of the toxic partially activated metabolite AZT-MP concomitant with low concentrations of the active metabolite.
The comparison of the x-ray crystallographic structures of TmpK complexed with the physiological substrate thymidine monophosphate (dTMP) and with azidothymidine monophosphate (AZT-MP) revealed the structural cause behind the slow phosphorylation rate of the drug to its diphosphate form (AZT-DP). Now that we understand the reasons behind the slow catalytic rate of AZT-MP phosphorylation, we are taking a two-pronged approach to solving this bottleneck: (1) development of new molecules that are well phosphorylated by TmpK but retain the antiviral property of AZT, a process we call structure-based prodrug development, and (2) development of TmkP mutants that phosphorylate AZT at an enhanced rate that could potentially be used to increase the therapeutic index of AZT after their introduction via gene therapy.
The combination of x-ray crystallography to elucidate the structure of various enzyme-substrate complexes, the design, synthesis, and kinetic characterization of potential new substrates, and molecular biology to produce mutants with modified substrate specificity are the techniques used to achieve this goal.