Major
Interests: 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. |
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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.
Selected Publications:
Lavie A, Allen KN, Petsko GA, and Ringe D (1994). X-ray
crystallographic structures of D-xylose isomerase-substrate
complexes position the substrate and provide evidence
for metal movement during catalysis. Biochemistry. 33:5469-5480.
Lavie A, Vetter IR, Konrad M, Goody RS, Reinstein J,
and Schlichting I (1997). Crystal structure of thymidylate
kinase reveals the cause behind the limiting step in
AZT activation. Nature Structural Biology. 4:601-604.
Lavie A, Schlichting I, Vetter IR, Konrad M, Reinstein
J, and Goody RS (1997). The bottleneck in AZT activation.
Nature Medicine. 3:922-924.
Lavie A, Konrad M, Brundiers R, Goody RS, Schlichting
I, and Reinstein J (1998). Crystal structure of yeast
thymidylate kinase complexed with the bisubstrate inhibitor
P1- (51-adenosyl)P5-thymidyl) entaphosphate (TP5A) at
2.0 A: Implications for catalysis and AZT activation.
Biochemistry. 37:3677-3686.
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