Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

College of Pharmacy

Sub-Department

Pharmaceutical Science

First Advisor

Sondra H Berger

Abstract

The goal of this research is to develop new allosteric thymidylate synthase (TS) inhibitors in an effort to reduce cancer drug resistance. Resistance is associated with elevation in TS levels upon exposure to active site targeted drugs. Human TS exists in conformational equilibrium between active and inactive states. Exploiting the structural differences in different conformations of hTS, an alternate mechanism to combat resistance was hypothesized: allosteric inhibitors that stabilize the inactive form will minimize resistance by increasing translational repression and/or by decreasing protein stability. Glutarate (GLU), an allosteric inhibitor, caused downregulation in TS levels, which is the first TS inhibitor reported to decrease TS levels. To obtain novel allosteric TS inhibitors, two approaches were employed.

Firstly, a new nucleotide stress response (NSR) assay was developed and validated for providing a screening platform to identify lead allosteric molecules. The assay measures the uptake of a fluorescent nucleoside analog, which was shown to correlate with TS inhibition induced by direct and indirect inhibitors or with genetic loss of TS. Studies were conducted with raltitrexed (RTX) (active site targeted TS inhibitor) and GLU on paired isogenic cell lines that express either hTS or active stabilized hTS. RTX showed similar induction of fluorescent nucleoside uptake in both cell lines, while GLU induced nucleoside uptake preferentially in cells expressing hTS, indicating that NSR assay can be used to screen allosteric inhibitors.

Secondly, virtual screening was utilized to identify lead allosteric inhibitors. A library of 13,750 compounds were docked with the allosteric site of hTS. Among all the compounds docked, benzoic acid and 4-oxobutanoic acid derivatives showed the highest binding affinity, causing 30% - 60% inhibition of TS activity. Furthermore, they inhibited cell growth, which was reversed in the presence of thymidine, and downregulated TS protein, suggestive of selectivity towards TS. Based on the data two lead pharmacophores were generated.

Collectively, the studies suggest that the stress response assay is a new resource for screening for lead allosteric molecules by high-throughput approaches. Lead pharmacophores obtained from virtual screens provide a scaffold to create allosteric inhibitors of higher potency and druggability.

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