Biomolecular Study of Human Thymidylate Synthase Inactive Conformer Inhibitors as a New Chemotherapeutic Approach

Abstract

Thymidylate synthase is a well-validated target for the therapy of adult cancers, including gastrointestinal, breast, pancreatic, and head and neck cancers. At elevated levels, TS exhibits oncogenic behavior. Upon binding of inhibitors to TS, inhibitory ternary complexes are formed that are catalytically inactive, resulting in depletion of dTMP. Paradoxically, exposure to TS inhibitors is associated with elevation in TS levels. This is postulated to contribute to the resistance that is reported in patients receiving TS-targeted chemotherapy. High-resolution crystal structures provided evidence that hTS undergoes conformational switching. A mutant enzyme stabilized in an active conformation and designated R163K-hTS has previously been shown to exhibit approximately 133 % of the catalytic activity of hTS. It was predicted that a physiological role of conformational switching is related to protection of the catalytic Cys’s from oxidation. Moreover, it was suggested that bifunctional acidic ligands may have stronger propensity to stabilize the inactive conformer through ionic bonds with basic amino acids. Such binding inhibits the enzymatic activity and could overcome the previously reported clinical resistance to active site inhibitors that stabilize the enzyme in the active conformation. The present study was designed to determine the differential potencies of some PDPA isosteres on hTS and R163K-hTS enzyme activities. The more potent compounds were used to examine the prediction that they bind selectively to the inactive conformation of hTS. We also aimed to investigate the biological effects of TS conformational switching upon exposure to the active site inhibitor RTX, and whether the level of upregulation of nucleoside transporters will differ among both types of cell lines expressing either hTS or R163K-hTS or not. In addition, we examined the hypothesis that the native enzyme known to populate the inactive conformer is more resistant to oxidative stress relative to the active conformer. This final experiment was achieved using the potent prooxidant TBHP in conjunction with cell viability assays. The Escherichia coli bacteria expressing either hTS or R163K-hTS were cultivated to produce TS enzymes. The enzymes were purified by FPLC. The purified enzymes were used to examine the effects of PDPA and its isosteres on catalytic activity by tracing the release of tritiated water upon using radioactive dUMP and MTHF. Purified hTS and R163K-hTS were also used to examine the effect of selected test compounds on conformational switching by monitoring the change in IF of both enzymes upon exposure to these test compounds. Chinese hamster lung fibroblast cell lines expressing either hTS or R163K-hTS enzymes were used to assess the effect of RTX on TS levels by western blotting. Nucleoside uptake via ENT1 by either types of cell lines after exposure to RTX was also monitored by employing the fluorescent nucleoside analog ETMPR in an in-situ fluorescence assay. TBHP was used to examine the effect of conformational switching on cell viability as assessed by MTT assay. Finally, the effect of TBHP on the level of hTS versus that of R163K-hTS as well as, PARP and cleaved PARP levels in either types of cell lines were assessed by western blottings. 139 The results of the current work has unexpectedly shown that the effect of RTX on CHL-hTS was more pronounced than its effect on CHL-R163K. That is because CHL-hTS cells has shown higher level of hTS and higher nucleoside uptake upon exposure to RTX as compared to CHL-R163K. The results also demonstrated that CHL-R163K cells were more sensitive to higher concentrations of TBHP as demonstrated by its lower survival compared to CHL-hTS by the MTT assay. This was further supported by the ability of TBHP to cause a progressive reduction in the level of R163K-hTS and intact PARP proteins reltive to hTS. Meanwhile, CHL-R163K cells were not able to overcome the insult caused by TBHP as demonstrated by the more pronounced increase in the level of cleaved PARP. This data actually provide us with some evidence that hTS enzyme which populates the inactive conformer could play a protective role against oxidative stress. The activity inhibition studies revealed that glutaric acid, PDPA and diglycolic anhydride were able to inhibit TS activity with a higher selectivity towards the native enzyme relative to the active stabilized mutant R163KhTS, as demonstrated by their IC s. This is possibly due to an allosteric effect that stabilizes the enzyme in an inactive conformer. PDPA and diglycolic anhydride showed higher potency towards hTS relative to glutaric acid. the other hand, the effect of the active site inhibitor RTX was more pronounced towards the active stabilized mutant as compared to the wild type enzyme. Finally, N-(4-carboxyphenyl) succinamic acid was able to inhibit both hTS and R163K-hTS to similar levels, which might suggest a different mode of binding or possibly binding to two different sites. 140 The change in enzymatic activity was further reflected as a change in IF of the enzyme. We were able to demonstrate some preliminary data that IF could be used to measure the effectiveness of both allosteric inhibitors and active site inhibitors, as a convenient alternative to the activity inhibition study. In addition, our results suggest that IF could possibly be used to predict the mode of binding to hTS enzyme.