Biomolecular study of human thymidylate synthase conformer-selective inhibitors: New chemotherapeutic approach.

Hala O El-Mesallamy,Nadia M Hamdy,Sondra H Berger,James M Chapman,Hekmat M El Magdoub,Mona F Schaalan,Lamiaa N Hammad,Luis Menéndez-Arias

doi:10.1371/journal.pone.0193810

Hala O El-Mesallamy, Nadia M Hamdy + Show 6 more

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https://doi.org/10.1371/journal.pone.0193810

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Abstract

Thymidylate synthase (TS) is a well-validated target for the therapy of adult cancers. Propane-1,3-diphosphonic acid (PDPA) has significant inhibitory properties against human thymidylate synthase (hTS) relative to mouse TS which is not predicted to adopt an inactive conformer. The current research aims to identify novel, lead inhibitors of hTS and examine the prediction that they bind selectively to hTS enzymes existing in different conformational equilibria. Conformer-selectivity was evaluated through performing activity inhibition studies, as well as intrinsic fluorescence (IF) studies in comparison to the known orthosteric inhibitor raltitrexed (RTX). Human TS was isolated from recombinant bacteria expressing either native hTS, capable of conformational switching, or an actively stabilized mutant (R163K-hTS). The examined test compounds were rationally or virtually predicted to have inhibitory activity against hTS. Among these compounds, glutarate, N-(4-carboxyphenyl) succinamic acid, and diglycolic anhydride showed higher selectivity towards native hTS as compared to R163K-hTS. The active site inhibitor RTX showed significantly higher inhibition of R163K-hTS relative to hTS. Targeting hTS via conformational selectivity represents a future approach for overcoming reported resistance towards active-state TS analogs.

Highlights

Thymidylate synthase (TS) is a well-validated target for the therapy of adult cancers including gastrointestinal, breast, pancreatic, and head and neck cancers [1]
One goal of our research is to identify novel, lead inhibitors of human thymidylate synthase (hTS) that bind to hTS distinctly from active-state inhibitors such as RTX
The kcat of R163K-hTS was 129% that of native hTS, as expected of an enzyme stabilized in an active state

Summary

Introduction

Thymidylate synthase (TS) is a well-validated target for the therapy of adult cancers including gastrointestinal, breast, pancreatic, and head and neck cancers [1]. TS exhibits oncogenic behavior [2]. In the TS-catalyzed reaction, thymidylate (dTMP) is formed from deoxyuridylate (dUMP) using N5, N10 methylene tetrahydrofolate (mTHF) as the methyl donor. Analogs of TS substrates are utilized clinically as cancer chemotherapy, including, 5-fluorouracil, capecitabine, pemetrexed, and raltitrexed (RTX) [3]. Upon binding to TS, inhibitory complexes are formed that are catalytically inactive, resulting in depletion of dTMP. Such a thymine-less state is lethal to most actively dividing cells, and TS is an ideal target

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