Structural Insight into Effective Inhibitors’ Binding to Toxoplasma gondii Dihydrofolate Reductase Thymidylate Synthase

Abstract

Pyrimethamine (Pyr), a known dihydrofolate reductase (DHFR) inhibitor, has long been used to treat toxoplasmosis caused by Toxoplasma gondii (Tg) infection. However, Pyr is effective only at high doses with associated toxicity to patients, calling for safer alternative treatments. In this study, we investigated a series of Pyr analogues, previously developed as DHFR inhibitors of Plasmodium falciparum bifunctional DHFR-thymidylate synthase (PfDHFR-TS), for their activity against T. gondii DHFR-TS (TgDHFR-TS). Of these, a set of compounds with a substitution at the C 6 position of the pyrimidine ring exhibited high binding affinities (in a low nanomolar range) against TgDHFR-TS and in vitro T. gondii inhibitory activity. Three-dimensional structures of TgDHFR-TS reported here include the ternary complexes with Pyr, P39, or P40. A comparison of these structures showed the minor steric strain between the p-chlorophenyl group of Pyr and Thr83 of TgDHFR-TS. Such a conflict was relieved in the complexes with the two analogues, P39 and P40, explaining their highest binding affinities described herein. Moreover, these structures suggested that the hydrophobic environment in the active-site pocket could be used for drug design to increase the potency and selectivity of antifolate inhibitors. These findings would accelerate the development of new antifolate drugs to treat toxoplasmosis.

Investigating Antiprotozoal Chemotherapies with Novel Proteomic Tools – Chances and Limitations. A Critical Review

“…Luciferase assay [60] Calcium-dependent protein kinase 1 T. gondii Kinase assay Cocrystallization [61,62] Dihydrofolate reductase thymidylate synthase T. gondii Functional assay [63] Dihydrofolate reductase P. vivax…”

“…The antimalarial pyrimethamine inhibits the bifunctional wildtype DHFR-thymidylate synthases of Plasmodium sp. and other protozoans such as T. gondii [63]. Overexpressors of mutated enzymes are more resistant to pyrimethamine and can be selected for on pyrimethamine containing media.…”

Development of a Practical Synthetic Method for Clinical Candidate 3-(2-{3-[(2,4-Diamino-6-ethylpyrimidin-5-yl)oxy]propoxy} phenyl)propanoic acid (P218) and Its Hydroxylated Metabolites

“…Moreover, our development of these synthetic procedures will be a foundation for further derivatization and optimization of antimalarial drugs and other antifolate agents. [22][23][24] The synthesis of 2,4-diamino-6-ethyl-5-hydroxypyrimidine (5) began with a low-cost commercially available material, methyl propionate, using a chromatography-free synthetic method on a 60-gram scale, as depicted in Scheme 2. Methyl propionate underwent nucleophilic attack by acetonitrile in a presence of sodium hydride as a base in anhydrous tetrahydrofuran.…”

“…Moreover, our development of these synthetic procedures will be a foundation for further derivatization and optimization of antimalarial drugs and other antifolate agents. 22 23 24…”

Structure-Activity Relationship Analysis of Novel Compounds Against Drug-Resistant Plasmodium Falciparum Strains

“…In the context of antimalarial drug discovery, SAR analysis is crucial in identifying critical molecular features responsible for the efficacy of drug candidates against P. falciparum. Through this analysis, researchers aim to optimize the structural components of existing compounds or design entirely new molecules with enhanced antiparasitic properties [10].…”