Targeting the TS dimer interface in bifunctional Cryptosporidium hominis TS-DHFR from parasitic protozoa: Virtual screening identifies novel TS allosteric inhibitors

Abstract

Effective therapies are lacking to treat gastrointestinal infections caused by the genus Cryptosporidium, which can be fatal in the immunocompromised. One target of interest is Cryptosporidium hominis (C. hominis) thymidylate synthase-dihydrofolate reductase (ChTS-DHFR), a bifunctional enzyme necessary for DNA biosynthesis. Targeting the TS-TS dimer interface is a novel strategy previously used to identify inhibitors against the related bifunctional enzyme in Toxoplasma gondii. In the present study, we target the ChTS dimer interface through homology modeling and high-throughput virtual screening to identifying allosteric, ChTS-specific inhibitors. Our work led to the discovery of methylenedioxyphenyl-aminophenoxypropanol analogues which inhibit ChTS activity in a manner that is both dose-dependent and influenced by the conformation of the enzyme. Preliminary results presented here include an analysis of structure activity relationships and a ChTS-apo crystal structure of ChTS-DHFR supporting the continued development of inhibitors that stabilize a novel pocket formed in the open conformation of ChTS-TS.