Novel allosteric covalent inhibitors of bifunctional Cryptosporidium hominis TS-DHFR from parasitic protozoa identified by virtual screening

Victor Ruiz; Daniel J Czyzyk; Margarita Valhondo; William L Jorgensen; Karen S Anderson

doi:10.1016/j.bmcl.2019.03.022

Novel allosteric covalent inhibitors of bifunctional Cryptosporidium hominis TS-DHFR from parasitic protozoa identified by virtual screening

Bioorg Med Chem Lett. 2019 Jun 1;29(11):1413-1418. doi: 10.1016/j.bmcl.2019.03.022. Epub 2019 Mar 20.

Authors

Victor Ruiz¹, Daniel J Czyzyk¹, Margarita Valhondo², William L Jorgensen³, Karen S Anderson⁴

Affiliations

¹ Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
² Department of Chemistry, Yale University, 225 Prospect Street, PO Box 208107, New Haven, CT 06520-8107, USA.
³ Department of Chemistry, Yale University, 225 Prospect Street, PO Box 208107, New Haven, CT 06520-8107, USA. Electronic address: [email protected].
⁴ Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA; Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. Electronic address: [email protected].

Abstract

Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region "crossover helix" to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the non-active site pocket.

Keywords: Allosteric; Cryptosporidium hominis; DHFR; Glide; Non-active site; Virtual screen.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Allosteric Regulation / drug effects
Benzamides / chemistry
Benzamides / pharmacology*
Cryptosporidium / enzymology*
Dose-Response Relationship, Drug
Drug Design
Drug Evaluation, Preclinical
Enzyme Inhibitors / chemistry
Enzyme Inhibitors / pharmacology*
Humans
Models, Molecular
Molecular Structure
Multienzyme Complexes / antagonists & inhibitors*
Multienzyme Complexes / metabolism
Structure-Activity Relationship
Tetrahydrofolate Dehydrogenase / metabolism
Thiourea / chemistry
Thiourea / pharmacology*
Thymidylate Synthase / antagonists & inhibitors*
Thymidylate Synthase / metabolism

Substances

Benzamides
Enzyme Inhibitors
Multienzyme Complexes
thymidylate synthase-dihydrofolate reductase
benzamide
Tetrahydrofolate Dehydrogenase
Thymidylate Synthase
Thiourea

Abstract

Publication types

MeSH terms

Substances

Grants and funding