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

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 nonactive site pocket.

Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening

“…In the context of antifolate resistance, development of new generation antifolates based on novel chemotypes is warranted. Although the fragment-based screening approach has been successfully applied to DHFR enzymes in various bacteria and protozoa 41,42 , no similar study has yet been reported in the malaria parasite Plasmodium falciparum. Here, we describe the first fragment-based screening on PfDHFR.…”

Dihydrofolate reductase, thymidylate synthase, and serine hydroxy methyltransferase: successful targets against some infectious diseases

“…DHFR-TS In humans, the DHFR and TS enzymes exist as monomers, while in parasites, they exist as dimers on the TS-TS interface on the same polypeptide chain [ 22 ]. The protein–protein interface can serve as an essential target for small molecules.…”

“…It has been observed by the Ch DHFR-TS crystal structure that one DHFR domain of the dimer is linked to the other DHFR domain by the crossover helix making hydrophobic interactions. It moves back to the original monomer to complete the DHFR and TS domains [ 22 ]. From E. coli DHFR-folate-NADP + ternary complex, it was observed that the M20 loop adopts different confirmation as open-close or occluded according to the catalytic cycle, which is also the most flexible region of the DHFR [ 77 ].…”

“…The selective inhibitor (2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidin-methyl-phenyl- l -glutamic acid) for Ch TS was obtained, and X-ray structures with inhibitors were also resolved for human TS and Ch TS, which helped further in the selectivity of compounds [ 124 ]. In another study, a thiol-substituted 2-hydroxy-N-phenylbenzamide was found to show good activity against Ch DHFR-TS [ 22 ]. Methylenedioxyphenyl-aminophenoxypropanol analogues were discovered by virtual screening against Ch TS [ 125 ].…”

“…Apart from parasitic diseases, the role of these enzymes in several viral diseases cannot be ignored. As in the case of Flaviviruses which have played a significant role in the burst of infectious diseases like dengue, West Nile and Zika fevers, etc [ 19 – 22 ]. Zika virus infections have gained a lot of attention recently as it has caused abnormalities in the babies following maternal infections [ 23 ].…”