Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase–dihydrofolate reductase

Abstract

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.

Molecular Characteristics and Serodiagnostic Potential of Dihydrofolate Reductase from Echinococcus granulosus

“…DHFR has been studied extensively in a variety of organisms, including mammals 37 , protozoa 38 , bacteria 39 and several insects 27, 40 . Moreover, DHFR can be used as a drug target in the treatment of cancer and bacterial and parasitic infections 25 .…”

Treatment of Cryptosporidium: What We Know, Gaps, and the Way Forward

“…Licensed anti-bacterial and anti-protozoan drugs do not inhibit the cryptosporidium enzyme but, research teams are evaluating the activity of compounds designed to specifically block this key enzyme in the folate synthesis pathway in vitro. Published data has reported striking anti-cryptosporidial activity of these compounds within cell culture [56]. …”

A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium

“…8,17 C. hominis encodes and expresses TS and DHFR as a bifunctional enzyme in contrast to the monofunctional forms of the enzyme found in humans. 8 …”

“…The compound 906 was synthesized according to the previously reported methods 17 and the purified product was loaded into PLGA nanoparticles. In order to obtain high drug loading efficiencies, several different techniques were utilized for the preparation of several batches of compound 906 loaded nanoparticles (NP-906, Fig.…”

“…17 However, when compound 906 was evaluated in cell culture, the half maximal effective concentration (EC 50 ) required to significantly reduce C. parvum infection ranged between 1 and 5 μM. 17 Therefore, compound 906 was a good model drug to test the CP2 Ab-conjugated NP (CP2-NP-906) delivery strategy to improve its therapeutic efficiency in cell culture. Indeed, as shown in Figure 3, the anti-cryptosporidial activity of CP2-NP-906 on C. parvum sporozoites in cell culture revealed an EC 50 value in the 100–300 nM range, in agreement with results at the biochemical level.…”