Substituted pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis thymidylate synthase inhibitors

Area of Science:

• Biochemistry
• Drug Discovery
• Parasitology

Background:

• Cryptosporidiosis, caused by *Cryptosporidium hominis*, is a severe gastrointestinal illness, particularly in immunocompromised individuals.
• Current treatments like nitazoxanide and paromomycin lack sufficient clinical data for efficacy in this vulnerable population.
• Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are validated drug targets in other diseases, suggesting their potential in parasitic infections.

Purpose of the Study:

• To evaluate a novel series of antifolates as inhibitors of *Cryptosporidium hominis* thymidylate synthase (ChTS).
• To characterize the binding interactions of a potent ChTS inhibitor through crystal structure analysis.
• To identify opportunities for developing specific ChTS inhibitors based on structural insights.

Main Methods:

• Synthesis and evaluation of 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidine derivatives as ChTS inhibitors.
• Determination of the crystal structure of ChTS in complex with the most potent inhibitor (Compound 6).
• Analysis of molecular interactions (Van der Waals, hydrophobic, hydrogen bonds) between the inhibitor, protein residues, and substrate analog.

Main Results:

• A novel pyrrolo[2,3-d]pyrimidine derivative demonstrated potent inhibition of ChTS with a Ki of 8.83±0.67 nM.
• The crystal structure revealed key interactions, including those with non-conserved residues A287 and S290.
• Compound 6 was identified as a lead compound, with its structure providing a basis for further analog design.

Conclusions:

• Novel antifolates targeting ChTS represent a promising therapeutic avenue for cryptosporidiosis, especially in immunocompromised patients.
• The identified lead compound and its binding interactions offer a foundation for developing highly specific ChTS inhibitors.
• Structural insights into ChTS inhibition can guide the rational design of new anti-cryptosporidial drugs.