Thymidylate synthase-dihydrofolate reductase in protozoa

Area of Science:

• Biochemistry
• Parasitology
• Drug Discovery

Background:

• Protozoa possess a unique bifunctional thymidylate synthase (TS) and dihydrofolate reductase (DHFR) enzyme, crucial for folate metabolism.
• This enzyme exists as a dimer, with DHFR at the amino terminus and TS at the carboxy terminus, linked by a variable junction peptide.
• Studies often utilize antifolate-resistant Leishmania major strains due to gene amplification and overproduction of the TS-DHFR protein.

Purpose of the Study:

• To explore the structure-function relationship of protozoan TS-DHFR enzymes.
• To identify selective inhibitors targeting parasitic DHFR over human DHFR for therapeutic development.
• To understand mechanisms of antifolate resistance in key protozoan pathogens like Leishmania and Plasmodium.

Main Methods:

• Cloning and sequencing of TS-DHFR genes from protozoan species.
• Heterologous expression of recombinant Leishmania TS-DHFR for biochemical studies.
• Analysis of drug resistance mutations, such as point mutations in Plasmodium falciparum DHFR.

Main Results:

• Demonstrated extensive communication and substrate channeling between TS and DHFR domains.
• Identified that common antifolates are weak inhibitors of Leishmania major DHFR.
• Discovered selective inhibitors effective against Leishmania DHFR compared to human DHFR.
• Characterized a common point mutation in pyrimethamine-resistant Plasmodium falciparum strains affecting DHFR binding.

Conclusions:

• Protozoan TS-DHFR enzymes present unique structural and functional characteristics distinct from their human counterparts.
• Targeting parasitic DHFR with selective inhibitors offers a promising strategy for novel anti-infective drug development.
• Understanding resistance mechanisms, like specific mutations in P. falciparum DHFR, is vital for effective treatment strategies.