REVIEW ARTICLE Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase : structural basis for antifolate resistance and development of effective inhibitors Y. YUTHAVONG 1* , J. YUVANIYAMA 2 , P. CHITNUMSUB 1 , J. VANICHTANANKUL 1 , S. CHUSACULTANACHAI 1 , B. TARNCHOMPOO 1 , T. VILAIVAN 3 and S. KAMCHONWONGPAISAN 1 1 National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Science Park, 113 Phaholyothin Road, Pathumthani 12120, Thailand 2 Center for Protein Structure and Function and Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 3 Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand (Received 16 December 2003; revised 16 August 2004; accepted 17 August 2004) SUMMARY Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent anti- malarial activities against drug-resistant P. falciparum bearing the mutant enzymes. Key words: Plasmodium falciparum, malaria, dihydrofolate reductase, thymidylate synthase, antifolate, resistance, mutations, drug target, drug design. INTRODUCTION Drug resistance of the malaria parasites is one of the most important problems in malaria control. A major part of this problem is the resistance of the parasites to antifolates, inhibitors of dihydrofolate reductase (DHFR), a validated drug target which is a part of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) (Olliaro & Yuthavong, 1999 ; Hyde, 2002 ; Yuthavong, 2002). Not only have such inhibitors as pyrimethamine, cycloguanil and their derivatives been compromised by widespread resistance, but also their synergistic combinations with sulfa-drugs, which act on another enzyme, dihydropteroate synthase (DHPS) in the folate de novo synthesis pathway, are also under threat. Resistance to DHFR inhibitors is explained by the occurrence of point mutations in the enzyme, as shown by the changes in the base sequences of its gene, leading to changes in amino acids in certain positions of the enzyme (Cowman et al. 1988 ; Peterson, Walliker & Wellems, 1988). In order to develop new effective antifolates, it is important to know the molecular structure of DHFR-TS, how it interacts with inhibitors and substrates, and how mutations affect the interactions. DHFR-TS from malarial parasites consists of the DHFR domain and the TS domain, joined through the junction region (Bzik et al. 1987). The bifunc- tional enzyme from Plasmodium falciparum (Pf) has 608 amino acid residues, with 231 residues of the * Corresponding author : National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Science Park, 113 Phaholyothin Road, Pathumthani 12120, Thailand. Tel : +662 564 7000, ext 1426. Fax : +662 564 7000, ext 1427. E-mail : Yongyuth@nstda.or.th 249 Parasitology (2005), 130, 249–259. f 2005 Cambridge University Press DOI: 10.1017/S003118200400664X Printed in the United Kingdom