Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase Norbert Schormann a , Sadanandan E. Velu b , Srinivasan Murugesan b , Olga Senkovich a , Kiera Walker a , Bala C. Chenna b , Bidhan Shinkre b , Amar Desai a , Debasish Chattopadhyay a, * a Department of Medicine and Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA b Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA article info Article history: Received 20 February 2010 Revised 5 April 2010 Accepted 6 April 2010 Available online 9 April 2010 Keywords: Parasitic disease Chagas disease DHFR Antifolate Drug development Crystal structure Trypanosoma cruzi Protozoan Kinetoplast abstract Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for devel- oping drugs to treat Chagas’ disease. We have undertaken a detailed structure–activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cru- zi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme–ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60–70% of the total binding energy is contributed by the 2,4-dia- minoquinazoline scaffold. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Chagas’ disease is caused by the protozoan parasite Trypano- soma cruzi, which is primarily transmitted by an insect vector but also via blood transfusion, organ transplant, and from mother to child. The disease affects millions of people in Latin America where the vector and the parasite are endemic. 1 Nevertheless, since the infected hosts, if untreated can carry the parasite through their life increased travel and immigration give rise to an emerging threat in many countries outside the endemic regions. But the drugs used for the treatment of T. cruzi infection cause serious side effects and are not effective in the chronic stage of the disease. Therefore, validating potential drug targets and identifying novel drug candidates is of considerable global importance. Since the dihydrofolate reductase (DHFR) activity of T. cruzi (TcDHFR) is essential for the parasite, it represents a potential target for ra- tional drug design. DHFR has a proven track record as a drug target in cancer chemotherapy. More importantly, DHFR inhibitors are successfully used in the treatment of bacterial and parasitic infections. 2–5 While DHFR is a monofunctional protein in mam- mals, T. cruzi and other protozoan parasites carry a bifunctional form of the enzyme in which the DHFR domain is linked to the thy- midylate synthase (TS) domain with a linker sequence whose length varies from one parasite to the other. Earlier data from our laboratory showed that an inhibitor of TcDHFR, namely the antifolate drug trimetrexate (TMQ), inhibits the growth of amasti- gote and trypomastigote forms of T. cruzi in vitro. 6 However, very few, if any, high affinity inhibitors of TcDHFR have been reported. In order to facilitate rational design of a selective inhibitor of the T. cruzi enzyme we have initiated a comprehensive structure– activity study using various antifolate molecules through selective library search and limited chemical synthesis. As part of this study we previously characterized the structure of the biologically rele- vant bifunctional form of the parasitic enzyme, (TcDHFR–TS), and determined the structure of the enzyme with TMQ bound to the DHFR active site. 7 We also identified a number of highly active inhibitors of TcDHFR from libraries of antifolate compounds and developed a three-dimensional model for quantitative structure– activity (3D-QSAR) analysis of inhibitors of TcDHFR activity. 8 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.04.020 Abbreviations: TMQ, trimetrexate (5-methyl-6-{3,4,5-trimethoxyphenyl) amino]- methyl} quinazoline-2,4-diamine); CO4, 5-methyl-6-{[methyl(3,4,5-trime- thoxyphenyl)amino]methyl} pyrido[2,3-d] pyrimidine-2,4-diamine; DHF, dihydro- folate; NADP/NADPH, oxidized and reduced nicotinamide adenine dinucleotide phosphate; dUMP, deoxyuridine monophosphate; EDO, ethylene glycol. * Corresponding author. Tel.: +1 205 934 0124; fax: +1 205 934 0480. E-mail address: debasish@uab.edu (D. Chattopadhyay). Bioorganic & Medicinal Chemistry 18 (2010) 4056–4066 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc