Structures of Leishmania major Pteridine Reductase Complexes Reveal the Active Site Features Important for Ligand Binding and to Guide Inhibitor Design Alexander W. Schu ¨ ttelkopf 1 , Larry W. Hardy 2 , Stephen M. Beverley 3 and William N. Hunter 1 * 1 Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee Dundee DD1 5EH, UK 2 Sepracor Inc., 84 Waterford Drive, Marlborough, MA 01752 USA 3 Department of Molecular Microbiology, Washington University School of Medicine Campus Box 8230, 660 S. Euclid Avenue, St. Louis, MO 63110-1093, USA Pteridine reductase (PTR1) is an NADPH-dependent short-chain reductase found in parasitic trypanosomatid protozoans. The enzyme participates in the salvage of pterins and represents a target for the development of improved therapies for infections caused by these parasites. A series of crystallographic analyses of Leishmania major PTR1 are reported. Structures of the enzyme in a binary complex with the cofactor NADPH, and ternary complexes with cofactor and biopterin, 5,6-dihydrobiopterin, and 5,6,7,8- tetrahydrobiopterin reveal that PTR1 does not undergo any major conformational changes to accomplish binding and processing of substrates, and confirm that these molecules bind in a single orientation at the catalytic center suitable for two distinct reductions. Ternary complexes with cofactor and CB3717 and trimethoprim (TOP), potent inhibitors of thymidylate synthase and dihydrofolate reductase, respect- ively, have been characterized. The structure with CB3717 reveals that the quinazoline moiety binds in similar fashion to the pterin substrates/ products and dominates interactions with the enzyme. In the complex with TOP, steric restrictions enforced on the trimethoxyphenyl substituent prevent the 2,4-diaminopyrimidine moiety from adopting the pterin mode of binding observed in dihydrofolate reductase, and explain the inhibition properties of a range of pyrimidine derivates. The molecular detail provided by these complex structures identifies the important interactions necessary to assist the structure-based development of novel enzyme inhibitors of potential therapeutic value. q 2005 Elsevier Ltd. All rights reserved. Keywords: antifolates; dihydrofolate reductase; Leishmania; pterin; short- chain reductase *Corresponding author Introduction Infection by trypanosomatid protozoans, e.g. Trypanosoma and Leishmania species, causes a range of serious human diseases. 1 Current treat- ments of these infections involve drugs such as sodium stibogluconate and nifurtimox, which induce serious side-effects and this, together with an increase in drug-resistant parasites, has created an urgent requirement for new and more effective treatments. 2 The ideal enzyme targets for the development of such new antimicrobial drugs are those that are essential for the survival of the parasite, and either absent from the human host or display markedly differing substrate specificities. Our improved knowledge of trypanosomatid 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: ASA, accessible surface area; DPI, diffraction-component precision index; DHB, 2-amino-7, 8-dihydro-6-(1,2-dihydroxypropyl)pteridin-4(3H)-one or 7,8-dihydrobiopterin; DHFR, dihydrofolate reductase; E.S.R.F., European Synchrotron Radiation Facility; MTX, 4-amino-N10-methyl-pteroylglutamic acid or metho- trexate; NCS, non-crystallographic symmetry; pABA, para-aminobenzoic acid; PDB, Protein Data Bank; PTR1, pteridine reductase; SDR, short-chain dehydrogenase/ reductase; S.R.S., Synchrotron Radiation Source; TAQ, 2,4, 6-triaminoquinazoline; THB, 2-amino-5,6,7,8-tetrahydro- 6-(1,2-dihydroxypropyl)pteridin-4(3H)-one or 5,6,7,8- tetrahydrobiopterin; TOP, 5-((3,4,5-trimethoxyphenyl) methyl)-2,4-diaminopyrimidine or trimethoprim; TS, thymidylate synthase. E-mail address of the corresponding author: w.n.hunter@dundee.ac.uk doi:10.1016/j.jmb.2005.06.076 J. Mol. Biol. (2005) 352, 105–116