Using Fragment Cocktail Crystallography To Assist Inhibitor Design of Trypanosoma brucei Nucleoside 2-Deoxyribosyltransferase ² Ju¨rgen Bosch, X,‡,§ Mark A. Robien, X,‡ Christopher Mehlin, X,‡ Erica Boni, X,‡ Aaron Riechers, # Frederick S. Buckner, ‡,# Wesley C. Van Voorhis, ‡,# Peter J. Myler, | Elizabeth A. Worthey, | George DeTitta, ⊥ Joseph R. Luft, ⊥ Angela Lauricella, ⊥ Stacey Gulde, ⊥ Lori A. Anderson, X,‡ Oleksandr Kalyuzhniy, X,‡ Helen M. Neely, X,‡ Jenni Ross, X,‡ Thomas N. Earnest, ∞ Michael Soltis, × Lori Schoenfeld, X,‡ Frank Zucker, X,‡ Ethan A. Merritt, X,‡ Erkang Fan, X,‡ Christophe L. M. J. Verlinde, X,‡ and Wim G. J. Hol* ,X,‡,§ Department of Biochemistry, DiVision of Infectious Disease, and Howard Hughes Medical Institute, UniVersity of Washington, Seattle, Washington 98195, Structural Genomics of Pathogenic Protozoa (SGPP) Consortium, Seattle, Washington 98185, Seattle Biomedical Research Institute, Seattle, Washington 98109, Hauptmann-Woodward Institute, Buffalo, New York 14203, Physical Biosciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and SSRL, Stanford UniVersity, Stanford, California 94309 ReceiVed April 11, 2006 The 1.8 Å resolution de noVo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SAD a phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited “fragment cocktail soaks”. Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of ∼10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins. Introduction Trypanosomatids are parasitic unicellular eukaryotic organ- isms, infecting plants as well as animals. Several species within the family are responsible for serious, but largely neglected, diseases of humans and domestic animals. Most of these are distributed in tropical and subtropical areas of the world. In sub-Saharan Africa, Trypanosoma brucei, the causative agent of sleeping sickness, is transmitted by the tsetse fly. If untreated, this disease is fatal to humans. Estimations by the WHO indicate nearly 60 million people at risk. 1,2 In Central and South America Trypanosoma cruzi causes Chagas’ disease, with an estimated 100 million people at risk. Furthermore, some Trypanosoma brucei subspecies have an economic impact on cattle farming, since they cause a disease called “nagana”. 1,2 Nucleoside 2-deoxyribosyltransferase (NDRT, a EC 2.4.2.6) is essential for nucleoside recycling of purine and pyrimidine phosphorylases in organisms missing those enzymes. 3 It cata- lyzes the cleavage of the glycosidic bonds of 2-deoxyribo- nucleosides and transfers the deoxyribose to another base, thereby playing an important role in recycling nucleosides. Biochemical evidence of NDRT activity has been reported in the kinetoplastid organism Crithidia luciliae. 4 NDRTs can be divided into two classes depending on their substrate specific- ity: class I enzymes transfer deoxyribose between two purines, whereas class II enzymes transfer deoxyribose between either purines or pyrimidines. The structures of class I and class II enzymes are very similar to each other and differ mainly in one loop shielding the active site. 3,5 Both enzymes are found in various Lactobacillus species where the active enzyme consists of a trimer of dimers. 6 Trypanosoma brucei lacks enzymes of de noVo purine biosynthesis and, thus, is entirely dependent on scavenging purine nucleosides from the host. Scavenged nucleosides undergo metabolism by purine salvage/recycling enzymes in order to provide the necessary nucleotides for cellular function and replication. Since NDRT is involved in nucleoside salvage/ recycling, specific inhibition of this enzyme could have dramatic effects on the growth and replication of Trypanosoma. We therefore not only solved the unliganded structure of the enzyme from T. brucei but also employed a fragment-based cocktail screening in combination with crystal X-ray analysis in order to identify potential leads for future drug development. This work was performed as part of the Structural Genomics of Pathogenic Protozoa (SGPP) consortium effort to identify structural targets for the development of antiprotozoan thera- peutics (see also www.sgpp.org). Crystallographic screening methods have been used to sample large compound libraries in order to detect novel lead com- pounds for drug development. Ligands are detected by extra features in the electron density map compared to maps of unliganded protein. The crystallographic method reveals im- mediately the chemical environment of the bound ligand and hence can be utilized directly for lead optimization. The term “fragment” describes a low molecular weight compound (∼100- 300 Da), which typically is smaller than druglike molecules. Fragments cover a wider range of “chemical space” usually leading to higher hit rates and are therefore easier to exploit. An initial and probably first exploration of the “cocktail soak” crystallographic procedure was performed in the early 1990s at the University of Groningen. 7 More sophisticated procedures are currently a popular tool for early lead discovery strategies. 8-12 ² Coordinates and structure factors have been deposited with the PDB (accession codes 2A0K, 2F2T, 2F64, 2F62, 2F67). * To whom correspondence should be addressed. Phone: (206) 685- 7044. Fax: (206) 685-7002. E-mail: wghol@u.washington.edu. X Department of Biochemistry, University of Washington. ‡ Structural Genomics of Pathogenic Protozoa (SGPP) Consortium. § Howard Hughes Medical Institute, University of Washington. # Division of Infectious Disease, University of Washington. | Seattle Biomedical Research Institute. ⊥ Hauptmann Woodward Institute. ∞ Lawrence Berkeley National Laboratory. × Stanford University. a Abbreviations: SAD, Single-wavelength Anomalous Diffraction; NDRT, nucleoside 2-deoxyribosyltransferase; SeMet, selenomethionine. 5939 J. Med. Chem. 2006, 49, 5939-5946 10.1021/jm060429m CCC: $33.50 © 2006 American Chemical Society Published on Web 09/02/2006