A New Target for Shigellosis: Rational Design and Crystallographic Studies of Inhibitors of tRNA-guanine Transglycosylase Ulrich Gra È dler 1 , Hans-Dieter Gerber 1 , DeeAnne M. Goodenough- Lashua 3 , George A. Garcia 3 , Ralf Ficner 2 , Klaus Reuter 2 Milton T. Stubbs 1 and Gerhard Klebe 1 * 1 Institut fu È r Pharmazeutische Chemie, Philipps-Universita Èt Marburg, Marbacher Weg 6 35032 Marburg, Germany 2 Institut du È r Molekularbiologie und Tumorforschung, Philipps- Universita Èt Marburg, Emil- Mannkopff-Str. 2 35037 Marburg, Germany 3 Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor MI 48109-1065, USA Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hyper-modi®cation of cognate tRNAs leading to the exchange of G34 at the wobble position in the anticodon loop by preQ 1 (2-amino-5-(amino- methyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one) as part of the biosynthesis of queuine (Q). Mutation of the tgt gene in Shigella ¯exneri results in a sig- ni®cant loss of pathogenicity of the bacterium, revealing TGT as a new target for the design of potent drugs against Shigellosis. The X-ray struc- ture of Zymomonas mobilis TGT in complex with preQ 1 was used to search for new putative inhibitors with the computer program LUDI. An initial screen of the Available Chemical Directory, a database compiled from commercially available compounds, suggested several hits. Of these, 4- aminophthalhydrazide (APH) showed an inhibition constant in the low micromolar range. The 1.95 A Ê crystal structure of APH in complex with Z. mobilis TGT served as a starting point for further modi®cation of this initial lead. # 2001 Academic Press Keywords: rational drug design; Shigellosis; modi®ed tRNA nucleoside; LUDI; crystal structure *Corresponding author Introduction Shigellae are the causative agents of dysentery and effect some 600,000 infant deaths per year, mainly in developing countries with sub-standard hygiene and water supplies. The search for Shigella antigens for immunization programs has not yet produced a successful vaccine. Accordingly, infec- tions are usually treated by antibiotics such as azithromycin and cipro¯oxacin (Khan et al., 1997). The fast emergence of multidrug resistance makes the development of drugs using new therapeutic principles highly desirable. Extensive studies of Shigella ¯exneri have resulted in a detailed understanding of the patho- genesis, involving bacterial invasion into colonic epithelial cells followed by intracellular multipli- cation and spreading into adjacent cells (Sansonetti, 1997). The virulence was shown to be multifactorial and is under control of various genes encoded by the chromosome and the large virulence plasmid (Dorman & Porter, 1998). Characterization of chromosomal mutants of S. ¯exneri has resulted in the identi®cation of a gene, vacC, that contributes signi®cantly to the pathogenicity (Durand et al., 1994). The nucleotide sequence of the vacC gene is highly (>98 %) hom- ologous to the tgt gene of Escherichia coli, coding for tRNA-guanine transglycosylase (TGT, EC 2.4.2.29) (Okada & Nishimura, 1979). In eubacteria (such as E. coli), TGT is involved in the biosyn- Present address: U. Gra È dler, Byk-Gulden, Byk- Gulden-Straûe 2, 78467 Konstanz, Germany. Abbreviations used: TGT, tRNA-guanine transglycosylase; Q, queuine (2-amino-5-[[(4S,5R- dihydroxy-1-cyclopenten-3S- yl)amino]methyl]pyrrolo[2,3-d]pyrimidin-4(3H)-one); ACD, Available Chemical Directory; APH, 4-aminophthalhydrazide; preQ 1 , 2-amino-5- (aminomethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one; API, 4- aminophthalimide; 3,5-DAPI, 3,5-diaminophthalimide; 3,5-DAPH, 3,5-diaminophthalhydrazide; 3,5-DAPH-01, 5-amino-3-(1H-[1,2,4]triazole-3-yl-sulfanyl)- phthalhydrazide; 3,5-DAPH02, 5-amino-3-(5-amino-1H- [1,2,4]triazole-3-yl-sulfanyl)-phthalhydrazide; 5-ANH, 5- aminonaphthalene-2,3-hydrazide. E-mail address of the corresponding author: klebe@mailer.uni-marburg.de doi:10.1006/jmbi.2000.4256 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 455±467 0022-2836/01/030455±13 $35.00/0 # 2001 Academic Press