DOI: 10.1002/cmdc.200700113 Crystal Structure of the Bacterial Ribosomal Decoding Site Complexed with a Synthetic Doubly Functionalized Paromomycin Derivative: a New Specific Binding Mode to an A-Minor Motif Enhances in vitro Antibacterial Activity Jiro Kondo, [b] Kandasamy Pachamuthu, [a] Boris FranÅois, [b] Janek Szychowski, [a] Stephen Hanessian,* [a] and Eric Westhof* [b] Introduction Aminoglycosides are clinically important antibiotics targeting the bacterial ribosomal decoding site (A site) in the 30S ribo- some. [1] The molecular mechanisms of their action on protein biosynthesis have been well investigated during the past decade. [2] It has been revealed by X-ray analyses that several types of natural aminoglycosides such as 4,5-disubstituted (for example, paromomycin and neomycin), 4,6-disubstituted (for example, geneticin and tobramycin), and 4-monosubstituted (for example, apramycin) aminoglycosides specifically bind to and stabilize the decoding “on” state of the A site in a con- served manner. [3–8] In other words, the A site, which is a molec- ular switch monitoring correct Watson–Crick base pairings be- tween the mRNA codon and the tRNA anticodon, is always turned “on” in the presence of aminoglycosides, which eventu- ally induces misreading of codons. [9–12] Although they have been known as potent antibacterial agents, their widespread use in clinical practice has been com- promised because of the rapid emergence of drug-resistant strains of bacteria. [1,13–15] Resistance to aminoglycosides arises mainly from 1) chemical modification of aminoglycosides by resistance enzymes and 2) chemical modification and/or point mutations of A-site nucleotides. To overcome this problem, modified aminoglycosides that can delay or avoid acquired re- sistance by pathogenic bacteria are required. In preceding papers, [16–18] we reported several promising re- sults of our analogue design paradigm by taking advantage of the structural information available from X-ray analyses. In the course of these studies, we have discovered a compound with potent antibacterial activity in both Gram-negative and Gram- positive bacteria. Compound 1 is a paromomycin derivative with a g-amino-a-hydroxybutyryl (l-haba) group at position N1 of ring II, and a phenethylaminoethyl ether substituent group at position C2’’ of ring III. The former modification was first identified in the natural compound butirosin which may steri- cally disturb resistance enzyme recognition. [19] In addition, it has been revealed by X-ray analysis of the bacterial A site in complex with amikacin that the l-haba group can increase binding affinity of aminoglycosides to the A site through three [a] Dr. K. Pachamuthu, J. Szychowski, Prof. S. Hanessian Department of Chemistry, UniversitØ de MontrØal P.O. Box 6128, Station Centre-ville Montreal, P.Q., H3C3J7 (Canada) Fax:(+ 1)514-343-5728 E-mail:stephen.hanessian@umontreal.ca [b] Dr.J.Kondo,Dr.B.FranÅois,Prof.E.Westhof Architecture et RØactivitØ de l’ARN, UniversitØ Louis Pasteur Institut de Biologie MolØculaire et Cellulaire, CNRS 15 rue RenØ Descartes, 67084 Strasbourg (France) Fax:(+ 33)3-88-60-18-22 E-mail:E.Westhof@ibmc.u-strasbg.fr The crystal structure of the complex between oligonucleotide containing the bacterial ribosomal decoding site (Asite) and the synthetic paromomycin analogue 1, which contains the g-amino- a-hydroxybutyryl (l-haba) group at position N1 of ring II (2-DOS ring), and an ether chain with an O-phenethylaminoethyl group at position C2’’ ofringIII,isreported.Interestingly,nexttothepa- romomycin analogue 1 specifically bound to the A site, a second molecule of 1 with a different conformation is observed at the crystal packing interface which mimics the A-minor interaction between two bulged-out adenines from the A site and the codon–anticodon stem of the mRNA–tRNA complex. Improved antibacterial activity supports the conclusion that analogue 1 might affect protein synthesis on the ribosome in two different ways: 1) specific binding to the A site forces maintenance of the “on” state with two bulged out adenines, and 2) a new binding mode of 1 to an A-minor motif which stabilizes complex forma- tion between the ribosome and the mRNA–tRNA complex regard- less of whether the codon–anticodon stem is of the cognate or near-cognate type. ChemMedChem 2007,2,1631–1638 # 2007 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim 1631