Janek Szychowski

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

The crystal structure of the complex between oligonucleotide containing the bacterial ribosomal decoding site (A site) and the synthetic paromomycin analogue 1, which contains the γ‐amino‐α‐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′′ of ring III, is reported. Interestingly, next to the paromomycin 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 formation between the ribosome and the mRNA–tRNA complex regardless of whether the codon–anticodon stem is of the cognate or near‐cognate type.

Inhibition of aminoglycoside-deactivating enzymes APH(3')-IIIa and AAC(6')-Ii by amphiphilic paromomycin O2''-ether analogues.

Aminoglycoside antibiotics have been in clinical use for the last sixty years. Following the discovery of streptomycin in 1944, many other broad-spectrum aminoglycosides have been discovered (Figure 1). 2] Typically, aminoglycosides are classified according to the substitution pattern of the deoxystreptamine unit that forms the core of these antimicrobial compounds. 4,5-Disubstituted deoxystreptamine compounds are comprised in class A aminoglycosides (Figure 1 a), whereas 4,6disubstituted derivatives are in class B (Figure 1 b). The isolation of new aminoglycosides declined rapidly in the early seventies, when efforts were diverted to the preparation of semisynthetic analogues intended to counteract increasing bacterial resistance to these useful drugs. This led to dibekacin (11), a deoxygenated analogue of kanamycin B (7), and to arbekacin (12), by modifying the N1 group of dibekacin with the 2S-4-amino-2-hydroxybutanoyl moiety originally found in butirosin (2). Although it has been known for decades that aminoglycosides interfere with protein biosynthesis by binding to the prokaryotic ribosome, the lack of precise structural information hampered the identification of beneficial drug modification until the late nineties. A better understanding of the mode of action of aminoglycosides, exemplified by paromomycin (3), was obtained from biochemical and spectroscopic approaches, as well as by mass spectrometry and nuclear magnetic resonance. Definitive confirmation was provided by X-ray structures of the 30S ribosomal subunit bound to aminoglycosides, as well as kinetic studies of protein biosynthesis. 10] This long-awaited information led to an increase in structurebased modifications of aminoglycosides, leading to many of semisynthetic analogues from our laboratory and elsewhere. 12] Aminoglycoside therapy is usually limited to a clinical environment since parenteral injection of these highly hydrophilic drugs is required to obtain the desired plasma concentration in a patient. Their use is also limited by their otoand nephrotoxicity. Since well-studied dosage strategies are used to maximize their antibiotic potential while minimizing their toxicity, the future of these antibiotics will eventually be compromised by the emergence of bacterial resistance. In order to overcome this threat to human health, the structures of some other classes of antibiotics have also been substantially modified. For example, the b-lactam family has “evolved” remarkably since the first report of penicillin resistance. 14] However, clinically effective aminoglycosides have been only minimally modified since their first use. 2] Bacteria have developed two general strategies to resist aminoglycosides: 1) diminution of intracellular concentration of the antibiotic, mainly by efflux; and 2) chemical modification of the drug itself or its biological target. Fortunately, bacterial responses influencing aminoglycoside intracellular concentration, as well as the chemical modification of the ribosomal A-site, are still not widespread. However, modification of aminoglycosides by deactivating enzymes is a major threat to the continued clinical efficacy of these antibiotics. Aminoglycoside deactivating enzymes can be divided into three categories: nucleotidyltransferases (ANTs), acetyltransferases (AACs), and phosphotransferases (APHs). Once adenylated, acetylated or phosphorylated, the affinity of an aminoglycoside for its biological target is drastically attenuated. There are multiple ANTs, AACs and APHs, that can each target different amino or hydroxy groups on the various aminoglycosides. APH(3’)-IIIa mediates the phosphorylation of aminoglycosides at their 3’-OH position by a sequential mechanism where ATP binds first and ADP is the last species to leave the active site. X-ray structures of APH(3’)-IIIa bound to kanamycin A or neomycin B are available. The Enterococcus faecium enzyme AAC(6’)-Ii catalyzes the acetylation of most aminoglycosides at the 6’-N position. This isoform proceeds via an ordered bi bi mechanism, with acetyl coenzyme A (AcCoA) binding first. Crystal structures have been reported for AAC(6’)-Ii in complex with AcCoA, CoA, and some inhibitors. A number of inhibitors of AAC(6’)-Ii have been reported. Based on mechanistic and structural information regarding the mode of action of aminoglycosides as well as the enzymes that deactivate them, aminoglycoside analogues have recently been prepared in an attempt to overcome bacterial resistance. In this regard, we reported the preparation of paromomycin analogues with hydrophobic substituents at the O2’’ position. 23] The persistent antimicrobial activities of some of these amphiphilic O2’’ analogues compared to the parent pa[a] Dr. J. Szychowski, Prof. S. Hanessian, Prof. J. W. Keillor Department of Chemistry, Universit de Montr al C. P. 6128, Succ. Centre-Ville, Montr al, QC, H3C 3J7 (Canada) [b] Dr. J. Kondo, Prof. E. Westhof Architecture et R activit de l’ARN, IBMC-CNRS, Universit de Strasbourg 15 rue Ren Descartes, 67084 Strasbourg Cedex (France) [c] Dr. J. Kondo Department of Materials and Life Sciences Faculty of Science and Technology, Sophia University 7–1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554 (Japan) [d] O. Zahr, Prof. K. Auclair Department of Chemistry, McGill University 801 Sherbrooke Street West, Montreal, QC, H3A 2K6 (Canada) [e] Prof. J. W. Keillor Current address : Department of Chemistry, University of Ottawa 10 Marie-Curie, Ottawa, ON, K1N 6N5 (Canada) E-mail : jkeillor@uottawa.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100346.

Robust polymeric nanoparticles for the delivery of aminoglycoside antibiotics using carboxymethyldextran-b-poly(ethyleneglycols) lightly grafted with n-dodecyl groups

Aminoglycoside antibiotics are effective in the treatment of infections caused by aerobic Gram negative bacilli, but their widespread use is hampered by serious side effects that may be alleviated through the use of tailored delivery systems. Robust polyion complex (PIC) micelles, incorporating up to 50 weight % drug, were prepared using two aminoglycosides: paromomycin and neomycin, and a dihydrophilic block copolymer consisting of a poly(ethyleneglycol) (PEG) chain linked to a carboxymethyldextran fragment (CMD) lightly grafted with n-dodecyl groups. The micelles were stable under physiological conditions (pH 7.4, 150 mM NaCl), in contrast to micelles formed by the unmodified CMD-PEG and the aminoglycosides or their guanidinylated derivatives. The aminoglycosides were released from the n-dodecyl-CMD-PEG micelles in a pharmacologically active form as indicated by their ability to kill test micro-organisms in culture. This study opens up new opportunities in the biomedical applications of PIC micelles with inherently enhanced stability.