ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 60 AN EXPANDING VIEW OF AMINOGLYCOSIDE–NUCLEIC ACID RECOGNITION BY BERT WILLIS AND DEV P. ARYA Department of Chemistry, Clemson University, Clemson, SC 29634, USA I. Introduction 263 1. Early Years 264 II. Mechanism of Action 265 1. Pre-Ribosomal Binding: Cellular Membrane Permeation 265 2. Ribosomal RNA Binding 267 III. Major Issues in Aminoglycoside Therapeutic Applications 272 1. Toxicity 272 2. Combating Toxicity 275 3. Aminoglycoside Resistance 275 4. Fighting Resistance with Aminoglycoside Derivatives 277 IV. Experimental Techniques for Probing Aminoglycoside–RNA Interactions 281 1. NMR 282 2. X-Ray Crystallography 283 3. Isothermal Titration Calorimetry 284 4. Surface Plasmon Resonance 286 V. Novel Targets for Aminoglycoside Recognition 286 1. RNA 287 2. DNA Triplex 295 3. DNA/RNA Hybrids 296 4. A-Form Nucleic Acids 298 5. B-DNA 298 6. Aminoglycosides as Cleaving Agents 300 7. Other Aminoglycoside Targets: The Anthrax Lethal Factor 301 VI. Conclusion 303 I. INTRODUCTION The origin of aminoglycoside antibiotics began with streptomycin 60 years ago.1 Isolated from Actinomyces griseus, streptomycin immediately found applications ISBN: 0-12-007260-2 DOI: 10.1016/S0065-2318(06)60006-1 263 r 2006 Elsevier Inc. All rights reserved 264 B. WILLIS AND D. P. ARYA TABLE I Aminoglycoside Antibiotic Classes and their Source Organism3 Aminoglycoside Organism Kanamycin Streptomyces kanamyceticus Streptomycin S. griseus Gentamicin Micromonospora purpurea Spectinomycin S. spectabilis Butirosin Bacillus circulans Tobramycin S. tenebrarius Neomycin S. fradiae Amikacin Semisynthetic derivative of kanamycin Netilmicin Semisynthetic derivative of sisomicin Isepamicin Semisynthetic derivative of gentamicin B Note: Reprinted with permission, Copyright 1997 Elsevier. for the treatment against the tuberculosis-causing microorganism Mycobacterium tuberculosis.2 This was the second antibiotic (after penicillin) to be used clinically. Throughout the coming years, a number of aminoglycosides were discovered (Table I),3 with varying potencies for treating infections. However, therapeutic applications were diminished by the emergence of resistance to aminoglycosides by bacteria. Coupled with adverse side effects, such as renal toxicity and ototoxicity, aminoglycoside antibiotic applications toward infectious diseases were placed on the back burner in favor of less harmful antibiotics such as b-lactams. Only within the past decade has the area become increasingly intriguing again, due largely to chemical derivatization, a deeper understanding of resistance mechanisms, and structural information on aminoglycoside activity. Also, though widely known for binding to prokaryotic ribosomal RNA, aminoglycosides have more recently shown affinity for other nucleic acid targets. This opens the door to a new area of therapeutic applications. Outlined in the forthcoming paragraphs is the history of aminoglycosides, from their discovery, structural elucidation, and mechanism of action, to current knowledge of mechanisms of action and resistance, toxicity, and novel nucleic acid targets. Owing to a number of recent reviews pertaining to some of these areas, particular emphasis will be placed on the recently discovered aminoglycoside targets and their potential therapeutic applications. 1. Early Years Early structural studies of aminoglycosides relied on chemical degradation ex- periments.4 The chemical structure of streptidine (the central aglycon in strep- tomycin) was determined in 1946.5 Some years later, the streptose moiety was characterized.6 The absolute configuration was confirmed during this period,7 and AN EXPANDING VIEW OF AMINOGLYCOSIDE–NUCLEIC ACID 265 the absolute structure completed the structural investigations by 1968.8 The de- velopment of such powerful techniques as NMR, X-ray crystallography, and mass spectrometry paved the way for structural elucidation of the large population of aminoglycosides known to date. All natural or semisynthetic aminoglycosides share a similar structural motif. Consisting of two to four rings of cyclitols (sat- urated six-membered carbon rings), pentoses, or hexoses linked by glycosidic bonds, aminoglycosides are unique in the carbohydrate class by their substitutions of amino groups at various ring positions. The amino groups are responsible for the basic nature of the aminoglycoside. Most amines have pKa values greater than 7, protonating them at physiological pH. The pH of each amine varies, as illus- trated by NMR studies of the neomycin class.9,10 The ionic nature, coupled with the presence of hydroxyl groups at other ring positions, is also responsible for the high hydrophilicity of aminoglycosides. A number of reviews on the structural characteristics and chemistry of aminoglycosides are available.11,12 Representative structures of aminoglycosides are illustrated in Fig. 1. After its discovery, streptomycin became the first antibiotic for tuberculosis treatment.13 Early on, it was known to be a basic molecule with nucleic acid- binding properties.14 It was eventually shown to prevent protein synthesis,15 but its bactericidal properties were yet to be explained. In 1959, it was proposed that ribosomes were the primary target for streptomycin.16 However, this was con- troversial due to beliefs that membrane damage was the sole process.17,18 Davies and others were later able to show that streptomycin binds within the 30S subunit of the 70S ribosome,19,20 illustrating that indeed the mechanism of ac- tion was probably ribosome related. Davies quickly thereafter showed resound- ing evidence that streptomycin disrupts the fidelity of translation between the ribosome and mRNA, producing proteins of inefficient function.21 It was pro- posed that this ‘‘flooding’’ of non-productive proteins affects other cellular functions, which leads to cell death. To this day, a complete understanding of the bactericidal effect is lacking, though much progress has been made (dis- cussed in a later section). The in vitro studies of the 1960s were punctuated with evidence of in vivo codon misreading by the late 1970s.22 II. MECHANISM OF ACTION 1. Pre-Ribosomal Binding: Cellular Membrane Permeation Before eliciting action on its ribosomal target, aminoglycosides must first penetrate the cell membrane, which, upon evaluation of the ionic properties of 266 B. WILLIS AND D. P. ARYA R R II O II HO 2' O HO 1' HO 2' H2N I HO 1' N 3 H2N I H N 3 O 1 H O NH2 O 1 H H O NH2 HO OH HO OH O O R NH2 1'' NH Neamine OH III III 1'' 2 OH Paromamine IV 2''' 3'' O OH 3'' O HO OH 1''' R H2N OH R NH Neomycin 2 NH Ribostamycin OH Paromomycin 2 NH2 NHR2 II R1 O HO 2' O R 1' II 2' 1 H2N I 1' H N R 3 2 I 2 N 3 O NH2 H O O NH2 1 H HO O O 1 III OH R1 R2 1'' III R1 R2 1'' HO O O OH HO CH3 OH OH Kanamycin A 2'' 2'' NH2 CH3 CH3 Gentamicin C1 H N OH NH2 Kanamycin B CH3 H Gentamicin C2 OH HNH2 Tobramycin H H Gentamicin C1a HN NH2 HN I 4 H O OH N CHO HO 1 NH2 II O OH 1' HN H3C 2' OH O 1'' Streptomycin HO III O NHCH3 HO 2'' HO FIG. 1. Representative chemical structures of aminoglycoside antibiotics. aminoglycosides, would seem unlikely due to the lipophilic nature of the cell wall. Like many steps in the bactericidal activity of aminoglycosides, a detailed picture of the uptake of these cationic ligands is incompletely resolved. Amino- glycoside uptake is believed to involve a self-promoted process23–25 character- ized by three steps.26 The first phase is characterized by outer-membrane binding by the aminoglycoside, most probably to negatively charged pockets of AN EXPANDING VIEW OF AMINOGLYCOSIDE–NUCLEIC ACID 267 lipopolysaccharides, phospholipids, or other membrane proteins possessing negatively charged residues (in Gram-negative bacteria). Membrane binding in Gram-positive bacteria probably involves phospholipid or teichoic acid inter- actions in the first phase. It is this surface binding that is believed to displace divalent cations that bridge adjacent lipopolysaccharide molecules, causing a disruption in the membrane integrity, and an enhancement in permeability for the aminoglycoside.27 The second phase is proposed to involve protrusion of the inner membrane, which is concentration-dependent due to the requirement of a threshold poten- tial for the transmembrane step.28,29 This step is also energy-dependent, and requires a proton motive force (Dp) according to the chemiosmotic hypothesis.30 Since aerobically grown bacteria generate a Dp by a membrane-bound respi- ratory chain with oxygen as the terminal acceptor, anaerobic bacteria lack an acceptor, therefore compromising the requirement of energy for uptake to oc- cur. Numerous results have shown that anaerobic bacteria display an absence of aminoglycoside uptake.29,31,32 Alternative terminal electron acceptors (such as nitrates) in the presence of anaerobic bacteria have been shown to promote aminoglycoside uptake.33,34 The third step, also energy-dependent, is believed to involve minimal uptake of aminoglycoside into the cell, which results in rib- osomal binding and non-functional protein synthesis.35 The incorporation of these newly synthesized proteins into the inner membrane potentially disrupts the membrane integrity, sparking further aminoglycoside ‘‘leaking’’ into the cell, which triggers more ribosome binding and eventual membrane disruption for further ligand uptake.36 The ultimate consequence of this uptake phase is an irreversible saturation of all ribosomes, which leads to cell death. A model of the uptake mechanism and lethal action is shown in Fig. 2.35 2. Ribosomal RNA Binding As mentioned in the introductory paragraph, the mechanism of aminoglyco- side (streptomycin) action toward bacteria was found to involve ribosome binding. In their landmark paper, Davies and coworkers showed that amino- glycosides such as streptomycin, kanamycin, and neomycins induce misreadings during polypeptide synthesis.21 This finding led to the general belief that ami- noglycosides binding to the ribosome disrupts the rRNA conformation in such a way that accurate recognition between the codon of mRNA and the anticodon of tRNA is lost. 268 B.