C H A P T E R

T W E N T Y- O N E

Aminoglycosides: Redesign Strategies for Improved Antibiotics and Compounds for Treatment of Human Genetic Diseases
Varvara Pokrovskaya, Igor Nudelman, Jeyakumar Kandasamy, and Timor Baasov Contents
1. Introduction 2. Aminoglycoside Antibiotics: Their Mode of Action and Major Drawbacks 3. Strategies Toward Development of Improved Antibiotics 3.1. Alteration of neomycin B at C500 position to prevent APH(30 ) resistance 3.2. Dual activity of C500 -modified neomycin B derivatives against Bacillus anthracis 3.3. 30 ,40 -Methylidene protected aminoglycosides: A strategy to reduce toxicity and overcome resistance enzymes 3.4. Neomycin B-based hybrid antibiotics: A strategy to delay resistance development 4. Aminoglycosides as Readthrough Inducers for the Treatment of Genetic Diseases 4.1. Development of new variants of aminoglycosides with improved readthrough activity and reduced toxicity 5. Concluding Remarks and Future Perspectives Acknowledgments References 438 440 443 444 447 449 450 454 455 458 458 459

Abstract
Aminoglycosides are highly potent, broad-spectrum antibiotics that kill bacteria by binding to the ribosomal decoding site and reducing the fidelity of protein synthesis. The emergence of bacterial strains resistant to these drugs, as well
The Edith and Joseph Fischer Enzyme Inhibitors Laboratory, Schulich Faculty of Chemistry, TechnionIsrael Institute of Technology, Haifa, Israel Methods in Enzymology, Volume 478 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)78021-6
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2010 Elsevier Inc. All rights reserved.

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as their relative toxicity, have inspired extensive searches toward the goal of obtaining novel molecular designs with improved antibacterial activity and reduced toxicity. In recent years, a new therapeutic approach that employs the ability of certain aminoglycosides to induce mammalian ribosomes to readthrough premature stop codon mutations has emerged. This new and challenging task has introduced fresh research avenues in the field of aminoglycosides research. In this chapter, our recent observations and current challenges in the design of aminoglycosides with improved antibacterial activity and the treatment of human genetic diseases are discussed.

1. Introduction
The discovery of streptomycin by Selman Waksman in 1944 was a landmark not just in antibiotic history but also in a revolutionary recognition of complex carbohydrates as an important class of natural products (Schatz et al., 1944). Streptomycin was the first aminoglycoside to be isolated from a bacterial source and the first effective antibiotic against Mycobacterium tuberculosis. In the following decades, several milestone aminoglycoside drugs (Fig. 21.1), such as neomycin, kanamycin, tobramycin, and others, were isolated from soil bacteria by intense search for natural products with antibacterial activity (Umezawa and Hooper, 1982). However, the rapid spread of antibiotic resistance to this family of antibacterial agents in pathogenic bacteria (Vakulenko and Mobashery, 2003), along with their relative toxicity to mammals, have stimulated medicinal chemistry approaches toward the development of improved antibiotics. Earlier studies on direct chemical modification of existing aminoglycoside drugs, with the aim of circumventing the resistance mechanisms, have yielded several semisynthetic drugs such as amikacin, dibekacin, netilmicin, and isepamicin that were introduced into clinical use in the 1970s and 1980s (Kondo and Hotta, 1999). More recent advancements in studies of resistance mechanisms (Wright, 2008), high-resolution structures of aminoglycosides in complex with their ribosomal RNA (rRNA) target (Francois et al., 2005; Ogle and Ramakrishnan, 2005), along with the identification and characterization of biosynthetic enzymes for certain aminoglycosides (Llewellyn and Spencer, 2006), have stimulated the development of innovative chemical (Li and Chang, 2006; Silva and Carvalho, 2007) and chemoenzymatic strategies (Llewellyn and Spencer, 2008; Nudelman et al., 2008) toward improved aminoglycoside derivatives and mimetics (Chittapragada et al., 2009; Hermann, 2007). Although the prokaryotic selectivity of action is critical to the therapeutic utility of aminoglycosides as antibiotics, they are not entirely selective to bacterial ribosome; they also bind to the eukaryotic rRNA (Bottger et al., 2001)

4, 5-Disubstituted

4, 6-Disubstituted 6' NH2 O 1' R3 O 4 HO

6' R1 I R1 6' R2 O R3 HO O 1' HO R4 1' 4 NH2 II H2N H2N O 4 NH2 O 1 NHR2 NH2 O HO HO 5 OH 6O 5'' O Neamine O Me paromamine III OH NH2 HO NH OH O OH O Me HN Ribostamycin OH IV butirosin B R1 Neamine Ribostamycin Neomycin B Paromomycin Paromamine Butirosin B NH2 NH2 NH2 OH OH NH2 R2 H H H H H AHB Gentamicin C1 Gentamicin C1a Gentamicin C2 Gentamicin C2a Gentamicin C2b Geneticin (G418) R1 NHCH3 H NH2 CH3 NHCH3 CH3 R2 CH3 NH2 CH3 NH2 H OH

R1 R2

NH2
1

6O

NHR4 OH OH R3 OH NH2 OH NH2 NH2 N2H R4 H H AHB H H AHB

O HO H2N R 1 R2 Kanamycin A Kanamycin B Amikacin Tobramycin Debekacin Arbekacin AHB = OH OH OH OH OH OH OH H H H H H OH

NH2 Neomycin family Gentamicin family O Kanamycin family

Figure 21.1 Chemical structures of 4,5- and 4,6-disubtituted 2-DOS containing natural and synthetic aminoglycosides.

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and promote mistranslation (Eustice and Wilhelm, 1984). The use of this disadvantage of aminoglycoside antibiotics for the possible treatment of human genetic diseases caused by premature nonsense mutations is extremely challenging (Zingman et al., 2007). There are more than 1800 inherited human diseases caused by nonsense mutations, that is, alterations in the genetic code that prematurely stop the translation of proteins. Aminoglycosides have emerged as vanguard pharmacogenetic agents in treating such genetic disorders due to their unique ability to induce mammalian ribosomes to readthrough premature stop codon mutations. In numerous preclinical and pilot clinical studies, this new therapeutic approach shows promise in phenotype correction by promoting otherwise defective protein synthesis (Kellermayer et al., 2006). However, severe side effects of existing aminoglycoside drugs, including high toxicity to mammals and the reduced readthrough efficiency at subtoxic doses, have inspired extensive searches toward the goal of obtaining novel molecular designs with improved readthrough activity and reduced toxicity (Hainrichson et al., 2008; Hermann, 2007). During the past few years, several comprehensive review articles that cover traditional areas of aminoglycoside use as antibiotics, including development of novel semisynthetic aminoglycoside derivatives (Chittapragada et al., 2009; Zhou et al., 2007), molecular mechanism of action, mechanisms of resistance (Wright, 2008), and toxicity (Guthrie, 2008), have been published. In addition, the book titled Aminoglycoside Antibiotics: From Chemical Biology to Drug Discovery was published in 2007 by John Wiley & Sons, Inc., providing an excellent overview of recent advances in the field (Arya, 2007). Therefore, in this chapter we focus mostly on our recent efforts to develop new designs with improved activity against resistant and pathogenic bacteria, new designs that are active against drug-resistant bacteria and exhibit reduced potential for generating new bacterial resistance, and new designs with improved premature stop codon suppression activity and reduced toxicity.

2. Aminoglycoside Antibiotics: Their Mode of Action and Major Drawbacks

The majority of aminoglycosides consist of a central aminocyclitol ring, usually 2-deoxystreptamine (2-DOS) linked to one or more amino sugars by glycosidic bonds. Depending on the substitution pattern on the 2-DOS ring, aminoglycosides can be divided into two major classes: 4,5and 4,6-disubstituted 2-DOS aminoglycosides (Fig. 21.1). The nomenclature usually refers to ring I as primed and corresponds to the amino sugar

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linked at position 4. Ring II is unprimed and corresponds to the central 2-DOS ring. Ring III is referred to as the doubly primed and corresponds to the substituent in position 5 or 6 of the 2-DOS. Rings with sequential numbers (IV, V) are usually attached to ring III (Fig. 21.1). Neomycin B, a representative of 4,5-disubstituted 2-DOS subclass, is used topically in the form of creams and lotions for the treatment of bacterial infections caused from skin burns, wounds, and dermatitis ( Jana and Deb, 2006). Paromomycin, another representative of 4,5-disubstituted 2-DOS, is used therapeutically against intestinal parasites ( Jana and Deb, 2006) and in the treatment of a variety of tropical diseases, including leishmaniasis and certain types of fungal infection (Sundar and Chakravarty, 2008). On the other hand, the 4,6-disubstituted 2-DOS subclass antibiotics, including gentamicin, amikacin, and tobramycin, have important clinical applications in the treatment of serious Gram-negative bacterial infections, especially in cases of opportunistic bacteria accompanying cystic fibrosis (CF), AIDS, and cancer. Both the 4,5- and 4,6-disubstituted subclasses of aminoglycosides exert their antibacterial activity by targeting the phylogenetically conserved decoding site (A-site) of bacterial 16S rRNA in the 30S ribosomal subunit (Moazed and Noller, 1987) and interfering with decoding and global translation processes. During the recent years, several studies on NMR and crystal structures of aminoglycosides bound to bacterial A-site oligonucleotide models (Fourmy et al., 1998; Francois et al., 2005), along with crystal structures of the bacterial 30S and 70S ribosomal particles (Carter et al., 2000; Selmer et al., 2006) with and without the bound aminoglycoside, have provided fascinating insights into our understanding of the decoding mechanism in prokaryote cells and of how 2-DOS aminoglycosides induce the deleterious misreading of the genetic code. These structures revealed that upon binding to the 30S subunit, aminoglycosides displace the two noncomplementary adenines, A1492 and A1493, at the A-site of the ribosome and lock them into so-called on state orientation, similar to that observed during mRNA decoding. As a result, during the codonanticodon interaction and the proofreading process not only the cognate tRNA is stabilized, but near-cognate tRNA is stabilized as well which causes the misreading process, the accumulation of truncated and nonfunctional proteins and eventually leading to bacterial cell death. While this mechanism of action is now well accepted for the majority of 2-DOS aminoglycosides, the recent crystallographic investigation of a series of aminoglycosides bound to the A-site oligonucleotide models (Francois et al., 2005; Vicens and Westhof, 2003) suggest that the actual molecular mechanism of this molecular switch system is more complex and that additional thermodynamic and kinetic factors are likely to govern the impact of aminoglycosides on prokaryotic translation (Ogle and Ramakrishnan, 2005). Indeed, recent

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study to characterize the energetics and dynamics associated with the aminoglycosiderRNA interaction demonstrated that the aminoglycosideinduced reduction in the mobility of the A1492 residue is an important determinant of antibacterial activity (Kaul et al., 2006). Resistance to the aminoglycosides occurs by three methods: (1) decrease of intracellular drug concentration (import and efflux), (2) modification of the target rRNA and ribosomal proteins, and (3) enzymatic drug modification (Wright, 2008). The latter is the most prevalent mechanism in clinical isolates of resistant bacteria. Three distinct classes of aminoglycoside-modifying enzymes are known: the aminoglycoside phosphotransferases (APHs), the aminoglycoside-acetyltransferases (AACs), and the aminoglycoside-adenyltransferases (ANTs; Fig. 21.2). These modifications reduce the binding affinity of aminoglycosides to the A-site and thus significantly decrease their antibacterial potential. Members of each of these classes of enzymes are widespread in both Gram-negative and Gram-positive bacteria, and 3D crystal structures of representative proteins from each class have recently been solved (Magnet and Blanchard, 2005; Wright, 2008). Among these three enzyme classes, aminoglycoside 30 -phosphotransferases [APH (30 )s], of which seven isozymes are known, are most widely represented. These enzymes catalyze transfer of g-phosphoryl group of ATP to the 30 -hydroxyl of many aminoglycosides, rendering them inactive. Although the enzymes of all three classes are typically monofunctional enzymes, the recent emergence of genes encoding bifunctional aminoglycoside-modifying enzymes, for example, the bifunctional AAC(60 )/APH(200 ) enzyme, is another level of sophistication relevant to the clinical use of aminoglycosides (Zhang et al., 2009).
AAC(6)APH(2)

ANT(4) 4

6 NH2

AAC(3) AAC(1) 3 NH2 1 NH 2 OH

APH(3)

O HO HO 3 H2N O O HO 5 O OH NH2 3 O O OH OH

H2N AAC(6)APH(2)

Neomycin B

Figure 21.2 Aminoglycoside-modifying enzymes and their modification sites are highlighted on the structure of neomycin B.

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Another main drawback of aminoglycoside antibiotics is their relatively high toxicity to humans. The aminoglycosides-induced toxic effects include nephrotoxicity, ototoxicity (vestibular and auditory), and, rarely, neuromuscular blockade and hypersensitivity reactions (Nagai and Takano, 2004; Talaska and Schacht, 2007). The first two types of toxicities were suggested to result from a combination of several mechanisms, such as inhibition of phospholipases, interaction with phospholipids, as well as formation of free radicals (Forge and Schacht, 2000). Nephrotoxicity receives the most attention, perhaps because of easier documentation of reduced renal function, but it is usually reversible. Ototoxicity is usually irreversible and it is believed to result from aminoglycoside binding to phospholipids and from disrupting mitochondrial protein synthesis due to accumulation of drug in the inner ear (Hobbie et al., 2008a,b). Numerous studies suggested that the interference between aminoglycosides and some steps of calciummediated acetylcholine release at the level of presynaptic structures is the main cause of the neuromuscular blockage induced by aminoglycosides (Albiero et al., 1978).

3. Strategies Toward Development of Improved Antibiotics

The problems of bacterial resistance and inherent toxicity of aminoglycosides have inspired continuous attempts for the development of improved aminoglycoside variants by using series of diverse approaches (Fig. 21.3). In general, these approaches can be divided into two different categories: (1) the modifications of existing drugs and (2) the design of aminoglycosides mimetics. The first category includes either the direct

Design strategies for aminoglycoside antibiotics improvement

Modifications of the existing drugs

Aminoglycoside mimetics

Direct modifications

Glycosylation strategies

Total-synthetic mimetics

Semisynthetic mimetics

Figure 21.3 Design strategies employed for development of improved aminoglycoside antibiotics.

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modification of the intact aminoglycoside by attachment of various appendages at different locations, or glycosylation of the selected aminoglycoside scaffolds, usually the pseudodisaccharides-like neamine and paromamine, with various natural or unnatural sugars, or a combination of both. The second category considers the generation of aminoglycoside mimetics either by further minimization of the original aminoglycoside entity to one ring, usually rings I or II, to which different appendages are attached at different locations, or rational design of the completely new entities. Using these strategies, many semisynthetic analogs of natural aminoglycosides (Chittapragada et al., 2009; Li and Chang, 2006; Wang and Chang, 2007; Zhou et al., 2007) and aminoglycoside mimetics (Chittapragada et al., 2009; Hermann, 2007) have been reported during recent years. Some of these designs were found to be effective against aminoglycoside-resistant bacterial strains. Little progress, however, has been made toward the discovery of new aminoglycoside derivatives with diminished toxicity (Shitara et al., 1995), which indeed is one of the remaining and perhaps the most challenging task. In addition, the latest semisynthetic aminoglycoside introduced into human therapy was two decades ago (arbekacin, Fig. 21.1, a kanamycin B derivative used in Japan since 1990; Kondo and Hotta, 1999), while the resistance to all the currently available aminoglycosides is increasing in prevalence. Clearly, a novel aminoglycoside derivative(s) with reduced toxicity demonstrated efficiency against the current generation of resistant pathogens, and preferably with reduced potential for generating bacterial resistance should be an important addition for the treatment of infectious diseases. To address the need of such designs, the following sections summarize our recent efforts to develop new aminoglycoside antibiotics by using four different strategies (Fig. 21.4): (1) the designs which in addition to targeting rRNA also resist to aminoglycoside-modifying enzyme(s); (2) the designs that target both the toxigenic bacterium and its lethal toxin; (3) the designs which in addition to resisting to aminoglycoside-modifying enzyme(s) and targeting rRNA, also exhibit reduced toxicity; (4) the designs of hybrid antibiotics which in addition to resisting to existing aminoglycoside-modifying enzymes and targeting rRNA, also delay the development of new resistance.

3.1. Alteration of neomycin B at C500 position to prevent APH(30 ) resistance

In the presence of APH(30 ) enzymes the majority of aminoglycoside drugs undergo phosphorylation at C30 -OH, but neomycin B (NeoB) undergoes phosphorylation at two distinct positions: C30 -OH and C500 -OH (Fig. 21.2). We hypothesized that by attaching an extra rigid sugar ring at C500 -OH of NeoB, in addition to blocking this position from initial phosphorylation may also inhibit the formation of a precise ternary complex

O O O 3 H2N O HO HO

NH2 NH2 NH2 O O OH Toxicity reduction Overcoming APH(3 ) H2N B. anthracis inhibition

HO HO H2N HO 5

NH2 O O O OH Neomycin B O NH2 NH2 OH

HO Overcoming APH(3 ) resistance HO

HO HO O H2N O

NH2 O NH2 O O NH2

HN

3 , 4 -Methylidene protected pseudotrisaccharide NH2 OH O HO HO

OH NH2 O O OH

5 O HO O n(NH2) H 2N O OH HO O H2N OH

OH

Delay in resistance development

Pseudopentasaccharides

NH2 NH2 O HO O HO H2N O O 5 S HO S O O 5 O H 2N O NH2 H 2N OH OH O O OH H2N H2N OH O HO NeoB dimer H2N

NH2 NH2 OH HOOC

F O N N H2N N X

HO HO N N N

NH2 O H2N Y 5 NH2 OO O OH NH2

OH

OH NH2 O O OH

NeoBCiprofloxacin hybrids

Figure 21.4 Redesign of neomycin B (NeoB) for improved antibacterial performance by using four different strategies.

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1, R = 11, R = OH H2N HO O NH2 O OH O HO HO

OH O OH O

2, R = H2N HO

OH O OH O

O OH

MIC = >512 (kcat / Km) = 8.3

MIC = 40 (kcat / Km)=5.1 3, R = OH HO H2N NH2 O OH O

MIC = 64 (kcat / Km) = 6.7 10, R = H2N O S HO HO

NH2 O H2N

HO

OH

O O

MIC = 512 (kcat / Km) = 13 9, R = H2N O H2N

R 5'' OH O OH NH2 O

NH2 OH

MIC = 128 (kcat / Km) = 9.7 OH O O NH2

OH

4, R = HO HO

MIC = 40 (kcat / Km) = 12 5, R = H2N HO 6, R = O HO HO NH2 O O NH2 O OH O

HO

OH

R = OH; neomycin B MIC = 64 (kcat / Km) = 28 O 7, R = H2N OH O NH2 OH MIC = 128 (kcat / Km) = 12

MIC = 40 (kcat / Km) = 3.0 8, R = HO

MIC = 32 (kcat / Km) = 2.5

HO

OH

NH2 MIC = 32 (kcat / Km) = 6.7

MIC = 64 (kcat / Km) = 5.4

Figure 21.5 Comparative antibacterial activity against P. aeruinosa and specificity constants (kcat/Km values in 104 M 1s 1) with APH(30 )-IIIa enzyme of NeoB and its 500 -modified derivatives 111. Minimal inhibitory concentration (MIC) values are given in mg/mL.

required for the phosphorylation of the C30 -OH by APH(30 )s, while the affinity of the resulting derivative to the target rRNA would not change or even increase. Using this strategy, a series of branched derivatives of NeoB, compounds 111 (Fig. 21.5), were synthesized. All these compounds were assembled by employing the general synthetic approach shown in Scheme 21.1. Initially, the NeoB was converted into the common acceptors, to which various donors were attached, followed by a two-step deprotection to yield the target C500 -branched derivatives. All new structures keep the whole antibiotic constitution intact as a recognition element to the rRNA, while the extended sugar rings (ring V in structure 110, and rings V and VI in 11) of each structure was designed in a manner that incorporates the potential functionalities directed for the recognition of the phosphodiester bond of RNA. The designed structures (compounds 111) exhibited similar or better antibacterial activities to that of the parent NeoB against selected bacterial

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N3 AcO AcO Neomycin B O N3 O HX O O 5 OAc N3 N3 O OAc X = O, S Common NeoB acceptor O OAc H2N X = O, S N3
n(NH2)

NH2 O LG HO HO O Deprotection steps

n(NH2)

O H2N X 5 NH2 O OH NH2 O O O NH2 OH

N3 OAc

Donor Coupling step

OH O

OH Pseudopentasaccharides

Scheme 21.1 General synthetic strategy for the assembly of C500 -derivatives of NeoB (LG leaving group).

strains, including pathogenic and resistant strains, and especially good activities were observed against Pseudomonas aeruginosa (Fridman et al., 2003; Hainrichson et al., 2005; Fig. 21.5). The specificity constant values (kcat/ Km) of 111 with APH(30 )-IIIa enzyme were lower than that of NeoB, implying that these derivatives are poorer substrates of the enzyme than the parent NeoB. Since the strains of P. aeruginosa harbor a chromosomal APH (30 )-IIb-encoding gene (Hainrichson et al., 2007), the observed superior activity of new derivatives to that of NeoB in this bacterium could be ascribed because their inferior substrate activity for the APH(30 )-IIb resistance enzyme.

3.2. Dual activity of C500 -modified neomycin B derivatives against Bacillus anthracis
Anthrax is an infectious disease caused by toxigenic strains of the Grampositive bacterium B. anthracis. It has been well established that the anthrax toxins (protective antigen, PA, edema factor, EF, and lethal factor, LF) play a major role from the very beginning of infection to death of the host. Among them, LF is considered the dominant virulence factor of anthrax and therefore an intensive search for specific inhibitors of LF has been performed during the last decade (Forino et al., 2005; Shoop et al., 2005). Unlike this strategy, we anticipated that it would be highly beneficial if the developed material were bifunctional, with the ability to inactivate the released LF toxin and, in parallel, to function as an antibiotic. Indeed, in the earlier experiments, Wong and coworkers (Numa et al., 2005) tested a library of approximately 3000 compounds, over 60 of which were synthetic and commercial aminoglycosides, and have demonstrated that NeoB is the most potent inhibitor of LF with the apparent Ki value in the low nanomolar concentration range. To improve the inhibitory effect of NeoB derivatives and in parallel to address the need of such dual activity designs, in collaboration with Wongs

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laboratory, initially we tested the C500 -modified NeoB derivatives 1, 2, 411 (Fig. 21.5) as inhibitors of LF along with against B. anthracis (Sterne strain) (Fridman et al., 2005). Most of the compounds exhibited significant antibacterial activity against B. anthracis and displayed activity levels comparable to that of NeoB. At low ionic strength assay conditions, the compounds 5, 6, and 9 exhibited the Ki values in the range of 0.21.3 nM, indicating them as predominantly better inhibitors of LF than the NeoB (Ki 37 nM). However, an increase in the ionic strength from 0 to 150 mM KCl (best resembles the physiological ionic strength in many cell types) drastically shifted the measured Ki values of all the tested compounds by a factor of $150053,000 towards higher concentrations, indicating that the predominant interaction between LF protein and the aminoglycosides is electrostatic in origin. Since at these conditions several C500 -derivatives were only two- to fivefold better inhibitors than NeoB, we attempted to further increase this gap and developed the disulfide dimer than NeoB, compound 12 (Fig. 21.6). At both low and high salt concentrations (150 mM KCl), compound 12 showed 53-fold higher affinity to LF relative to that of NeoB, indicating that twice the number of charged groups in 12 is probably responsible for the increased affinity. Compound 12 also displayed significant antibacterial activity against B. anthracis. Thus, the design strategy employed in this particular study provided a new direction for the development of novel antibiotics that target both the toxigenic bacterium and its released lethal toxin.
NH2 OH O HO HO NH2 O H2N HO O O OH NH2 O OH O OH NeoB Ki = 37 nM Ki (150 mM KCl) = 59 mM NH2 NH2 OH O NH2 HO HO H2N S 5'' NH2 O O O O NH2 NH2 OH

HO HO

HO H2N H2N

O O O

5''

S NH2

O H2N

OH OH O H2N HO O OH OH

H2N

12

H2N

Ki = 0.7 nM Ki (150 mM KCl) = 1.1 mM

Figure 21.6 Comparative apparent inhibition constants (Ki values) of NeoB and its disulfide dimer 12 against anthrax lethal factor (LF) toxin activity at low and high salt conditions.

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3.3. 30 ,40 -Methylidene protected aminoglycosides: A strategy to reduce toxicity and overcome resistance enzymes
It is highly noteworthy that, although a new synthetic variant may exhibit favorable antibacterial activity, high toxicity can prevent its clinical application. Therefore, the design of novel variants of aminoglycosides, which in addition to resisting to aminoglycoside-modifying enzymes and targeting rRNA, will also exhibit reduced toxicity is of high urgency. To address the need of such designs, a new pseudodisaccharide 13 with a 30 ,40 -protection was designed and its properties were evaluated in comparison to other two structurally related pseudodisaccharides, compounds 14 and 15 (Fig. 21.7; Chen et al., 2008). We anticipated that (1) the preservation of 30 ,40 -oxygens in 13 should keep the lower basicity of the 20 -NH2 group and subsequently the lower toxicity than those of the 30 ,40 -dideoxy analog 15; (2) the methylidene group should also protect 13 from the action of various APH(30 ) and ANT(40 ) resistance enzymes; (3) the 30 ,40 -methylidene protection is supposed to be substantially stable under both acid and base conditions usually used in carbohydrate chemistry, and should easily be constructed from the corresponding 30 ,40 -diol. The observed data in this study demonstrated a relationship between the basicity of the 20 -amine group and the estimated LD50 values in mice: the increase in the basicity of the 20 -amino functionality is associated with the acute toxicity increase of an aminoglycoside (Fig. 21.7), with 13 being least toxic. Similar results have been obtained by replacement of the 5-OH with 5-fluorine in kanamycin B and its several clinical derivatives (Shitara et al., 1995). The toxicities of the resulting fluoro analogs were significantly lower than the parent compounds and this phenomenon again was attributed to basicity reduction of the 3-NH2 group induced by the strongly electronwithdrawing 5-fluorine. Thus, significantly high acute toxicity of the clinical drugs such as tobramycin (30 -deoxy), gentamicin (30 ,40 -dideoxy), dibekacin (30 ,40 -dideoxy), and arbekacin (30 ,40 -dideoxy) could be ascribed to the increased basicity of 20 -NH2 group (ring I) in these drugs caused

O O

NH2 O NH2 O HO NH2 NH2 OH <

HO HO

NH2 O NH2 NH2 NH2 OH 14: Neamine LD50 = 161 2'-N pKa = 7.2 O HO <

NH2 O NH2 O HO NH2 NH2

13 LD50 = 303 2'-N pKa = 6.0

OH 15: Gentamine C1A LD50 = 104 2'-N pKa = 8.4

Figure 21.7 Correlation between basicity change of 20 -amine group (pKa values) and toxicity (estimated LD50 values (mg/kg) in mice) change in compounds 13, 14, and 15.

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Bacterial strain B. subtilis ATCC 6633 E. coli ATCC 25922 P. aeruginosa ATCC 27853 P. aeruginosa 584 / 5 Acute toxicity, LD50 (mg/kg)

Genta 16 C1A MIC (mg / ml) 12 6 96 12 48 70 48 24 24 226

NH2 O O H2N O HO O NH2 O O HN 16 OH

NH2 4 O 3 H2N O HO HO NH2 O O HN OH Gentamicin C1A

NH2

NH2

HO

Figure 21.8 Comparative acute toxicity (in mice) and antibacterial activity data between gentamicin C1A (Genta C1A) and compound 16.

mainly because of the lack of 30 -hydroxyl or 30 ,40 -hydroxyl groups, respectively. Compounds 1315 were also evaluated for their substrate/inhibitory activities against APH(30 )-IIb enzyme encoded in P. aeruginosa. As per design, while the compound 14 functioned as a substrate (Km 10.3 0.5 mM and kcat 1.7 0.1 s 1), the compounds 13 and 15 exhibited no substrate activity and demonstrated only poor inhibitory activities with the estimated apparent Ki values of 642 52 mM and 650 62 mM, respectively. Based on the data obtained with the model structures 1315, the new pseudotrisaccharide 16 (also called NB45) was constructed, and its properties were evaluated in comparison with the structurally closer analog gentamicin C1A (Fig. 21.8; Chen et al., 2008). It was found that both compound 16 and gentamicin C1A display very similar spectrum of antibacterial activity against Gram-negative and Gram-positive bacteria, including resistant and pathogenic strains, and exhibit similar antitranslational activities on prokaryotic protein synthesis (IC50 values of 38 nM and 59 nM, respectively). Kinetic analysis of compound 16 with APH(30 )-IIb enzyme confirmed the lack of substrate activity and only poor inhibitory activity. Most importantly, compound 16 exhibited significantly lower acute toxicity in mice (LD50 226 mg/kg) than gentamicin C1A (LD50 70 mg/kg), indicating that the 30 ,40 -methylidene protection in 16 not just provokes its low toxicity and protects it from the deactivation by APH(30 ) enzymes, but it also does not perturb its interaction with the ribosomal target or its penetration through the bacterial membrane that could influence its antibacterial activity.

3.4. Neomycin B-based hybrid antibiotics: A strategy to delay resistance development

A series of distinct examples in the previous sections highlight that particular drawback of aminoglycosides, such as bacterial resistance caused by individual type of enzymatic modification, toxicity, and combination of both, can be

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managed/overcome by reasonably designed semisynthetic aminoglycosides. However, unfortunately, as it happened to the previously introduced semisynthetic aminoglycosides, such as amikacin, dibekacin, and arbekacin (Davis et al., 2010; Ishino et al., 2004), there is a high probability that some new resistance will also emerge soon after the introduction of new design to the clinic. Therefore, more advance redesign strategy capable of producing new derivatives, that are active against known and spreading resistance mechanisms, should be highly beneficial. One such strategy that has been pursued in recent years employs a combination of two different drugs in one molecule (Bremner et al., 2007). With this strategy, each drug moiety is designed to bind independently to two different biological targets and synchronously accumulate at both target sites. Such dual action drugs, or hybrid drugs, offer the possibility to overcome the current resistance and in addition to reduce the appearance of new resistant strains (Long and Marquess, 2009). The underlying hypothesis is that treatments that inhibit multiple targets in the bacterial cell might delay and decrease the pathogens ability to accumulate simultaneous mutations that affect the multiple targets. Several successful applications of hybrid drugs approach have been reported (Barbachyn, 2008; Bremner et al., 2007). To address the need of such designs, a series of 17 new hybrid structures (compounds 19aq, Scheme 21.2) containing fluoroquinolone ciprofloxacin

F O N HOOC N H2N 7 N
SPACER 2

NH2 O HO HO H2N O N3 +
SPACER 1

Ciprofloxacin azido derivatives 17ai

SPACER 2

OH 5'' O NH2 OH O OH O OH NeoB alkyne derivatives 18ac

SPACER 1

NH2 NH2

(CH2)n, n = 2 6,

OH

Cu(I) MW, 40 s

O , N H

O N H

F O N HOOC N H2N 19aq NeoB-Cipro hybrid compounds 7 N

SPACER 1

N N N

NH2 O HO HO H2N O
SPACER 2

NH2 NH2 OH

5H' OH NH2 O O OH

O OH

Scheme 21.2 General synthetic strategy for the synthesis of NeoB-Cipro hybrids (19aq).

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(Cipro) and aminoglycoside (NeoB) antibiotics linked via 1,2,3-triazole moiety were designed, synthesized, and their antibacterial activities were determined against both Gram-negative and Gram-positive bacteria, including resistant strains (Pokrovskaya et al., 2009). The spacers X and Y were selected to vary both the length and chemical nature of the linkage between the two pharmakophores Cipro and NeoB. The key coupling reaction between NeoB-alkyne derivatives (compounds 18ac) and Cipro-azide derivatives (compounds 17ai) was performed under microwave irradiation ($40 s) in the presence of organic base (7% Et3N in water) and the Cu(I) catalyst to ensure the production of a single (anti)stereoisomer at the triazole moiety with almost quantitative yield. The majority of hybrids was significantly more potent than the parent NeoB, and overcome most prevalent types of resistance [including APH (30 )-Ia, APH(30 )-IIIa, and AAC(60 )/APH(200 ) enzymes] associated with aminoglycosides. For example, one of the leads, the hybrid 19i (Fig. 21.9), was 10-fold more potent than NeoB against several susceptible Escherichia coli strains, 128-fold more potent against E. coli AG100B and E. coli AG100A strains expressing APH(30 )-I aminoglycoside-resistant enzyme, and over 100-fold better against methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43300). In addition, selected hybrids inhibited bacterial protein synthesis with the potencies similar to or better than that of NeoB, and were up to 32-fold more potent inhibitors than ciprofloxacin for the fluoroquinolone targets, DNA gyrase, and toposiomerase IV, indicating a balanced dual mode of action. While the approach of hybrid antibiotics shares many of the possible advantages of a coformulation combination, like a cocktail of two different

N N HOOC O
19i

N F

N N N

NH2 O HO HO H2N H OO O N O HO NH2 O OH

NH2 NH2 OH

H2N

O OH

Antibacterial activity MIC (mg/ml)

E.coli E.coli E.coli R477-100 ATCC 25922 AG100B NeoB 19i 24 1.5 48 3 384 3 E.coli MRSA AG100A ATCC 43300 96 0.75 384 3

Dual mode of action on both targets IC50 (mM)

DNA gyrase Cipro = 1.3 19i = 0.085 TopoIV Cipro = 10.8 19i = 0.55 Protein synthesis NeoB = 10.5 19i = 16.7

Figure 21.9 Structure of hybrid 19i and its biological evaluation data in comparison to NeoB and Cipro.

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drugs, it is apparent that antagonistic/suppressive activity of the hybrid relative to the sum of its components appears to be a key element for the delay of resistance development. Indeed, recent studies on various physical combinations of antibiotics of different classes have demonstrated that, while synergistic combinations of antibiotics resulted in an increase in the selection of drug-resistant mutants, suppressive combinations greatly slowed the rate of accumulation of drug-resistant mutants (Chait et al., 2007; Palmer et al., 2010; Yeh et al., 2009). The possible explanation in the latter situation is that when the combination of two drugs (AB) is less inhibitory to the bacterial growth than each drug A and B separately (suppressive combination, Fig. 21.10), it is not beneficial for the bacteria to develop resistance against either A or B because in each situation the resistant strain will face a more deleterious condition than in the case of AB; the strain that acquires resistance to one drug (either against A or B) in the cocktail loses in competition with the sensitive strains because second drug is a stronger antibiotic alone than in combination. Consequently, a large delay in resistance development can occur. The observed low frequency of 19i-resistant mutations development in both Gram-negative (E. coli) and Gram-positive (B. subtilis) (Table 21.1), relative to that of each drug separately or their 1:1 mixture, is consistent to the above discussed observations with the cocktails (Fig. 21.10). To our knowledge, this study provided the first demonstration of the ability of hybrid aminoglycoside-based conjugate to delay the emergence of resistance development in both Gram-positive and Gram-negative bacteria. In sum, this class of aminoglycoside-based hybrids provides a promising new pharmacophore with an unusual dual mechanism of action, potent activity against aminoglycosides-resistant pathogens, and most importantly, reduced potential for generating bacterial resistance.
Bacterial growth inhibition (%)

Resistance Resistance to B to A Drug A A+B Drug B

100 75 50 25 0

Figure 21.10 Bacterial growth inhibition by drugs A, B, and their suppressive combination AB. Dashed arrows illustrate that each single-drug resistance steps are unfavorable.

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Table 21.1 Comparative study on the emergence of resistance in E. coli and B. subtilis after 15 serial passages in the presence of Cipro, NeoB, Cipro NeoB mixture (1:1 molar ratio) and hybrid structure 19i MIC (mg/mL) B. subtilis ATCC 6633 Ratioa 15th Passage 1st Passage E. coli ATCC 35218 Ratioa 15th Passage 1st Passage Compound

37.5 7.6 8 1
a

0.75 0.38 6 3

0.02 0.05 0.75 3

75 20 4 1

0.75 0.2 48 3

0.01 0.01 12 3

Cipro Cipro NeoB NeoB 19i

The ratio was calculated by dividing the MIC after 15th passage to the initial MIC value.

4. Aminoglycosides as Readthrough Inducers for the Treatment of Genetic Diseases

Compelling evidence is now available that certain aminoglycoside structures can induce mammalian ribosomes to readthrough premature stop codon mutations and generate full-length functional proteins. However, the major concern that arises with the potential use of aminoglycosides for translational therapy is their toxicity to the organism, preventing their repeated long-term administration required for the treatment of genetic diseases (Hainrichson et al., 2008). To make things even more complicated, (1) to date there is still no clear answer to the question why some aminoglycosides induce termination suppression while others do not, and (2) the identity of the stop codon and the sequence context surrounding it influence the readthrough activity differently among the various aminoglycosides that do have this activity (Howard et al., 2000; Manuvakhova et al., 2000). Despite its high toxicity, the clinical drug gentamicin was/is frequently used for proof-of-concept experiments in various disease models and in clinical trials, and no systematic study has been performed to tune aminoglycosides structures for better readthrough activity and lower toxicity. In addition, while the recent X-ray crystal structures of the bacterial ribosome in complex with aminoglycosides shed light on the mechanism of aminoglycosides action as antibiotics (Carter et al., 2000; Selmer et al., 2006), no crystal structure of human ribosome is presently available and the mechanism of aminoglycoside-induced readthrough is still obscure (Hainrichson et al., 2008; Kondo et al., 2007). Clearly, the challenges in

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6' HO HO

OH O

H2N

O HO

NH2 1 NH2 5 6 OH

Paromamine

6' HO HO

OH O HO HO NH2 OH

6' NH2

OH O OH NH2

H2N H2N 5''

O O 5 O

H2N H2N

O O 5 O

NH2

H 1 N O

OH

AHB

Me HO 6' O HO HO NH2 H2N O 1 NHR O H 2N 5 OH O

HO

OH

HO

OH

HO

OH

20 (NB30) In vitro (CF, USH, DMD, HS) Cell culture (CF, USH, DMD, HS) Cell toxicity Acute toxicity (mice) Ototoxicity (cochlear explants)

21 (NB54) In vitro (CF, USH, DMD, HS) Cell culture (CF, USH, DMD, HS) Cell toxicity Acute toxicity (mice) Ototoxicity (cochlear explants)

22 R = H (NB74) 23 R = AHB (NB84) In vitro (CF, USH, DMD, HS) Cell culture (CF, USH, DMD, HS) Cell toxicity

Figure 21.11 Structures and list of the performed biological tests for the new developed lead compounds 2023. Important structural elements for readthrough activity including 60 -OH, AHB, and (R)-60 -Me groups are highlighted.

this emerging field should be at the development of new design strategies to meet novel molecular designs with improved suppression activity and reduced toxicity. To address the need of such designs, recently we hypothesized that by separating the structural elements of aminoglycosides that induce readthrough from those that affect toxicity, we might reach potent derivatives with improved readthrough activity and reduced toxicity (Hainrichson et al., 2008; Nudelman et al., 2006, 2009). Using this strategy, we have systematically developed the lead compounds 20 (Nudelman et al., 2006), 21 (Nudelman et al., 2009), 22, and 24 (Nudelman et al., 2010; also named NB30, NB54, NB74, and NB84, respectively, Fig. 21.11), which are discussed in the following section.

4.1. Development of new variants of aminoglycosides with improved readthrough activity and reduced toxicity
Following key factors/observations guided us for the development of new designs. First, earlier in vitro studies on suppression activity in mammalian system have shown that aminoglycosides with a 60 -OH group on ring I (such as G418 and paromomycin, Fig. 21.1) are generally more effective than those with an amine at the same position (Howard et al., 2004;

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Manuvakhova et al., 2000). Therefore, as a key structural element for the improvement of readthrough activity of a new variant, we selected the presence of 60 -OH group on ring I (Fig. 21.11). Second, although the molecular basis for aminoglycosides-induced toxicity is still controversial, several lines of evidence point to the inhibition of mitochondrial protein synthesis machinery as the Achilles heel of aminoglycosides toxicity; (1) aminoglycosides were shown to inhibit translation and induce miscoding in chicken embryo mitochondria (Kurtz, 1974), and (2) by replacing a 34-nucleotide portion of bacterial 16S rRNA helix 44 with its mitochondrial homolog, it has been demonstrated that there is a correlation between the aminoglycoside-induced antitranslational activity and its demonstrated ototoxicity (Hobbie et al., 2008a,b). Therefore, as a key element/sign for the reduced toxicity of a new design, we selected the lack of its antibacterial activity; since the bacterial protein synthesis machinery is in many aspects very close to that of mitochondrial machinery, the reduced impact of a drug on bacterial ribosome is likely to be associated with its reduced action on mitoribosome and subsequently with its diminished toxicity. Using this strategy, initially, we have identified the pseudo-disaccharide paromamine as a minimal core structure (consists of only two rings I and II of the natural antibiotic paromomycin) exhibiting significant readthrough activity, and by attaching 5-amino ribose as a ring III have spotted compound 20 as the first lead structure (Nudelman et al., 2006). Compound 20 exhibited significantly reduced cell, cochlear, and acute toxicities in comparison to gentamicin and paromomycin (Nudelman et al., 2006, 2009), and promoted dose-dependent suppression of nonsense mutations of the PCDH15 gene, one of the underlying causes of type 1 Usher syndrome (USH1) (Rebibo-Sabbah et al., 2007). Compound 20 displayed no significant antibacterial activity and was about 10-fold poorer inhibitor of prokaryotic translation than paromomycin and gentamicin, in agreement with the toxicity results and with the design hypothesis. However, its binding affinity to the eukaryotic A-site model oligonucleotide (Kondo et al., 2007) along with its suppression potency was significantly lower relative to that of the parent drug paromomycin and gentamicin (Nudelman et al., 2006, 2009). In attempts to further improve the suppression efficiency and reduce the toxicity of 20, we borrowed the known pharmacophore, (S)-4-amino-2hydroxybutanoic acid (AHB) moiety from the butirosin/amikacin and by installing it on 20 have developed the compound 21 as a second-generation lead structure (Fig. 21.11; Nudelman et al., 2009). Compound 21 exhibited significantly reduced cell, cochlear, and acute toxicities, and has substantially higher stop codon readthrough potency in both in vitro and ex vivo studies than those of gentamicin and paromomycin (Nudelman et al., 2009). The superior in vitro readthrough efficiency of 21 to that of compound 20, gentamicin, and paromomycin was demonstrated in seven different

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nonsense DNA-constructs underlying the genetic diseases CF, Duchenne muscular dystrophy (DMD), USH1, and Hurler syndrome (HS). Importantly, compound 21 (as well as compound 20) lacks significant antibacterial activity and exhibited substantially reduced prokaryotic antitranslational activity in comparison to gentamicin and paromomycin. In continuation to the systematic fine-tuning of the developed leads, most recently, we have discovered a new pharmacophore, (R)-60 -methyl group, which we borrowed from the natural aminoglycoside G418 (Fig. 21.1), and by installing it on 20 and 21 we spotted the third-generation lead structures 22 and 23, respectively (Fig. 21.11; Nudelman et al., 2010). Both leads (22 and 23) exhibited significantly reduced cell toxicity and superior readthrough efficiency than those of gentamicin. The evidence for the superior readthrough efficiency of 22 and 23 over that of gentamicin was demonstrated in vitro on six different DNA fragments derived from the mutant PCDH15, CFTR, Dystrophin, and IDUA genes carrying nonsense mutations and representing the underlying causes for the genetic diseases USH1, CF, DMD, and HS, respectively, and ex vivo in cultured cell lines on three different DNA fragments that model the genetic diseases USH1, CF, and HS. Furthermore, compound 23 also exhibited several-fold higher suppression activity than that of 21, while both (23 and 21) displayed similar cytotoxicity. Essentially, the same trend was also observed in comparative study between the compounds 22 and 20, suggesting that the installation of (R)-60 -methyl group on aminoglycoside structure to yield the (R)-60 -secondary alcohol on ring I, significantly increases suppression activity while has no significant influence on the cell toxicity of the resulted derivative. In summary of this part, the above-described results validate our design strategy that by separating the structural elements of aminoglycosides that induce readthrough from those that affect toxicity, we can reach potent derivatives with improved readthrough activity and reduced toxicity. Thus, although the strict consideration of lack of antibacterial activity as a key parameter for the initial sign of reduced toxicity potential is rather a naive parameter of the design, it could be used as a quick test for the initial selection of the hits. The accompanied in vitro protein translation inhibition tests, in both bacterial and eukaryotic cytoplasmic systems, by using luciferase reporter system can validate the antibacterial data. It will be highly informative to add to these tests also the mitochondrial protein translation inhibition test. However, unfortunately, an in vitro system for the quantitative assessment of mitochondrial protein translation inhibition, analogous to the bacterial and cytoplasmic systems, is not available to date, and the offered tests employing selective radiolabeling of the mitochondrial proteins (e.g., see McKee et al., 2006) is not easily accessible for the majority of chemists dealing with the design and synthesis. Nevertheless, the observed continued inability of the new designs, 20, 21, 22, and 23, to show significant antibacterial activity in conjunction with their decreased

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prokaryotic ribosome specificity is striking and remain to be further investigated. Of greatest concern is whether these data are truly linked or not to the observed relatively reduced cell toxicity of 20, 21, 22, and 23. Clearly, further structural and biochemical studies are needed to understand this issue satisfactorily.

5. Concluding Remarks and Future Perspectives

To date, the majority of strategies to redesign aminoglycosides for improved antibacterial performance have been driven by the goal of finding new designs active against resistant or recalcitrant bacterial pathogens, and relatively little effort has been put into attempts to redesign aminoglycoside structure to reduce toxic responses or to delay a potential for generating bacterial resistance. Selected examples detailed here within the antibiotic field of aminoglycosides highlight our recent attempts to address those issues. Aminoglycosides that target unique bacterial rRNA A-site with high selectivity and specificity in comparison to that of human cytoplasmic and mitochondrial A-sites should guide the rational for the development of new designs with strong antibacterial activity and low toxicity. The data on aminoglycoside hybrids holds significant promise and opens an additional avenue for future research towards novel designs with reduced potential for generating new bacterial resistance. As to the redesign of aminoglycosides for the treatment of genetic diseases, although the discovery of an ideal readthrough inducer is still a challenging task, the redesign strategy and the data detailed here illustrate that this may be an achievable goal. In this avenue of research, the human toxicity of aminoglycosides should be placed as a central problem. As a potential solution to this problem, the therapeutic window between the cytoplasmic and mitochondrial decoding sites can be exploited for the development of new designs; those structures exhibiting extensive specificity and selectivity for the cytoplasmic rRNA A-site can decrease the functional dosing ranges and subsequently decrease the anticipated toxicity, making them potential drugs for the treatments of human genetic disorders.

ACKNOWLEDGMENTS
We thank the USIsrael Binational Science Foundation (grant no. 2006/301), the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities (grant no. 515/07), and the Mitchel Fund (grant no. 2012386) for their generous support of our research work.

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REFERENCES
Albiero, L., Bamonte, F., Ongini, E., and Parravicini, L. (1978). Comparison of neuromuscular effects and acute toxicity of some aminoglycoside antibiotics. Arch. Int. Pharmacor. dyn. The 233, 343350. Arya, D. P. (ed.) (2007). Aminoglycoside Antibiotics: From Chemical Biology to Drug Discovery, Wiley, Hoboken, New Jersey. Barbachyn, M. R. (2008). Recent advances in the discovery of hybrid antibacterial agents. Annu. Rep. Med. Chem. 43, 281290. Bottger, E. C., Springer, B., Prammananan, T., Kidan, Y., and Sander, P. (2001). Structural basis for selectivity and toxicity of ribosomal antibiotics. EMBO Rep. 2, 318323. Bremner, J. B., Ambrus, J. I., and Samosorn, S. (2007). Dual action-based approaches to antibacterial agents. Curr. Med. Chem. 14, 14591477. Carter, A. P., Clemons, W. M., Brodersen, D. E., Morgan-Warren, R. J., Wimberly, B. T., and Ramakrishnan, V. (2000). Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407, 340348. Chait, R., Craney, A., and Kishony, R. (2007). Antibiotic interactions that select against resistance. Nature 446, 668671. Chen, L., Hainrichson, M., Bourdetsky, D., Mor, A., Yaron, S., and Baasov, T. (2008). Structure-toxicity relationship of aminoglycosides: Correlation of 20 -amine basicity with acute toxicity in pseudo-disaccharide scaffolds. Bioorg. Med. Chem. 16, 89408951. Chittapragada, M., Roberts, S., and Ham, Y. W. (2009). Aminoglycosides: Molecular insights on the recognition of RNA and aminoglycoside mimics. Perspect. Med. Chem. 3, 2137. Davis, M. A., Baker, K. N., Orfe, L. H., Shah, D., Besser, T. E., and Call, D. R. (2010). Discovery of a gene conferring multiple aminoglycoside resistance in Escherichia coli. Antimicrob. Agents Chemother. 54(6), 26662669. Eustice, D. C., and Wilhelm, J. M. (1984). Fidelity of the eukaryotic codon-anticodon interaction: Interference by aminoglycoside antibiotics. Biochemistry 23, 14621467. Forge, A., and Schacht, J. (2000). Aminoglycoside antibiotics. Audiol. Neurootol. 5, 322. Forino, M., Johnson, S., Wong, T. Y., Rozanov, D. V., Savinov, A. Y., Li, W., Fattorusso, R., Becattini, B., Orry, A. J., Jung, D., Abagyan, R. A., Smith, J. W., et al. (2005). Efficient synthetic inhibitors of anthrax lethal factor. Proc. Natl. Acad. Sci. USA 102, 94999504. Fourmy, D., Yoshizawa, S., and Puglisi, J. D. (1998). Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA. J. Mol. Biol. 277, 333345. Francois, B., Russell, R. J., Murray, J. B., Aboul-ela, F., Masquida, B., Vicens, Q., and Westhof, E. (2005). Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: Role of the number of rings and positive charges in the specific binding leading to miscoding. Nucleic Acids Res. 33, 56775690. Fridman, M., Belakhov, V., Yaron, S., and Baasov, T. (2003). A new class of branched aminoglycosides: Pseudo-pentasaccharide derivatives of neomycin B. Org. Lett. 5, 35753578. Fridman, M., Belakhov, V., Lee, L. V., Liang, F. S., Wong, C. H., and Baasov, T. (2005). Dual effect of synthetic aminoglycosides: Antibacterial activity against Bacillus anthracis and inhibition of anthrax lethal factor. Angew. Chem. Int. Ed. Engl. 44, 447452. Guthrie, O. W. (2008). Aminoglycoside induced ototoxicity. Toxicology 249, 9196. Hainrichson, M., Pokrovskaya, V., Shallom-Shezifi, D., Fridman, M., Belakhov, V., Shachar, D., Yaron, S., and Baasov, T. (2005). Branched aminoglycosides: Biochemical studies and antibacterial activity of neomycin B derivatives. Bioorg. Med. Chem. 13, 57975807.

460

Varvara Pokrovskaya et al.

Hainrichson, M., Yaniv, O., Cherniavsky, M., Nudelman, I., Shallom-Shezifi, D., Yaron, S., and Baasov, T. (2007). Overexpression and initial characterization of the chromosomal aminoglycoside 30 -O-phosphotransferase APH(30 )-IIb from Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 51, 774776. Hainrichson, M., Nudelman, I., and Baasov, T. (2008). Designer aminoglycosides: The race to develop improved antibiotics and compounds for the treatment of human genetic diseases. Org. Biomol. Chem. 6, 227239. Hermann, T. (2007). Aminoglycoside antibiotics: Old drugs and new therapeutic approaches. Cell. Mol. Life Sci. 64, 18411852. Hobbie, S. N., Akshay, S., Kalapala, S. K., Bruell, C. M., Shcherbakov, D., and Bottger, E. C. (2008a). Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity. Proc. Natl. Acad. Sci. USA 105, 2088820893. Hobbie, S. N., Bruell, C. M., Akshay, S., Kalapala, S. K., Shcherbakov, D., and Bottger, E. C. (2008b). Mitochondrial deafness alleles confer misreading of the genetic code. Proc. Natl. Acad. Sci. USA 105, 32443249. Howard, M. T., Shirts, B. H., Petros, L. M., Flanigan, K. M., Gesteland, R. F., and Atkins, J. F. (2000). Sequence specificity of aminoglycoside-induced stop condon readthrough: Potential implications for treatment of Duchenne muscular dystrophy. Ann. Neurol. 48, 164169. Howard, M. T., Anderson, C. B., Fass, U., Khatri, S., Gesteland, R. F., Atkins, J. F., and Flanigan, K. M. (2004). Readthrough of dystrophin stop codon mutations induced by aminoglycosides. Ann. Neurol. 55, 422426. Ishino, K., Ishikawa, J., Ikeda, Y., and Hotta, K. (2004). Characterization of a bifunctional aminoglycoside-modifying enzyme with novel substrate specificity and its gene from a clinical isolate of methicillin-resistant Staphylococcus aureus with high arbekacin resistance. J. Antibiot. 57, 679686. Jana, S., and Deb, J. K. (2006). Molecular understanding of aminoglycoside action and resistance. Appl. Microbiol. Biotechnol. 70, 140150. Kaul, M., Barbieri, C. M., and Pilch, D. S. (2006). Aminoglycoside-induced reduction in nucleotide mobility at the ribosomal RNA A-site as a potentially key determinant of antibacterial activity. J. Am. Chem. Soc. 128, 12611271. Kellermayer, R., Szigeti, R., Keeling, K. M., Bedekovics, T., and Bedwell, D. M. (2006). Aminoglycosides as potential pharmacogenetic agents in the treatment of Hailey-Hailey disease. J. Invest. Dermatol. 126, 229231. Kondo, S., and Hotta, K. (1999). Semisynthetic aminoglycoside antibiotics: Development and enzymatic modifications. J. Infect. Chemother. 5, 19. Kondo, J., Hainrichson, M., Nudelman, I., Shallom-Shezifi, D., Barbieri, C. M., Pilch, D. S., Westhof, E., and Baasov, T. (2007). Differential selectivity of natural and synthetic aminoglycosides towards the eukaryotic and prokaryotic decoding A sites. Chembiochem 8, 17001709. Kurtz, D. I. (1974). Fidelity of protein synthesis with chicken embryo mitochondrial and cytoplasmic ribosomes. Biochemistry 13, 572577. Li, J., and Chang, C. W. (2006). Recent developments in the synthesis of novel aminoglycoside antibiotics. Anti-infective Agents Med. Chem. 5, 255271. Llewellyn, N. M., and Spencer, J. B. (2006). Biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics. Nat. Prod. Rep. 23, 864874. Llewellyn, N. M., and Spencer, J. B. (2008). Chemoenzymatic acylation of aminoglycoside antibiotics. Chem. Commun. (32), 37863788. Long, D. D., and Marquess, D. G. (2009). Novel heterodimer antibiotics: A review of recent patent literature. Future Med. Chem. 1(6), 10371050.

Designer Aminoglycosides

461

Magnet, S., and Blanchard, J. S. (2005). Molecular insights into aminoglycoside action and resistance. Chem. Rev. 105, 477498. Manuvakhova, M., Keeling, K., and Bedwell, D. M. (2000). Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. RNA 6, 10441055. McKee, E. E., Ferguson, M., Bentley, A. T., and Marks, T. A. (2006). Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrob. Agents Chemother. 50, 20422049. Moazed, D., and Noller, H. F. (1987). Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 327, 389394. Nagai, J., and Takano, M. (2004). Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity. Drug Metab. Pharmacokinet. 19, 159170. Nudelman, I., Rebibo-Sabbah, A., Shallom-Shezifi, D., Hainrichson, M., Stahl, I., BenYosef, T., and Baasov, T. (2006). Redesign of aminoglycosides for treatment of human genetic diseases caused by premature stop mutations. Bioorg. Med. Chem. Lett. 16, 63106315. Nudelman, I., Chen, L., Llewellyn, N. M., Sahraoui, E. H., Cherniavsky, M., Spencer, J. B., and Baasov, T. (2008). Combined chemical-enzymatic assembly of aminoglycoside derivatives with N-1-AHB side chain. Adv. Synth. Catal. 350, 16821688. Nudelman, I., Rebibo-Sabbah, A., Cherniavsky, M., Belakhov, V., Hainrichson, M., Chen, F., Schacht, J., Pilch, D. S., Ben-Yosef, T., and Baasov, T. (2009). Development of novel aminoglycoside (NB54) with reduced toxicity and enhanced suppression of disease-causing premature stop mutations. J. Med. Chem. 52, 28362845. Nudelman, I., Glikin, D., Smolkin, B., Hainrichson, M., Belakhov, V., and Baasov, T. (2010). Repairing faulty genes by aminoglycosides: Development of new derivatives of geneticin (G418) with enhanced suppression of diseases-causing nonsense mutations. Bioorg. Med. Chem. 18(11), 37353746. Numa, M. M., Lee, L. V., Hsu, C. C., Bower, K. E., and Wong, C. H. (2005). Identification of novel anthrax lethal factor inhibitors generated by combinatorial Pictet-Spengler reaction followed by screening in situ. Chembiochem 6, 10021006. Ogle, J. M., and Ramakrishnan, V. (2005). Structural insights into translational fidelity. Annu. Rev. Biochem. 74, 129177. Palmer, A. C., Angelino, E., and Kishony, R. (2010). Chemical decay of an antibiotic inverts selection for resistance. Nat. Chem. Biol. 6, 105107. Pokrovskaya, V., Belakhov, V., Hainrichson, M., Yaron, S., and Baasov, T. (2009). Design, synthesis, and evaluation of novel fluoroquinolone-aminoglycoside hybrid antibiotics. J. Med. Chem. 52, 22432254. Rebibo-Sabbah, A., Nudelman, I., Ahmed, Z. M., Baasov, T., and Ben-Yosef, T. (2007). In vitro and ex vivo suppression by aminoglycosides of PCDH15 nonsense mutations underlying type 1 Usher syndrome. Hum. Genet. 122, 373381. Schatz, A., Bugie, E., and Waksman, S. A. (1944). Streptomycin, a substance exhibiting antibiotic activity against gram-positive and gram-negative bacteria. Proc. Soc. Exp. Biol. Med. 55, 6669. Selmer, M., Dunham, C. M., Murphy, F. V., 4th, Weixlbaumer, A., Petry, S., Kelley, A. C., Weir, J. R., and Ramakrishnan, V. (2006). Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313, 19351942. Shitara, T., Umemura, E., Tsuchiya, T., and Matsuno, T. (1995). Synthesis of 5-deoxy-5epifluoro derivatives of arbekacin, amikacin, and 1-N-[(S)-4-amino-2-hydroxybutanoyl] tobramycin (study on structure-toxicity relationships). Carbohydr. Res. 276, 7589. Shoop, W. L., Xiong, Y., Wiltsie, J., Woods, A., Guo, J., Pivnichny, J. V., Felcetto, T., Michael, B. F., Bansal, A., Cummings, R. T., Cunningham, B. R., Friedlander, A. M., et al. (2005). Anthrax lethal factor inhibition. Proc. Natl. Acad. Sci. USA 102, 79587963.

462

Varvara Pokrovskaya et al.

Silva, J. G., and Carvalho, I. (2007). New insights into aminoglycoside antibiotics and derivatives. Curr. Med. Chem. 14, 11011119. Sundar, S., and Chakravarty, J. (2008). Paromomycin in the treatment of leishmaniasis. Expet. Opin. Investig. Drugs 17, 787794. Talaska, A. E., and Schacht, J. (2007). Adverse effects of aminoglycoside therapy. In Aminoglycoside antibiotics: from chemical biology to drug discovery, (D. P. Arya, ed.), pp. 255266. Wiley, Hoboken, New Jersey. Umezawa, H., and Hooper, I. R. (eds.), (1982). Aminoglycoside Antibiotics, Springer, New York, Heidelberg. Vakulenko, S. B., and Mobashery, S. (2003). Versatility of aminoglycosides and prospects for their future. Clin. Microbiol. Rev. 16, 430450. Vicens, Q., and Westhof, E. (2003). Molecular recognition of aminoglycoside antibiotics by ribosomal RNA and resistance enzymes: An analysis of x-ray crystal structures. Biopolymers 70, 4257. Wang, J., and Chang, C. W. T. (2007). Design, chemical synthesis, and antibacterial activity of kanamycin and neomycin class aminoglycoside antibiotics. In Aminoglycoside Antibiotics: From Chemical Biology to Drug Discovery, (D. P. Arya, ed.), 2nd edn. pp. 141180. Wiley, Hoboken, New Jersey. Wright, G. D. (2008). Mechanisms of aminoglycoside antibiotic resistance. In Bacterial Resistance to Antimicrobials, (K. Lewis, A. A. Salyers, H. W. Taber, and R. G. Wax, eds.), 2nd edn. pp. 71101. CRC press, Taylor & Francis group, Boca Raton, FL. Yeh, P. J., Hegreness, M. J., Aiden, A. P., and Kishony, R. (2009). Drug interactions and the evolution of antibiotic resistance. Nat. Rev. 7, 460466. Zhang, W. L., Fisher, J. F., and Mobashery, S. (2009). The bifunctional enzymes of antibiotic resistance. Curr. Opin. Microbiol. 12, 505511. Zhou, J., Wang, G., Zhang, L. H., and Ye, X. S. (2007). Modifications of aminoglycoside antibiotics targeting RNA. Med. Res. Rev. 27, 279316. Zingman, L. V., Park, S., Olson, T. M., Alekseev, A. E., and Terzic, A. (2007). Aminoglycosideinduced translational read-through in disease: Overcoming nonsense mutations by pharmacogenetic therapy. Clin. Pharmacol. Ther. 81, 99103.