Overcoming Aminoglycoside Enzymatic Resistance: Design of Novel Antibiotics and Inhibitors

molecules

Review Overcoming Aminoglycoside Enzymatic Resistance: Design of Novel Antibiotics and Inhibitors

Sandra G. Zárate 1, M. Luisa De la Cruz Claure 2, Raúl Benito-Arenas 3, Julia Revuelta 3, Andrés G. Santana 3,* and Agatha Bastida 3,*

1 Facultad de Tecnología-Carrera de Ingeniería Química, Universidad Mayor Real y Pontiﬁcia de San Francisco Xavier de Chuquisaca, Regimiento Campos 180, Casilla 60-B, Sucre, Bolivia; [email protected] 2 Facultad de Ciencias Químico Farmacéuticas y Bioquímicas, Universidad Mayor Real y Pontiﬁcia de San Francisco Xavier de Chuquisaca, Dalence 51, Casilla 497, Sucre, Bolivia; [email protected] 3 Departmento de Química Bio-Orgánica, Instituto de Química Orgánica General (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain; [email protected] (R.B.-A.); [email protected] (J.R.) * Correspondence: [email protected] (A.G.S.); [email protected] (A.B.); Tel: +34-915-612-800 (A.B.)

Received: 7 November 2017; Accepted: 26 January 2018; Published: 30 January 2018

Abstract: Resistance to aminoglycoside antibiotics has had a profound impact on clinical practice. Despite their powerful bactericidal activity, aminoglycosides were one of the ﬁrst groups of antibiotics to meet the challenge of resistance. The most prevalent source of clinically relevant resistance against these therapeutics is conferred by the enzymatic modiﬁcation of the antibiotic. Therefore, a deeper knowledge of the aminoglycoside-modifying enzymes and their interactions with the antibiotics and solvent is of paramount importance in order to facilitate the design of more effective and potent inhibitors and/or novel semisynthetic aminoglycosides that are not susceptible to modifying enzymes.

Keywords: antibiotic resistance; combination therapy; bi-substrate inhibitors; decoy acceptors

1. Introduction The continuous appearance of bacterial strains resistant to most antibiotics already known, along with the scarce perspective of new bactericidal agents coming out in the near future, have placed bacterial multi-drug resistance (MDR) among the most pressing issues for worldwide health [1–3]. Furthermore, the increasing number of hospital MDR infections has boosted the interest for new aminoglycoside antibiotics for clinical use, and consequently research on this topic has drawn renewed attention. Aminoglycosides are a group of natural antibiotics from Streptomyces that have been used in clinical practice for more than 50 years [4]. Their potent bactericidal activity relies upon binding specifically to the 16S rRNA of the 30S ribosomal subunit, thus interfering with protein synthesis [5,6]. However, the first resistant bacterial strains began to appear in the 1960s due to a high rate of dissemination via R-plasmids, transposons, and integrons [7,8]. In an attempt to overcome this emerging resistance to natural antibiotics, the first semi-synthetic aminoglycosides, such as amikacin, dibekacin, isepamicin, and netilmicin, were introduced during the 1970s [9,10]. The discovery of gentamicins, a newer family of aminoglycosides isolated from Micromonospora, contributed to a come-back of this class of bactericidal agents in clinical practice [11]. These antibiotics were very active against Pseudomonas aeruginosa, a tougher micro-organism less susceptible to the original aminoglycosides, which unfortunately soon after developed resistance through the ANT(200) enzyme [12,13]. Butirosine, another aminoglycoside discovered later, was able to avoid inactivation by APH(30) and ANT(200) enzymes [14]. There is a large number of aminoglycoside antibiotics, but the resistance mechanisms developed by micro-organisms increase with the frequency of their use.

Molecules 2018, 23, 284; doi:10.3390/molecules23020284 www.mdpi.com/journal/molecules Molecules 2018, 23, 284 2 of 18

Molecules 2018, 23, x FOR PEER REVIEW 2 of 18 2. Understanding the Modifying Enzymes 2. Understanding the Modifying Enzymes Tolerance towards antibiotics can be achieved through different mechanisms. However, the most prevalentTolerance in the towards clinic is antibiotics that due can to enzymaticbe achieved modiﬁcation through different that rendersmechanisms. aminoglycosides However, the of decreasedmost prevalent afﬁnity in forthe theirclinic naturalis that due primary to enzy target,matic modification 16S rRNA [that15–17 renders]. There aminoglycosides are three families of decreased affinity for their natural primary target, 16S rRNA [15–17]. There are three families of of aminoglycoside-modifying enzymes (AMEs): N-acetyltransferases (AACs) that acetylate an aminoglycoside-modifying enzymes (AMEs): N-acetyltransferases (AACs) that acetylate an amino group amino group using acetyl-Coenzyme A; O-nucleotidyltransferases (ANTs) that transfer an adenyl using acetyl-Coenzyme A; O-nucleotidyltransferases (ANTs) that transfer an adenyl group from ATP to group from ATP to a hydroxyl group of the antibiotic; and O-phosphotransferases (APHs), which a hydroxyl group of the antibiotic; and O-phosphotransferases (APHs), which phosphorylate a hydroxyl phosphorylate a hydroxyl group also employing ATP (Figure1). The emergence of bifunctional group also employing00 ATP (Figure0 1). The emergence of bifunctional enzymes (i.e., APH(2″)-AAC(6′)) enzymesthat are (i.e., able APH(2 to modify)-AAC(6 almost)) thatall aminoglycoside are able to modify antibiotics almost presents all aminoglycoside a huge challenge antibiotics that presents new a hugeaminoglycosides challenge that will new have aminoglycosides to overcome [18] will (Figure have 1). to overcome [18] (Figure1).

FigureFigure 1. 1.Susceptible Susceptible positionspositions in Kan B B to to AME AME modification. modiﬁcation.

AACsAACs are are found found both both in in Gram-positive Gram-positive andand Gram-negative bacteria bacteria and and are are able able to toreduce reduce the the affinity of the antibiotic for the receptor 16S rRNA by 4 orders of magnitude (Table 1). This family of affinity of the antibiotic for the receptor 16S rRNA by 4 orders of magnitude (Table1). This family enzymes is the largest within the AMEs, with 48 sequences identified so far, and they all weigh of enzymes is the largest within the AMEs, with 48 sequences identified so far, and they all weigh around 20–25 kDa and comprise a wide range of positions susceptible to modification (6′, 2′, N-1, and around 20–25 kDa and comprise a wide range of positions susceptible to modification (60, 20, N-1, N-3). Mutagenesis studies have shown that just the mutation of a single amino acid of the AAC can and N-3). Mutagenesis studies have shown that just the mutation of a single amino acid of the AAC modulate the specificity for the antibiotic. For example, AAC(6′)-I and AAC(6′)-II share the capacity can modulate the specificity for the antibiotic. For example, AAC(60)-I and AAC(60)-II share the to modify kanamycin but they differ in their propensity to acetylate amikacin or gentamicin C. The capacitystructural to modify gene of kanamycinthe AAC(3) butand theyAAC(6 differ′) is generally in their propensity found on transposable to acetylate amikacinelements [19]. or gentamicin AAC(3) 0 C.enzymes The structural were genethe first of theto AAC(3)be described and AAC(6to confer) is resistance generally foundto gentamicin on transposable (G), kanamycin elements (K), [19 ]. AAC(3)fortimicin enzymes (F), and were tobramycin the first to (T) be [20] described and can to acetylate confer resistance either N/O to groups gentamicin of the (G), aminoglycoside. kanamycin (K), fortimicinFive AAC(2 (F),′) and enzymes tobramycin are encoded (T) [ 20in ]Mycobacteria and can acetylate and one either has beenN/O crystalizedgroups of from the aminoglycoside.Mycobacterium 0 Fivetuberculosis. AAC(2 ) enzymesAAC(2′) from are encoded M. tuberculosis in Mycobacteria catalytically and and one enthalpically has been crystalizedfavors those from aminoglycosidesMycobacterium tuberculosis.with amine/hydroxylAAC(20) from groupsM. tuberculosis at the 2′ positioncatalytically and shows and enthalpically an increase favorsin affinity those for aminoglycosides all antibiotics withwhen amine/hydroxyl CoA is present. groups This AME at the exhibits 20 position a high andtolerance shows towards an increase the antibiotic in affinity molecule for all (4,5- antibiotics and when4,6-substitution), CoA is present. and Thisit is thought AME exhibits to be mo a highdulated tolerance by water towards molecules the antibioticthat mediate molecule between (4,5- andaminoglycoside 4,6-substitution), and side and chain it is thought carboxylate to be groups modulated of the receptor. by water Moreover, molecules side that chain mediate reorientation between aminoglycosiderelative to the andapo-enzyme side chain upon carboxylate binding groupsof the subs of thetrates receptor. also contributes Moreover, to side the chainplasticity reorientation of this relativeenzyme to [21]. the apo-enzymeAAC(6′) enzymes upon are binding the most of the prevalent substrates in clinical also contributes strains, conferring to the plasticity resistance of to this enzymeamikacin [21 ].(A), AAC(6 gentamicin0) enzymes (G), kanamycin are the most (K), neomyc prevalentin (N), in clinicaldibekacin strains, (D), sisomicin conferring (S), isepamicin resistance to amikacin(I), and (A),tobramycin gentamicin (T) and (G), has kanamycin an ordered (K), kinetic neomycin mechanism. (N), dibekacin Three-dimensional (D), sisomicin (3D) (S),structures isepamicin of (I),AAC(6 and tobramycin′), AAC(3), (T)and and AAC(2 has′ an) have ordered been kineticdescribed mechanism. with a remarkable Three-dimensional conservation (3D) of the structures overall of structure, albeit with low amino acid sequence identity [22,23]. AAC(60), AAC(3), and AAC(20) have been described with a remarkable conservation of the overall APHs are the second-most abundant family of AMEs and confer resistance to aminoglycosides structure, albeit with low amino acid sequence identity [22,23]. in Enterococcus and Staphylococcus strains (Table 2). There are seven types of APHs with 30 kDa mass APHs are the second-most abundant family of AMEs and confer resistance to aminoglycosides and high sequence homology (20–40%) in their C-terminal end [24]. APH(3′)-IIIa is usually used as a in Enterococcus and Staphylococcus strains (Table2). There are seven types of APHs with 30 kDa mass resistance marker and its 3D structure has been described several times [25,26].0 APH(3′)-IIIa andpromiscuity high sequence seems homology to be governed (20–40%) by disordered in their C-terminal elements that end adopt [24]. well-defined APH(3 )-IIIa conformations is usually used 0 aswhen a resistance the aminoglycoside marker and is its bound 3D structure [27]. APH(3 has′)-IIa been from described Enterococcus several faecalis times is able [25 to,26 phosphorylate]. APH(3 )-IIIa

Molecules 2018, 23, 284 3 of 18 promiscuity seems to be governed by disordered elements that adopt well-defined conformations when the aminoglycoside is bound [27]. APH(30)-IIa from Enterococcus faecalis is able to phosphorylate the 30 and 50-OH groups, giving rise to di-phosphorylated aminoglycosides [28] through an ordered sequential mechanism in which the binding of ATP is followed by the antibiotic [29]. An interesting property of this enzyme is that it is competitively inhibited by tobramycin, which one would expect not to be a substrate because it lacks a free 30-hydroxylgroup. APH(200) is a very promiscuous enzyme based on the range of susceptible positions in the antibiotic skeleton (200, 30, 300, and 500); it represents an important resistance element in Gram-positive bacteria, and this enzyme uses Guanosine Triphosphate (GTP) as the most efficient donor substrate over ATP. Oddly enough, many other APHs have been identified, such as APH(6), APH(300), APH(9), APH(4), and APH(7), but their occurrence is strikingly uncommon in the clinic. APH(9)-Ia exhibits a similar folding to that of the APH(30) and APH(200) enzymes, but it differs significantly in its substrate binding area and in the fact that it undergoes a conformational change upon ligand binding.

Table 1. N-acetyltransferase (AAC)-modifying enzymes.

Enzyme Resistance Proﬁle Bacterial Source Pdb Number I (a–d,e,f–z) T, A, N, D, S, K, I Salmonella enterica 1S60, 2VBQ, 1S3Z, 1S5K, 2QIR II T, G, N, D, S, K Enterococcus faecium 2A4N, 5E96 Acinetobacter haemolyticus 4F0Y, 4EVY, 4F0Y AAC(60) Acinetobacter baumannii 4E80 Escherichia coli 6BFF, 6BFH, 1V0C, 2BUE, 2VQY Staphylococcus warneri 4QC6 I (a–b) G, S, F Serratia marcesans 1B04 II (a–c) T, G, N, D, S Pseudomonas aeruginosa 4YFJ AAC(3) III (a–c) T, G, D, S, K, N, P, L Klebsiella pneumoniae, IV T, S, N, D, S, A Campylobacter jejuni VII G Actinomycetes I (a–c) T, S, N, D, Ne Providencia stuartii 5US1 AAC(20) Mycobacterium tuberculosis 1M44, 1M4D, 1M4G, 1M41 Ia P, L, R, AP E. coli AAC(1) Campylobacter spp. Abbreviations: A, amikacin; AP, apramycin; D, dibekacin; F, fortimicn; H, hygromycin; I, isepamicin; G, gentamicin; K, kanamycin; L, lividomycin; N, netilmicin; Ne, neomycin; P, paromomycin; R, ribostamycin; S, sisomicin; T, tobramycin.

Table 2. O-phosphotransferase (APH)-modifying enzymes.

Enzyme Resistance Proﬁle Bacterial Source Pdb I (a–d) K, Ne, R, L, P Acinetobacter baumannii 4FEV II K, Ne, B, P, R Stenotrophomonas maltophilia 0 APH(3 ) III (a–b) K, Ne, P, B, L, R, B, A, I IV K, Ne, B, P, R V Ne, P, R VI K, Ne, P, R, B, A, I Bacillus circulans I-a K, G, T, S, D APH(2”) I-(b,d) K, G, T, N, D Escherichia coli 4DCA II-(a–b) K, G, T Enterococcus faecium 3HAM, 3HAV 5C4K, 5C4L, 4N57, 4DT8, 4DT9, IVa G, K, S Enterococcus cassaliﬂavus 4DTA, 4DTB, 3SG8, 3SG9 I (a–b) St Acinetobacter baumannii 4EJ7, 4FEU, 4FEV, 4FEX, 4FEW APH(3”) III a St Enterococcus faecalis 2BKK APH(7) I a H Streptomyces hygroscopicus APH(4) I-(a–b) H Escherichia coli 3W0O, 3TYK, 3W0M, 3W0N APH(6) I-(a–d) St Streptomyces griseus APH(9) I-(a–b) Sp Legionella pneumophila 3I0O, 3I0Q, 3I1A, 3Q2M Abbreviations: A, amikacin; D, dibekacin; H, hygromycin; I, isepamicin; G, gentamicin; K, kanamycin; L, lividomycin; N, netilmicin; Ne, neomycin; P, paromomycin; R, ribostamycin; S, sisomicin; T, tobramycin; Sp, spectinomycin; St, streptomycin. Molecules 2018, 23, 284 4 of 18

ANTs are the smallest family of AMEs, with four crystalized structures, ANT(200), ANT(300), ANT(40), and ANT(60), as can be seen in the following table (Table3)[30–35].

Table 3. O-nucleotidyltransferase (ANT)-modifying enzymes.

Enzyme Resistance Proﬁle Bacterial Host Pdb Number ANT(200) K, T, G, D, S Pseudomonas aeruginosa 4XJE, 5CFT, 5CFS, 5CFU Klebsiella pneumoniae 4WQK, 4WQL, 5KQJ ANT(300) St, Sp Salmonella enterica 4CS6, 5G4A ANT(40) K, Ne, T, A, D, I Pseudomonas aeruginosa 4EBJ, 4EBK Staphylococcus aureus 1KNY ANT(6) St Bacillus subtilis 2PBE, 1B87 ANT(9) Sp Enterococcus avium Abbreviations: A, amikacin; D, dibekacin; I, isepamicin; G, gentamicin; K, kanamycin; Ne, neomycin; S, sisomicin; T, tobramycin; Sp, spectinomycin; St, streptomycin.

Genes coding for ANT enzymes are found in plasmids, transposons, and chromosomes. ANT(200)- Ia is able to modify gentamicin, tobramycin, dibekacin, sisomicin, and kanamycin [30], while ANT(40)-Ia is active against almost all aminoglycosides and other aminoglycosides with 40/400-OH groups [33]; ANT(300) inactivates streptomicyn and spectinomycin [32], while ANT(6) and ANT(9) can modify only streptomycin and spectomycin, respectively [35,36]. ANT(2”)-Ia and ANT(40)-Ia are clinically relevant proteins and only share 27% amino acid homology. ANT(40)-Ia is a dimeric enzyme with two active sites, and recognizes all nucleotides triphosphate and almost all 4,5 or 4,6-aminoglycosides except those from the streptomycin family [37,38]. The 3D structure of ANT(40)-Ia was the first one to be described, and its complex with kanamycin revealed that several active site residues interact via hydrogen bonds with the glucosamine and 2-deoxystreptamine moiety, but relatively fewer residues interact with the 30-aminoglucose sugar, this being important knowledge for the design of antibiotics or inhibitors. The antibiotic binding pocket is covered by negatively charged residues, where Glutamic 145 acts as a general base for the activation of the 40-OH group of kanamycin, thus preparing the antibiotic for the attack of the Mg-ATP stabilized by lysine 149, which facilitates the nucleophilic attack. Overall, the dominant role of electrostatics in aminoglycoside recognition, in combination with the enzyme anionic regions, confers to the protein/antibiotic complex a highly dynamic character. The kinetic mechanism is an ordered process, where the antibiotic binds first to the active site and later on to the Mg-ATP. Determination of the order of product release revealed that PPi is discharged first, followed by the AMP-aminoglycoside, all in agreement with an ordered Bi-Bi mechanism [39]. ANT(200)-Ia from Klebsiella pneumoniae has an ordered sequential mechanism, where the presence of the two substrates at the same time is essential (Mg-ATP binds before the aminoglycoside) [40–43]. The observed promiscuity of ANT(200)-Ia from Pseudomonas aeruginosa is not only due to binding cleft size, but also it can be controlled by ligand modulation on dynamic, disordered, and thermodynamic properties of ANT under cellular conditions [44]. ANT(60), a 37 kDa protein, transfers an adenyl group from ATP to the 60-hydroxy function on the aminoglycoside, thus leading to a sharp decrease in the drug affinity for its target RNA. The conformational behavior of streptomycin, both in the free and the protein-bound states, was studied by NMR experiments given that the 3D structure is in a free form [45,46]. The streptomycin is characterized by a high degree of flexibility in solution, but this equilibrium is clearly altered upon binding to the enzyme. ANT(60) showed a clear specificity for nucleotides that incorporate a purine ring, and regarding the aminoglycoside counterpart, it is very specific: it only recognizes streptomycin. In contrast, the 3D structure and the kinetic mechanism of ANT(9) has not been resolved so far. It was in 1986 when the first bifunctional enzyme was described, AAC(60)-Ie-APH(200)-Ia from Enterococcus faecalis [47]; a structure-function analysis with various aminoglycosidic substrates revealed an enzyme with a broad specificity in both enzymatic activities catalyzing N- and O-acetylation. The AAC(60)-APH(2”) from Staphylococcus aureus enzyme confers resistance to gentamicin, kanamycin, tobramycin, and, when overexpressed, to amikacin, presenting a dramatic negative impact on clinical Molecules 2018, 23, 284 5 of 18 therapy [48]. Later on, other bifunctional enzymes were also described: ANT(300)-Ii-AAC(60)-IId from Serratia marcescens [49] and AAC(3)-Ib-AAC(60)-Ib from Pseudomonas aeruginosa [50] (Table4).

Table 4. Bifunctional modifying enzymes.

Enzyme Resistance Proﬁle Bacterial Source Pdb Number Enterococcus faecalis AAC(60)-Ie-APH(200)-IVa G, K, T, A Staphylococcus aureus 4ORQ APH(200)-Id-APH(200)-IVa K, G, T, S, D Enterococcus casseliﬂavus 4DBX, 4DE4, 4DFB APH(200)-Ia-APH(60)-Ie K, G, T, S, D, St Staphylococcus aureus 5IQF ANT(3)-Ib-AAC(60)-IId T, A, N, D, S, K, St, Sp Serratia marcescens AAC(3)-Ib-AAC(60)-Ib G, S, F, T, A, N, D, K, I Pseudomonas aeruginosa Abbreviations: A, amikacin; D, dibekacin; F, fortimicn; I, isepamicin; G, gentamicin; K, kanamycin; N, netilmicin; S, sisomicin; T, tobramycin; Sp, spectinomycin; St, streptomycin.

The adenyltransferase domain of ANT(300)-Ii-AAC(60)-IId appears to be highly specific for the aminoglycoside, while the acetyltransferase domain shows a broad substrate tolerance [51]. Kinetic analysis of the mechanism of ANT(300)-Ii points towards a Theorell–Chance type of reaction, with ATP binding to the active site before the aminoglycoside, and once the reaction has occurred, the product is the last to be released. However, the AAC(60)-IId domain follows an ordered Bi-Bi mechanism in which the antibiotic is the first to bind in the active site, and CoA is released prior to the modified aminoglycoside. Also, structural studies have revealed that this enzyme contains dynamic segments that modulate before and after aminoglycoside binding. In the case of AAC(3)-Ib-AAC(60)-Ib, both domains follow a sequential ordered kinetic mechanism in which the acetyl-CoA binds first to the active site, followed by the aminoglycoside, and the CoA is the last product to be released [52]. Despite this interesting behavior, to date only the crystal structure of some APH(2”) domains have been described.

3. Semi-Synthetic Aminoglycoside Derivatives The emergence of resistance towards the first generation of aminoglycosides led to increased efforts to identify similar antibiotics that were not susceptible to resistance [53–58]. From a conceptual point of view, one of the most simple and straightforward strategies to generate new derivatives that are not susceptible to enzymatic inactivation relies on the modification or elimination of those functional groups (OH/NH2) that can be altered by the enzyme. However, this should only be taken into consideration when such functional groups are not involved in key contacts with the receptor (16S rRNA) so that their biological activity is maintained. Tobramycin (30-deoxy-kanamycin B) showed high activity against strains expressing APH(30), and it is a good competitive inhibitor of this enzyme [59]. Dibekacin (30,40-dideoxy-kanamycin B) was the first rationally designed semi-synthetic aminoglycoside based on the 30 phosphorylation of kanamycin B, being effective against Staphylococcus and Pseudomonas, but unfortunately still susceptible to ANT(200)[60] and the bifunctional enzyme AAC(60)-APH(200)[61]. In contrast, fewer examples of deoxygenation on neomycin-related antibiotics have been reported [62]. The number and positions of amino groups play a significant role in the aminoglycoside activity. Methylation of the N-60 and N-300 positions in kanamycin B yielded a compound (60,300-di-N-methyl-kanamycin B) that displayed activity against resistant strains, but showed a weaker bactericidal activity than its natural antibiotic [63] (Figure2). The observation that butirosin, a N-1 derivative of the 2-deoxystreptamine moiety, is poorly modified by APH(3’), prompted the synthesis of several kanamycin and neomycin derivatives bearing an N-1 modification. Kanamycin derivatization at this position with a (S)-4-amino-2-hydroxybutyryl (AHB) group gave rise to amikacin, which has proven to be a very effective aminoglycoside antibiotic in clinic [64] (Figure3). This compound was able to arrest the cell growth of strains expressing the AAC(1), APH(30)-Ia, and ANT(20) enzymes [65]. The N-1 modification of dibekacin with an AHB group provided arbekacin, which was used in clinic against Pseudomonas and Staphylococcus [66] (Figure3). Arbekacin is a substrate of the bifunctional enzyme AAC(60)-APH(2”), but cannot be modified by the Molecules 2018, 23, 284 6 of 18

Molecules0 20182018,, 2323,, xx FOR0FOR PEERPEER REVIEWREVIEW 66 ofof 1818 APH(3Molecules) or 2018 ANT(4, 23, x FOR) present PEER REVIEW in some Methicillin-resistant Staphylococcus aureus (MRSA) strains6 [ of67 18,68 ]. The trend of antibacterial activity of these aminoglycoside derivatives with an N-1 AHB group is (MRSA)(MRSA) strainsstrains [67,68].[67,68]. TheThe trendtrend ofof antibacterialantibacterial activity of these aminoglycosidecoside derivativesderivatives withwith similar to a 30-deoxygenation in the antibiotic skeleton. Some other functionalities at the N-1 position an(MRSA) N-1-1 AHBAHB strains groupgroup [67,68]. isis similarsimilar The tototrend aa 33′′ -deoxygenation-deoxygenationof antibacterial in inactivity thethe antibioticantibiotic of these skeleton.skeleton. aminogly SomeSomecoside otherother derivatives functionalitiesfunctionalities with wereatan the alsoN-1 N AHB-1 studied,-1 positionposition group but wereiswere weresimilar alsoalso much to studied,studied, a 3 less′-deoxygenation but activebut werewere against muchmuch in theP. lessless aeruginosa. antibiotic activeactive againstagainst skeleton. P. aeruginosa.Some other functionalities at the N-1 position were also studied, but were much less active against P. aeruginosa.

0 00 FigureFigure 2. 2.Structures StructuresStructures of ofof Kanamycin KanamycinKanamycin B B and and 6’,3’’-di-N-Methyl6’,3’’-di-N-Methyl 6 ,3 -di-N-Methyl KanamycinKanamycin Kanamycin B.B. B. Figure 2. Structures of Kanamycin B and 6’,3’’-di-N-Methyl Kanamycin B.

FigureFigure 3. 3.Structure StructureStructure ofofof kanamycinkanamycin classclass ofof of aminoglycosides.aminoglycosides. aminoglycosides. Figure 3. Structure of kanamycin class of aminoglycosides. Further elaboration upon these N-1 derivatives yielded two new derivatives, JLN027 and etimicin, FurtherFurther elaboration elaboration upon upon thesethese NN-1-1 derivativesderivatives yielded yielded two two new new derivatives, derivatives, JLN027 JLN027 and and etimicin, etimicin, that areFurther more elaboration effective than upon gentamicin, these N-1 amikacinderivatives, and yielded tobramycicn two new [69–71], derivatives, but still JLN027 have andencountered etimicin, thatthat are are more more effective effective than than gentamicin, gentamicin, amikacin,amikacin, and tobramycicn tobramycicn [69–71], [69–71 ],but but still still have have encountered encountered resistancethatresistance are more toto somesome effective MRSAMRSA than strainsstrains gentamicin, [72–75][72–75] amikacin (Figure(Figure , 4). 4).and tobramycicn [69–71], but still have encountered resistanceresistance to to some some MRSA MRSA strains strains [ 72[72–75]–75] (Figure (Figure4 4).).

Figure 4. AminoglycosidesAminoglycosides withwith N-1-1 kanamycinkanamycin derivatives.derivatives. FigureFigure 4. 4.Aminoglycosides Aminoglycosides with N-1-1 kanamycin kanamycin derivatives. derivatives.

Molecules 2018, 23, x FOR PEER REVIEW 7 of 18

Modification of the N-6′, N-2′, N-3, and N-1 positions through the synthesis of deaminated aminoglycosides (neamine or kanamycin B) has shown a dramatic loss of enzymatic susceptibility towards APH(3′)-Ia/IIa while still retaining the antibacterial activity, probably due to a decreased overall positive charge on the antibiotic. AMEs do not show activity against pyranmycins, which are Moleculesa group2018 of, 23 semisynthetic, 284 derivatives of aminoglycosides that differ from regular aminoglycosides7 in of 18 that they contain a pyranose in place of a furanose at the O-5 position of neamine, giving rise to a Molecules 2018, 23, x FOR PEER REVIEW 7 of 18 derivative with low cytotoxicity (TC005 derivative) [76] (Figure 5). In order to improve the 0 0 derivatives,Modiﬁcation Chang’s of the groupN-6 , Nprepared-2 , N-3, some and N3-1′,4′-dideoxygenated positions through pyranmycin the synthesis and of kanamycin deaminated Modification of the N-6′, N-2′, N-3, and N-1 positions through the synthesis of deaminated aminoglycosidesderivatives giving (neamine resistance or kanamycinto AME (RR501) B) has [77, shown78] (Figure a dramatic 5). These loss simplified of enzymatic skeletons susceptibility proved aminoglycosides (neamine or kanamycin B) has shown a dramatic loss of enzymatic susceptibility highly effective0 against several pathogenic bacterial strains, such as P. aeruginosa and S. aureus. towardstowards APH(3 APH(3)-Ia/IIa′)-Ia/IIa whilewhile stillstill retainingretaining the the antibacterial antibacterial activity, activity, probably probably due due to toa adecreased decreased overalloverall positive positive charge charge on on the the antibiotic. antibiotic. AMEs AMEs do do not notshow show activityactivity against pyranmycins, which which are are a groupa group of semisynthetic of semisynthetic derivatives derivative ofs aminoglycosides of aminoglycosides that that differ differ from from regular regular aminoglycosides aminoglycosides in in that theythat contain they contain a pyranose a pyranose in place in of place a furanose of a furanose at the O at-5 positionthe O-5 position of neamine, of neamine, giving rise giving to a rise derivative to a withderivative low cytotoxicity with low (TC005 cytotoxicity derivative) (TC005 [76 ]deriva (Figuretive)5). In[76] order (Figure to improve 5). In theorder derivatives, to improve Chang’s the 0 0 groupderivatives, prepared Chang’s some 3 ,4group-dideoxygenated prepared some pyranmycin 3′,4′-dideoxygenated and kanamycin pyranmycin derivatives and giving kanamycin resistance to AMEderivatives (RR501) giving [77, 78resistance] (Figure to5). AME These (RR501) simpliﬁed [77,78] skeletons (Figure proved5). These highly simplified effective skeletons against proved several pathogenichighly effective bacterial against strains, several such pathogenic as P. aeruginosa bacterialand strains,S. aureus such. as P. aeruginosa and S. aureus.

Figure 5. Structure of kanamycin B analogs (Pyranmycin).

Selective modification of the N-3″ amino of kanamycin A into a guanidine group gave N-3″- guanidino kanamycin A (Figure 6), which presented protection against inactivation performed by ANT(4′), APH(3′), and AAC(6′), while maintaining its antibiotic activity [79]. The protecting effect of the guanidine group at the 3″-position was rationalized in terms of a binding hindrance with the respective inactivating enzymes, which presumably does not take place within the A-site. Plazomicin (PLZ, formerly ACHN-490) represents another example of amino modification, FigureFigure 5. 5.Structure Structure ofof kanamycinkanamycin B analogs (Pyranmycin). (Pyranmycin). where the N-6’, N-1, and N-3″ positions have been substituted with different ramifications (Figure 6). This aminoglycoside is not affected by any known AME except for AAC(2′), making it one of the few SelectiveSelective modiﬁcation modification of of the theN N-3-300″ aminoamino of kanamycin A A into into aa guanidine guanidine group group gave gave N-3N″-3- 00- examples that is currently in clinical use, retaining activity against most of the clinical isolates guanidinoguanidino kanamycin kanamycin A A (Figure (Figure6 ),6), which which presented presented protectionprotection against inactivation inactivation performed performed by by (Minimum Inhibitory Concentration, MIC 4 μg/mL) [80–82]. These new aminoglycosides constitute ANT(4ANT(40),′ APH(3), APH(30),′), and and AAC(6 AAC(60′), while maintaining maintaining its its antibiotic antibiotic activity activity [79]. [79 The].The protecting protecting effect effect of our most promising defense alternative for the treatment of resistant MRSA strains. PLZ is currently the guanidine group at the 3″00-position was rationalized in terms of a binding hindrance with the of thein Phase guanidine 3 clinical group trials at for the patients 3 -position with wasbloodstream rationalized infections in terms or nosocomial of a binding pneumonia hindrance and with it is the respective inactivating enzymes, which presumably does not take place within the A-site. respectivean important inactivating new weapon enzymes, in the which pipeline presumably to fight antibiotic does not resistance. take place within the A-site. Plazomicin (PLZ, formerly ACHN-490) represents another example of amino modification, where the N-6’, N-1, and N-3″ positions have been substituted with different ramifications (Figure 6). This aminoglycoside is not affected by any known AME except for AAC(2′), making it one of the few examples that is currently in clinical use, retaining activity against most of the clinical isolates (Minimum Inhibitory Concentration, MIC 4 μg/mL) [80–82]. These new aminoglycosides constitute our most promising defense alternative for the treatment of resistant MRSA strains. PLZ is currently in Phase 3 clinical trials for patients with bloodstream infections or nosocomial pneumonia and it is an important new weapon in the pipeline to fight antibiotic resistance.

00 FigureFigure 6. 6.Structure Structure of of the theN N-3-3’’-Guanidino-Guanidino Kanamyc Kanamycinin A A and and Plazomicin. Plazomicin.

PlazomicinThe aminosugar (PLZ, formerly ring in aminoglycosides ACHN-490) represents provides another these examplemolecules of with amino high modiﬁcation, affinity for wherethe theprokaryoticN-60, N-1, A-site, and N since-300 positions it penetrates have deeply been into substituted the major with groove, different thus displacing ramiﬁcations A1492 (Figure of 16S6 ). ThisrRNA, aminoglycoside giving the ribosome is not affected a continuous by any “on” known state during AME exceptthe translation for AAC(2 process.0), making Structural it one studies of the fewof examplesthe interactions that is between currently aminoglycosides in clinical use, retainingand the RNA activity or AMEs against involved most of in the their clinical enzymatic isolates

(Minimum Inhibitory Concentration, MIC 4 µg/mL) [80–82]. These new aminoglycosides constitute our most promisingFigure defense 6. Structure alternative of the for N-3’’-Guanidino the treatment Kanamyc of resistantin A and MRSA Plazomicin. strains. PLZ is currently in Phase 3 clinical trials for patients with bloodstream infections or nosocomial pneumonia and it is an importantThe newaminosugar weapon ring in the in pipelineaminoglycosides to ﬁght antibiotic provides resistance.these molecules with high affinity for the prokaryoticThe aminosugar A-site, since ring it in penetrates aminoglycosides deeply into provides the major these groove, molecules thus displacing with high A1492 afﬁnity of for 16S the prokaryoticrRNA, giving A-site, the since ribosome it penetrates a continuous deeply “on” into state the during major groove, the translation thus displacing process. Structural A1492 of 16S studies rRNA, givingof the the interactions ribosome abetween continuous aminoglycosides “on” state during and the the RNA translation or AMEs process. involved Structural in their studies enzymatic of the

Molecules 2018, 23, 284 8 of 18 interactionsMolecules 2018 between, 23, x FOR aminoglycosides PEER REVIEW and the RNA or AMEs involved in their enzymatic inactivation8 of 18 haveMolecules been 2018 performed, 23, x FOR PEER to identify REVIEW the molecular nature of these recognition processes. The8 of 4,5 18 or inactivation have been performed to identify the molecular nature of these recognition processes. The 4,6-disubstituted aminoglycosides speciﬁcally bind to the A-site with several conserved contacts, 4,5inactivation or 4,6-disubstituted have been aminoglycosidesperformed to identify specifically the mo bindlecular to thenature A-site of thesewith severalrecognition conserved processes. contacts, The the majority of them corresponding to the pseudo-disaccharide neamine [83–85]. The synthesis the4,5 ormajority 4,6-disubstituted of them corresponding aminoglycosides to specificallythe pseudo-disaccharide bind to the A-site neamine with several [83–85]. conserved The synthesis contacts, of of hybrid (4,5/4,6-) aminoglycosides, based on the superimposition of crystallographic structures, hybridthe majority (4,5/4,6-) of them aminoglycosides, corresponding based to the onpseu thedo-disaccharide superimposition neamine of crysta [83–85].llographic The synthesis structures, of originated a new family of 4,5,6-aminoglycoside derivatives with a better prognosis against AMEs originatedhybrid (4,5/4,6-) a new aminoglycosides,family of 4,5,6-aminoglycoside based on the derivatives superimposition with a betterof crysta prognosisllographic against structures, AMEs thanthanoriginated the the corresponding corresponding a new family natural natural of 4,5,6-aminoglycoside antibiotics antibiotics [[86]86] (Figure(Fig derivativesure7 7).). Another with approach approacha better prognosis ba basedsed on on structuralagainst structural AMEs data data showedshowedthan the that thatcorresponding the the antibiotic antibiotic natural scaffold scaffold antibiotics presents presents [86] different differ (Figenture conformations conformations 7). Another approach when when bound bound based to toon the the structural A-site A-site or ordata to to the AME.theshowed AME. This that realization This the realization antibiotic translated scaffoldtranslated into presents theinto design the differ design ofent a conformations conformationallyof a conformationally when locked bound locked aminoglycoside to aminoglycoside the A-site or to that retainedthatthe AME.retained the antibioticThis the realizationantibiotic activity activity translated but but was was into not not susceptiblethe susceptible design of to toa enzymatic enzymaticconformationally modiﬁcationmodification locked [87,88] [87 aminoglycoside,88 ](Figure (Figure 7).7 ). that retained the antibiotic activity but was not susceptible to enzymatic modification [87,88] (Figure 7).

FigureFigure 7. 7.Structure Structure of of the the4,5,6-Neomycin 4,5,6-Neomycin derivative and and constrained constrained neomycin. neomycin. Figure 7. Structure of the 4,5,6-Neomycin derivative and constrained neomycin. In order to increase the receptor binding affinity, aminoglycoside dimers were conceived and In order to increase the receptor binding afﬁnity, aminoglycoside dimers were conceived and synthesized.In order Crystallographic to increase the recept studiesor indicatedbinding affinity, that neither aminoglycosi the 6″-OHde groupdimers in were kanamycin conceived nor andthe synthesized. Crystallographic studies indicated that neither the 6”-OH group in kanamycin nor the 5-OHsynthesized. group inCrystallographic neamine were essential studies indicatedfor RNA binding; that neither thus, the they 6″ -OHwere groupselected in askanamycin anchoring nor points the 5-OH group in neamine were essential for RNA binding; thus, they were selected as anchoring points for5-OH the group synthesis in neamine of various were dimers essential equipped for RNA with binding; different thus, linkers they [89] were (Figure selected 8). asKanamycin anchoring dimers points for the synthesis of various dimers equipped with different linkers [89] (Figure8). Kanamycin dimers (Figurefor the synthesis 8) exhibited of various the same dimers activity equipped than the with pa differentrent compound, linkers [89] but (Figure were also 8). Kanamycininactivated dimersby the (Figuresame8 AMEs) exhibited (ANT(4 the′), sameAPH(3 activity′), and AAC(6 than the′)). In parent the case compound, of the neamine but weredimers also [90] inactivated (Figure 8), these by the (Figure 8) exhibited0 the same0 activity than the0 parent compound, but were also inactivated by the samepresentedsame AMEs AMEs the (ANT(4 (ANT(4 same),′ ),biological APH(3APH(3′),), andactivity and AAC(6 AAC(6 (lower′)).)). In ththe Inan thecase neomycin case of the of neamine theor kanamycin), neamine dimers dimers [90] but (Figure as [90 well] (Figure8), thesewere8 ), theseinactivatedpresented presented the by the samethe same AMEs.biological biological However, activity activity these(lower (lower compounds than than neomycin neomycin are interestingor orkanamycin), kanamycin), probes but for but as strains aswell well were not were inactivatedexpressinginactivated by AMEs,by the the AMEs. since AMEs. However,they However,exhibit these higher these compounds affinity compounds for are the interesting receptorare interesting RNA. probes probes for strains for notstrains expressing not AMEs,expressing since theyAMEs, exhibit since higherthey exhibit afﬁnity higher for the affinity receptor for the RNA. receptor RNA.

Figure 8. Structure of the Neamine and kanamycin dimers. FigureFigure 8. 8.Structure Structure ofof thethe NeamineNeamine and kanamycin kanamycin dimers. dimers.

Molecules 2018,, 2323,, 284x FOR PEER REVIEW 9 of 18

The molecular recognition of aminoglycosides with their receptors, in most cases, is stabilized by a Thesignificant molecular number recognition of salt-bridges of aminoglycosides and polar withcontacts, their but receptors, it seems in mostto be cases, promoted is stabilized by CH/ byπ astacking significant interactions number ofinvolving salt-bridges aromatic and polar residues contacts, of the but protein/RNA it seems to bewith promoted the antibiotic. by CH/ So,π stacking the role interactionsplayed by CH/ involvingπ stacking aromatic interactions residues ofin thethe protein/RNA molecular recognition with the antibiotic. of aminoglycosides So, the role playedby its byreceptors CH/π stackinghas been interactions evaluated inand the molecularproven to recognitionbe significant of aminoglycosides [91]. The modification by its receptors of natural has beenaminoglycosides evaluated and is a proven promising to be direction significant to search [91]. The for modificationnovel aminoglycosides of natural aminoglycosideswith potency against is a promisingresistant strains. direction to search for novel aminoglycosides with potency against resistant strains. Recently, CrichCrich etet al.al. preparedprepared aa semisyntheticsemisynthetic paromomycinparomomycin derivativederivative that displaysdisplays similarsimilar 0 00 antibacterial activityactivity toto the parent compoundcompound againstagainst clinicalclinical strainsstrains ofof E.E. coli and MRSA (ANT(4′,4″), 0 00 0 APH-(3APH-(3′,5″),), and and AAC(6 AAC(6′)).)). The The enhanced enhanced activity activity on on the the ribosome has been demonstrated to dependdepend onon 0 thethe equatorialequatorial hydroxyl group atat thethe 66′-position, thereby providing support for the crystallographically derived models ofof aminoglycoside–ribosomeaminoglycoside–ribosome interactionsinteractions [[92]92] (Figure(Figure9 9).).

OH O O O HO NH H N NH2 H2N O O O NH2 HO O 2 OH OH O OH NH2

O H2N 2 OH O OH Paromomycin derivative Paromomycin derivative Figure 9. Structure of the Paromomycin derivative4. Inhibitors of Aminoglycoside Modifying Enzymes. Figure 9. Structure of the Paromomycin derivative4. Inhibitors of Aminoglycoside Modifying Enzymes.

Another wayway to to evade evade aminoglycoside aminoglycoside resistance resistance by AMEs by AMEs is through is through the use ofthe speciﬁc use of inhibitors specific ofinhibitors these enzymes. of these enzymes. Some attempts Some attempts have been have made been tomade produce to produce inhibitors inhibitors of one of orone more or more of the of AMEsthe AMEs [93– [93–95].95]. Bi-substrate Bi-substrate analogs analogs have have been been synthesized synthesized as as inhibitors inhibitors of of AAC AAC and and ANT/APH ANT/APH enzymes based on thethe proposedproposed kinetickinetic mechanism.mechanism. For instance, the crystallographiccrystallographic structure of AAC(6AAC(60′)-Ii)-Ii inin complexcomplex with with kanamycin kanamycin B-CoA B-CoA provided provided a new a new insight insight into into chemical chemical optimization optimization [96] (Figure[96] (Figure 10). This10). This adduct adduct is a goodis a good inhibitor inhibitor of the of AACthe AAC family, family, but unfortunatelybut unfortunately does does not havenot have any bacterialany bacterial activity activity due todue a poorto a poor permeability permeability of the of cell the wallcell wall [97]. [97].

Figure 10. Structure of Kanamycin –CoA inhibitor to AME.

Using thethe samesame approach,approach, aa nucleotide–aminoglycosidenucleotide–aminoglycoside complexcomplex forfor thethe inhibitioninhibition ofof APHs/ANTsAPHs/ANTs has been described (Figure 1111).). Such a tethered bi-substratebi-substrate design contains a neamine core with the 30′-OH linked to adenosine via a non-hydrolyzable linkerlinker inin placeplace ofof thethe triphosphatetriphosphate groupgroup [[98].98].

Molecules 2018, 23, 284 10 of 18 Molecules 2018, 23, x FOR PEER REVIEW 10 of 18

Figure 11. Structure of nucleotide-neamine complex as inhibitor of APHs and ANTs. Figure 11. Structure of nucleotide-neamine complex as inhibitor of APHs and ANTs.

Regarding the the modification modiﬁcation of discrete of discrete functional functional groups groups targeted targeted by enzymatic by enzymatic resistance, resistance, different aminoglycosidedifferent aminoglycoside analogs have analogs been have synthesized, been synthesized, which turn which into suicide turn into inactivators suicide inactivators upon enzymatic upon 0 phosphorylation.enzymatic phosphorylation. Such is the Suchcase of is the2-nitro- case2’-deaminokanamycin, of 2-nitro-2 -deaminokanamycin, a good inhibitor a good of inhibitorAPH(3′)-Ia of 0 0 andAPH(3 APH(3)-Ia′)-IIa and APH(3[99] (Figure)-IIa 12), [99] which (Figure generates 12), which in situ generates a nitro-alkene in situ aintermediate nitro-alkene susceptible intermediate to Michaelsusceptible addition to Michael by close addition nucleophilic by close residues, nucleophilic thus residues, rendering thus a covalent rendering intermediate a covalent intermediate that blocks thethat active blocks site the and active abolishes site and the abolishes activity (Figure the activity 12). (FigureAnother 12 interesting). Another exam interestingple is the example synthesis is the of 0 3synthesis′-ketokanamycin, of 3 -ketokanamycin, where the keto where group the is keto known group to exist is known in equilibrium to exist in with equilibrium its ketal withform, its so ketal that theform, phosphorylated so that the phosphorylated ketal can be transformed ketal can back be transformed into a keto form back by into eliminating a keto form a dibasic by eliminating phosphate, a 0 anddibasic then phosphate, it can further and re-regenerate then it can further the ketal. re-regenerate Interestingly, the ketal.both 3 Interestingly,′-ketal- and 2 both′-nitro-kanamycin 3 -ketal- and 0 0 derivatives2 -nitro-kanamycin can inactivate derivatives the APH(3 can inactivate′) in an irreversible the APH(3 manner.) in an irreversible Most probably, manner. these Most compounds probably, arethese inhibitors compounds of other are inhibitorskinases too. of other kinases too.

Figure 12. Structure and mode of action of the 2’-nitro-2’deaminokanamycin B. Figure 12. Structure and mode of action of the 2’-nitro-2’deaminokanamycin B.

Allen et al. reported that α-hydroxytropolone-hydroxytropolone plus thethe appropriateappropriate aminoglycosideaminoglycoside substrates were active against resistant bacteria possessing the adenylyltransferase phenotype phenotype [100]. [100]. Recently, Recently, α-hydroxytropolone derivativesderivatives have have also also been been described described as as good good competitive competitive inhibitors inhibitors of ATPof ATP in the in thebinding binding site site of ANT(2 of ANT(200)-Ia″)-Ia [101 [101]] (Figure (Figure 13). 13).

Figure 13. StructureStructure of hydroxyt hydroxytropoloneropolone derivatives.

Garneau-Tsodikova etet al.al. havehave reported reported a a sulfonamide sulfonamide scaffold scaffold that that served served as as a pharmacophorea pharmacophore to togenerate generate inhibitors inhibitors of AAC(2 of AAC(200) from″) Mycobacteriumfrom Mycobacterium tuberculosis tuberculosis, whose, upregulation whose upregulation causes resistance causes resistanceto the aminoglycosides to the aminoglycosides [102] (Figure [102] 14 ).(Figure 14).

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Figure 14. Structure of a sulfonamide asas inhibitorinhibitor ofof thethe AAC(2”).AAC(2’’).

GivenGiven thatthat AMEsAMEs shareshare commoncommon bindingbinding featuresfeatures andand thatthat manymany ofof themthem alsoalso bindbind peptidespeptides andand proteins,proteins, cationiccationic peptidespeptides couldcould serveserve asas leadlead moleculesmolecules inin thethe developmentdevelopment ofof newnew inhibitorsinhibitors ofof thesethese enzymes. Therefore, Therefore, cationic cationic peptides peptides have have been been tested tested as asinhibitors inhibitors of APH(3 of APH(3′)-IIIa,0)-IIIa, AAC(6 AAC(6′)-Ii, 0and)-Ii, andAAC(6 AAC(6′)-APH(20)-APH(2″), and00), andthe theresults results showed showed that that the the indolicidin indolicidin moiety moiety and and its its analogs analogs have anan inhibitoryinhibitory effecteffect againstagainst bothboth ACCACC andand APHAPH enzymes,enzymes, albeitalbeit byby different mechanisms.mechanisms. TheseThese peptides constituteconstitute thethe ﬁrstfirst exampleexample ofof broad-spectrumbroad-spectrum inhibitorsinhibitors ofof AMEs,AMEs, butbut unfortunatelyunfortunately nonenone ofof themthem showedshowed anyany inhibitory inhibitory effect effectin vivoin vivo.. Known Known inhibitors inhibitors of eukaryotic of eukaryotic protein protein kinases kinases have been have studied been toostudied to determine too to determine whether they whether were activethey againstwere active APH(3 against0)-IIIa andAPH(3 AAC(6′)-IIIa0)-APH(2 and AAC(600) because′)-APH(2 of the″) structuralbecause of relation the structural found betweenrelation found these enzymesbetween [these95]. enzymes [95]. SomeSome aminoglycosideaminoglycoside dimers have alsoalso beenbeen usedused asas inhibitorsinhibitors ofof thethe AMEs.AMEs. NeamineNeamine dimersdimers werewere investigatedinvestigated for for their their antibacterial antibacterial activity activity and their and capability their capability to inhibit to the inhibit action ofthe bifunctional action of AAC(6bifunctional0)-APH(2 AAC(600), and′)-APH(2 were″ proven), and were to be proven active againstto be active clinically against isolated clinically strains isolated of P. aeruginosastrains of. However,P. aeruginosa the. However, synthesis the of one synthesis universal of one inhibitor universal for inhi all AMEsbitor for seems all AMEs still unreachable, seems still unreachable, since good inhibitionsince good relies inhibition on many relies mechanistic on many mechanistic and kinetic factorsand kinetic that canfactor varys that between can vary families between (AAC, families ANT, and(AAC, APH) ANT, and and between APH) typesand between too (i.e., types APH(3 too0)-I, (i.e., II). APH(3′)-I, II).

4.4. Combination TherapyTherapy TheThe use of of a a combination combination therapy therapy can can help help in insolving solving the theproblem problem of resistance of resistance by AMEs. by AMEs. This Thisapproach approach relies relies on the on rescue the rescue of original of original aminogly aminoglycosidescosides (gentamicin, (gentamicin, amikacin, amikacin, or etimicin) or etimicin) through throughthe co-administration the co-administration of the ofcorresponding the corresponding inhibitors inhibitors for foreach each enzyme. enzyme. Ideally, Ideally, the the adjuvantadjuvant compoundcompound (inhibitor)(inhibitor) would be targetedtargeted preferentiallypreferentially byby thethe resistanceresistance enzymes,enzymes, thusthus freeingfreeing thethe antibioticantibiotic toto bindbind thethe targettarget A-site.A-site. AnAn all-purposeall-purpose inhibitorinhibitor wouldwould bebe aa compoundcompound thatthat mimicsmimics thethe chargecharge andand shapeshape of of an an aminoglycoside aminoglycoside (common (common for for the the three three families families of AMEs), of AMEs), having having in mind in mind that allthat AMEs all AMEs have have a highly a highly negatively negatively charged charged surface surface in their in binding their binding sites such sites that such a must-have that a must-have feature shouldfeature beshould an overall be an positive overall charge.positive A charge. proof-of-concept A proof-of-concept for this strategy for this is strategy that the useis that of streptidine the use of alongstreptidine with streptomycinalong with streptomycin in the cell culturein the cell restores culture the restores activity the of activity the streptomycin of the streptomycin against ANT(6), against becauseANT(6), streptidinebecause streptidine competes competes for the binding for the site binding with thesitestreptomycin, with the streptomycin, acting as aacting “decoy as acceptor”a “decoy ofacceptor” the enzyme of the [103 enzyme] (Figure [103] 15 ).(Figure Thus, 15). streptidine Thus, stre couldptidine be a could good be starting a good compound starting compound for the design for the of moredesign efficient of more “decoy efficient acceptors” “decoy acceptors” of AMEs of targeting AMEs targeting streptomycin, streptomycin, or 2-deoxystreptamine or 2-deoxystreptamine for those for targetingthose targeting 4,5- or 4,5- 4,6-aminoglycosides or 4,6-aminoglycosides (Figure (Figure 15). 15). HybridHybrid antibioticsantibiotics havehave also also been been developed developed to to battle battle bacterial bacterial resistance resistance [104 [104–107].–107]. The The ability ability to slowto slow down down the the emergence emergence of of resistance resistance is is probably probably one one of of the the most most important important advantages advantages ofof hybridhybrid drugsdrugs [[108,109].108,109]. This strategy connects twotwo antibiotics thatthat havehave differentdifferent modesmodes ofof actionaction intointo aa singlesingle molecule.molecule. The main main difficulty difficulty so so far far has has been been to to fi findnd the the correct correct linker linker that that connects connects the the two two drugs drugs to toprovide provide better better inhibition inhibition on both on bothtargets. targets. The synthesis The synthesis of the Cipro-NeoB/Cipro-KanA, of the Cipro-NeoB/Cipro-KanA, which was which active wasagainst active a wide against range a wideof strains, range is ofone strains, of the most is one successful of the most examples successful of this examples approach of (Figure this approach 16). The (FigureMIC values 16). The of these MIC valueshybrids of thesewere hybridsthe same were against the same resistant against and resistant non-resistant and non-resistant strains (AAC(6 strains′), 0 0 0 00 (AAC(6APH(3′),), and APH(3 AAC(6), and′)/APH(2 AAC(6″) )/APH(2[110,111]. This)[110 kind,111]. of This aminoglyco kind of aminoglycosideside derivative, based derivative, on a hybrid based onstructure, a hybrid provides structure, a providespromising a promisingdrug with drugan unusual with an dual unusual mechanism dual mechanism of action, of a action, potent a potentprofile profileagainst against AMEs, AMEs,and reduced and reduced potential potential for generating for generating bacterial bacterial resistance. resistance.

Molecules 2018, 23, 284 12 of 18 MoleculesMolecules 20182018,, 2323,, xx FORFOR PEERPEER REVIEWREVIEW 1212 ofof 1818

FigureFigure 15. 15. StructureStructure of of the the streptomycin streptomycin and and streptidine streptidine asas “decoy“decoy acceptor”.acceptor”.

FigureFigure 16. 16. StructureStructure of of the the kanamycin kanamycin B-Cipro B-Cipro complex complex asas hybridhybrid antibiotic.antibiotic.

5.5. Conclusions Conclusions InIn thisthis this review,review, review, wewe we havehave have aimedaimed aimed toto covercover to cover thethe mostmost the rere mostlevantlevant relevant semi-syntheticsemi-synthetic semi-synthetic aminaminoglycosides,oglycosides, aminoglycosides, inhibitors,inhibitors, andinhibitors,and decoydecoy acceptorsacceptors and decoy ofof the acceptorsthe AMEs.AMEs. ofByBy the far,far, theAMEs.the mostmost By successfulsuccessful far, the chemicalchemical most successful approachapproach chemical toto modifymodify approach thethe naturalnatural to aminoglycosidesmodifyaminoglycosides the natural hashas aminoglycosides beenbeen thethe modificationmodification has been ofof the thethe modification NN-1/-1/NN-3-3″″ aminoamino of the groupsgroupsN-1/N -3 withwith00 amino thethe AHBAHB groups groupgroup with oror the aa guanidinoAHBguanidino group substituentsubstituent or a guanidino thatthat substituenthashas retainedretained that thethe has parentparent retained antibioticantibiotic the parent activity.activity. antibiotic AlkylationAlkylation activity. atat the Alkylationthe NN-6-6′′ positionposition at the ofNof-6 thethe0 position antibioticantibiotic of theresultedresulted antibiotic inin aa decrease resulteddecrease in ofof a affinityaffinity decrease forfor of resistantresistant affinity enzymes, forenzymes, resistant butbut enzymes, aa concomitantconcomitant but a concomitant decreasedecrease inin bacterialdecreasebacterial inactivityactivity bacterial hashas activity beenbeen observedobserved has been asas observed well.well. ChemicalChemical as well. deoxygenation Chemicaldeoxygenation deoxygenation ofof thethe 33′′/3/3 of′′,4,4′ the′-OH-OH 3 0 groups/3groups0,40-OH ofof aminoglycosidesgroupsaminoglycosides of aminoglycosides resultedresulted inin compounds resultedcompounds in compoundswithwith highhigh affinityaffinity with for highfor thethe affinity RNARNA andand for resistanceresistance the RNA against andagainst resistance MRSA.MRSA. Comparatively,againstComparatively, MRSA. Comparatively, fewerfewer inhibitorsinhibitors fewer oror decoydecoy inhibitors acceptorsacceptors or decoy ofof thethe acceptors AMEsAMEs havehave of the beenbeen AMEs described.described. have been described. OnOn thethe other other hand, hand, ANT(4ANT(4′0′)-Ia,)-Ia,)-Ia, ANT(2 ANT(2ANT(2″00″)-Ia,)-Ia, ANT(3 ANT(3″00″)(9),)(9), AAC(3)-IIIb,AAC(3)-IIIb, and andand APH(3 APH(3APH(3′0′)-IIIa)-IIIa)-IIIa have havehave been beenbeen describeddescribed as as highly highly dynamic dynamic enzymes enzymes and and display display prop propertiespropertieserties of of intrinsically intrinsically disordered disordered proteins proteins in in thethe absence absence of of the the aminoglycosidesaminoglycosides [112]. [[112].112]. The The degree degree of of promiscuity promiscuitypromiscuity by by AMEs AMEsAMEs is isis governed governed by byby the the dynamicsdynamics of of the the protein, protein, which which is is strongly strongly influenced influencedinfluenced by by the the ligand ligand and and the the cofactor, cofactor, as as well well as as its its interactioninteractioninteraction with with the the solvent, solvent, and and it it should should bebe takentaken intointo considerationconsideration inin thethethe designdesigndesign ofofof newnewnew drugs.drugs.drugs. StructuralStructural knowledge knowledge of of both both the the RNA- RNA- and and AME-aminoglycoside AME-aminoglycoside complexes complexes has has helped helped in in the the designdesign of of new new antibioticsantibiotics thatthat that cancan can escapeescape escape thethe the actionaction action ofof of thethe the enzymatienzymati enzymaticcc modificationmodification modification [113].[113]. [113 In].In Inorderorder order toto avoidtoavoid avoid oror inhibitinhibit or inhibit thethe activityactivity the activity ofof AMEs,AMEs, of AMEs, obtainingobtaining obtaining strustructuralctural structural andand mechanisticmechanistic and mechanistic informationinformation information isis ofof paramountparamount is of importance.paramountimportance. importance.

Acknowledgments:Acknowledgments: TheThe authors authors thank thank the the Spanish Spanish MinisterioMinisterio dede EconomíaEconomía Economía yy CompetitividadCompetitividad Competitividad (Grant(Grant (Grant MAT2015-MAT2015- MAT2015- 65184-C2-2-R and CTQ2016-79255-P) and the CSIC project iCOOPB20237 for their support. 65184-C2-2-R65184-C2-2-R andand CTQ2016-79255-P)CTQ2016-79255-P) andand thethe CSICCSIC projectproject iCOOPB20237iCOOPB20237 forfor theirtheir support.support. Conﬂicts of Interest: The authors declare no conﬂict of interest. ConflictsConflicts ofof Interest:Interest: TheThe authorsauthors declaredeclare nono conflictconflict ofof interest.interest.

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