Semisynthetic Aminoglycosides

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; zarate.sandra@usfx.bo
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; mariafeliz3@hotmail.com
3 Departmento de Química Bio-Orgánica, Instituto de Química Orgánica General (CSIC), Juan de la Cierva 3,
28006 Madrid, Spain; rbenito@iqog.csic.es (R.B.-A.); julia.revuelta@iqog.csic (J.R.)
* Correspondence: andres.g.santana@csic.es (A.G.S.); agatha.bastida@csic.es (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.
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Molecules 2018, 23, 284 2 of 18
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2. Understanding the Modifying Enzymes
2. Understanding the Modifying Enzymes
Tolerance towards antibiotics can be achieved through different mechanisms. However, the most
Tolerance
prevalent in the towards
clinic isantibiotics
that due can be achievedmodification
to enzymatic through different mechanisms.
that renders However, the of
aminoglycosides
most prevalent in the clinic is that due to enzymatic modification that renders aminoglycosides of
decreased affinity for their natural primary target, 16S rRNA [15–17]. There are three families
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 (Figure 1). The emergence of bifunctional
group also employing ATP (Figure 1). The emergence of bifunctional enzymes (i.e., APH(2″)-AAC(6′))
enzymes (i.e., APH(2 00 )-AAC(60 )) that are able to modify almost all aminoglycoside antibiotics presents
that are able to modify almost all aminoglycoside antibiotics presents a huge challenge that new
a huge challenge that
aminoglycosides willnew aminoglycosides
have to overcome [18]will have1).to overcome [18] (Figure 1).
(Figure
Figure1.1.Susceptible
Figure Susceptible positions
positions in Kan
Kan BB to
toAME
AMEmodification.
modification.
AACs
AACs areare foundboth
found bothininGram-positive
Gram-positive and and Gram-negative
Gram-negative bacteriabacteriaand andareareable
abletoto
reduce
reduce thethe
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 (Table 1). 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
cantomodulate the specificity for the antibiotic. For example, AAC(60 )-I and AAC(60 )-II share the
modify kanamycin but they differ in their propensity to acetylate amikacin or gentamicin C. The
capacity to modify
structural gene ofkanamycin
the AAC(3) but andthey
AAC(6′)differ in their propensity
is generally to acetylate amikacin
found on transposable elements [19].or gentamicin
AAC(3)
C. enzymes
The structural gene of the AAC(3) and AAC(6 0 ) is generally found on transposable elements [19].
were the first to be described to confer resistance to gentamicin (G), kanamycin (K),
AAC(3) enzymes
fortimicin (F), andwere the first to(T)
tobramycin be[20]
described
and cantoacetylate
confer resistance
either N/O togroups
gentamicin
of the(G), kanamycin (K),
aminoglycoside.
fortimicin (F), and tobramycin (T) [20] and can acetylate either N/O groups
Five AAC(2′) enzymes are encoded in Mycobacteria and one has been crystalized from Mycobacterium of the aminoglycoside.
Five AAC(20 ) enzymes
tuberculosis. AAC(2′) from are encoded in Mycobacteria
M. tuberculosis catalyticallyand andone has been crystalized
enthalpically favors thosefrom Mycobacterium
aminoglycosides
tuberculosis. 0
AAC(2 ) from M. tuberculosis catalytically and enthalpically
with amine/hydroxyl groups at the 2′ position and shows an increase favors those
in affinity foraminoglycosides
all antibiotics
withwhenamine/hydroxyl
CoA is present.groups This AME at the 20 position
exhibits a highand showstowards
tolerance an increase in affinity
the antibiotic for all (4,5-
molecule antibiotics
and
4,6-substitution), and it is thought to be modulated by water molecules
when CoA is present. This AME exhibits a high tolerance towards the antibiotic molecule (4,5- that mediate between
and aminoglycoside
4,6-substitution),and side
and chain carboxylate
it is thought groups
to be of the receptor.
modulated by water Moreover,
molecules side chain
that reorientation
mediate between
relative to the and
aminoglycoside apo-enzyme
side chain upon binding groups
carboxylate of the substrates also contributes
of the receptor. Moreover,toside thechain
plasticity of this
reorientation
relative to the apo-enzyme upon binding of the substrates also contributes to the plasticity of to
enzyme [21]. AAC(6′) enzymes are the most prevalent in clinical strains, conferring resistance this
amikacin
enzyme (A),
[21]. gentamicin
AAC(6 (G), kanamycin
0 ) enzymes are the most (K), neomycin
prevalent(N), dibekacin
in clinical (D), sisomicin
strains, conferring (S), isepamicin
resistance to
(I), and (A),
amikacin tobramycin
gentamicin(T) and (G),has an ordered
kanamycin kinetic
(K), mechanism.
neomycin Three-dimensional
(N), dibekacin (D), sisomicin(3D) (S),
structures of
isepamicin
(I),AAC(6′), AAC(3), and AAC(2′) have been described with a remarkable conservation of the overall
and tobramycin (T) and has an ordered kinetic mechanism. Three-dimensional (3D) structures of
structure, albeit with low amino acid sequence identity [22,23].
AAC(6 ), AAC(3), and AAC(20 ) have been described with a remarkable conservation of the overall
0
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 (Table 2). There are seven types of APHs with 30 kDa mass
resistance marker and its 3D structure has been described several times [25,26]. APH(3′)-IIIa
and high sequence
promiscuity seemshomology
to be governed (20–40%) in their C-terminal
by disordered elements that endadopt APH(30 )-IIIaconformations
[24]. well-defined is usually used
as when
a resistance marker andisits
the aminoglycoside 3D structure
bound has been
[27]. APH(3′)-IIa described
from several
Enterococcus times
faecalis [25,26].
is able APH(30 )-IIIa
to phosphorylate
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
APH(30 ) 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) Ia 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 (Table 3) [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] (Table 4).
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] (Figure 2).
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] (Figure 3). 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] (Figure 3).
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
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APH(3 0 ) or ANT(40 ) present in some Methicillin-resistant Staphylococcus aureus (MRSA) strains66[67,68].
Molecules 2018, 23, x FOR PEER REVIEW
of 18
of 18
The trend of
(MRSA) antibacterial
strains [67,68]. activity
The trend of
of these aminoglycoside
antibacterial activity of derivatives
these with an N-1
aminoglycoside AHB group
derivatives with is
(MRSA) strains 0 [67,68]. The trend of antibacterial activity of these aminoglycoside derivatives with
similar
(MRSA)
an N-1
N-1toAHB
astrains
3 -deoxygenation
group[67,68]. in
The to
is similar
similar the
trend
to antibiotic
of skeleton.
antibacterial theSome
activity other
of these functionalities
aminoglycoside at the N-1 position
derivatives with
an AHB group is aa 3′-deoxygenation
3′-deoxygenation in
in the antibiotic
antibiotic skeleton.
skeleton. Some other
Some other functionalities
functionalities
were
an also
N-1
at the
at the N-1studied,
AHB group
N-1 position but
is
position were were
were alsomuch
similar to a less
also studied,
studied, butactive against
3′-deoxygenation
but were
were muchin P.
the
much less aeruginosa.
antibiotic
less active skeleton.
active against
against P. Some other
P. aeruginosa.
aeruginosa. functionalities
at the N-1 position were also studied, but were much less active against P. aeruginosa.
Figure 2.Structures
Figure2.2.
Figure Structuresof
Structures ofKanamycin
of B and
Kanamycin B
Kanamycin 60 ,300 -di-N-MethylKanamycin
and 6’,3’’-di-N-Methyl
6’,3’’-di-N-Methyl Kanamycin
Kanamycin B.B.
B.
Figure 2. Structures of Kanamycin B and 6’,3’’-di-N-Methyl Kanamycin B.
Figure3.3.
Figure
Figure 3.Structure
Structureof
Structure of kanamycin
of kanamycin class
kanamycin class of
classof aminoglycosides.
ofaminoglycosides.
aminoglycosides.
Figure 3. Structure of kanamycin class of aminoglycosides.
Further elaboration
Further elaboration upon
upon these
these N-1
N-1 derivatives
derivatives yielded
yielded two
two new
new derivatives,
derivatives, JLN027
JLN027 and
and etimicin,
etimicin,
Further elaboration
Further elaboration upon
upon these
theseN-1
N-1 derivatives
derivatives yieldedtwo
yielded twonew
newderivatives,
derivatives,JLN027
JLN027andand etimicin,
etimicin,
that are
that are more
more effective
effective than
than gentamicin,
gentamicin, amikacin,
amikacin, and
and tobramycicn
tobramycicn [69–71],
[69–71], but
but still
still have
have encountered
encountered
that are
that more
are more effective than
effective gentamicin,
thanstrains
gentamicin, amikacin, and tobramycicn [69–71], but still have encountered
resistance
resistance to some
to some MRSA
MRSA strains [72–75]amikacin,
[72–75] and tobramycicn [69–71], but still have encountered
(Figure 4).
(Figure 4).
resistance to some MRSA strains [72–75] (Figure
resistance to some MRSA strains [72–75] (Figure 4). 4).
Figure 4.
Figure 4. Aminoglycosides
Aminoglycosides with
with N-1
N-1 kanamycin
kanamycin derivatives.
derivatives.
Figure4.4.Aminoglycosides
Figure Aminoglycosides with N-1 kanamycin
with N-1 kanamycinderivatives.
derivatives.
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
a group
Molecules of23,
2018, 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
derivative with low cytotoxicity (TC005 derivative) [76] (Figure 5). In order to improve the
Modification of thegroup 0
N-6 , N-2 0 , N-3, some
and N-1 positions throughpyranmycin
the synthesis
derivatives, Chang’s prepared 3′,4′-dideoxygenated andof kanamycin
deaminated
Modification of the N-6′, N-2′, N-3, and N-1 positions through the synthesis of deaminated
aminoglycosides
derivatives giving (neamine
resistanceor kanamycin
to AME (RR501) B) has shown
[77,78] a dramatic
(Figure loss
5). These of enzymatic
simplified susceptibility
skeletons proved
aminoglycosides 0
(neamine or kanamycin B) has shown a dramatic loss of enzymatic susceptibility
highly APH(3
towards effective)-Ia/IIa
againstwhile
severalstill
pathogenic
retainingbacterial strains, such
the antibacterial as P. aeruginosa
activity, probablyand dueS.to
aureus.
a decreased
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
overall positive charge on the antibiotic. AMEs do not show activity against pyranmycins, whichpyranmycins, whichare are a
group of semisynthetic derivatives of aminoglycosides that differ from regular aminoglycosides
a group of semisynthetic derivatives of aminoglycosides that differ from regular aminoglycosides in in that
they contain
that a pyranose
they contain in placeinofplace
a pyranose a furanose at the O-5
of a furanose at position of neamine,
the O-5 position giving rise
of neamine, to a rise
giving derivative
to a
with low cytotoxicity (TC005 derivative) [76] (Figure 5). In order to improve
derivative with low cytotoxicity (TC005 derivative) [76] (Figure 5). In order to improve the derivatives, Chang’s
the
group some 30 ,4group
preparedChang’s
derivatives,
0 -dideoxygenated pyranmycin and kanamycin derivatives giving resistance
prepared some 3′,4′-dideoxygenated pyranmycin and kanamycin
to AME (RR501)
derivatives [77,78]
giving (Figureto5).AME
resistance These simplified
(RR501) skeletons
[77,78] (Figure proved
5). Thesehighly effective
simplified against
skeletons several
proved
pathogenic bacterial
highly effective strains,
against several as P. aeruginosa
suchpathogenic S. aureus.
andstrains,
bacterial 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,Figure formerly ACHN-490) represents another example of amino modification,
Figure5.5.Structure
Structureof
of kanamycin
kanamycin B B analogs
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 by00any known AME except for AAC(2′), making it one of the few 00
Selective
Selective modificationofofthe
modification theN-3
N-3″ amino
amino of kanamycin
kanamycinAAinto intoaaguanidine
guanidinegroup
groupgave
gaveN-3″-
N-3 -
examples that is currently in clinical use, retaining activity against most of the clinical isolates
guanidino
guanidino kanamycinAA(Figure
kanamycin (Figure6), 6),which
which presented
presented protection
protection against
againstinactivation
inactivationperformed
performed byby
(Minimum
0 ), APH(3Inhibitory
0 ), and Concentration,
0 ), while MIC 4 μg/mL) [80–82]. These new aminoglycosides constitute
ANT(4′),
ANT(4 APH(3′), andAAC(6
AAC(6′), while maintaining
maintainingitsitsantibiotic activity
antibiotic [79].
activity TheThe
[79]. protecting effecteffect
protecting of
our most promising defense alternative for the treatment of resistant MRSA strains. PLZ is currently
the
of the guanidine
guanidine group
group at the
atfor 3″-position
00
thepatients
3 -position was rationalized in terms of a binding hindrance with the
in Phase 3 clinical trials with was rationalized
bloodstream in terms
infections of a binding
or nosocomial hindranceand
pneumonia with
it isthe
respective
respective inactivatingenzymes,
inactivating enzymes,which whichpresumably
presumably does
does not
not take
take place
place within
within the A-site.
the A-site.
an important new weapon in the pipeline to fight antibiotic resistance.
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 -Guanidino Kanamycin A and Plazomicin.

Figure 6.6.Structure
Figure theN-3
Structureofofthe N-3’’-Guanidino Kanamycin A and Plazomicin.
The aminosugar
Plazomicin ring in aminoglycosides
(PLZ, formerly ACHN-490) represents provides these example
another molecules of with
amino high affinity forwhere
modification, the
N-60 , N-1, A-site,
theprokaryotic and N-3 00 positions
since it penetrates
have deeply
been into the majorwith
substituted groove, thus displacing
different ramificationsA1492 of 16S 6).
(Figure
rRNA,
This giving the ribosome
aminoglycoside a continuous
is not affected by any“on” state during
known the translation
AME except for AAC(2process. Structural
0 ), making studies
it one of the
of the interactions between aminoglycosides and the RNA or AMEs involved in
few examples that is currently in clinical use, retaining activity against most of the clinical isolatestheir enzymatic
(Minimum Inhibitory Concentration, MIC 4 µg/mL) [80–82]. These new aminoglycosides constitute
our most promisingFiguredefense alternative
6. Structure of thefor the treatment
N-3’’-Guanidino of resistant
Kanamycin MRSA
A and strains. PLZ is currently
Plazomicin.
in Phase 3 clinical trials for patients with bloodstream infections or nosocomial pneumonia and it is an
Thenew
important aminosugar
weapon ringin the inpipeline
aminoglycosides provides resistance.
to fight antibiotic these molecules with high affinity for the
prokaryotic A-site, since
The aminosugar ring itinpenetrates deeply into
aminoglycosides the major
provides thesegroove, thus displacing
molecules with high A1492
affinityoffor
16Sthe
rRNA, giving
prokaryotic thesince
A-site, ribosome a continuous
it penetrates deeply “on”
intostate
the during the translation
major groove, process. Structural
thus displacing A1492 of 16Sstudies
rRNA,
of the
giving theinteractions
ribosome abetween
continuous aminoglycosides
“on” state duringand thetheRNA or AMEs
translation involved
process. in theirstudies
Structural enzymaticof the
Molecules 2018, 23, 284 8 of 18
Molecules 2018,
interactions 23, x FORaminoglycosides
between PEER REVIEW and the RNA or AMEs involved in their enzymatic inactivation 8 of 18
Molecules
have been2018, 23, x FOR PEER
performed REVIEW the molecular nature of these recognition processes. The8 of
to identify 4,518 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,
4,5 or 4,6-disubstituted
inactivation have been aminoglycosides specifically
performed to identify bind to the
the molecular A-site
nature with several
of these conserved
recognition contacts,
processes. The
the majority of them corresponding to the pseudo-disaccharide neamine [83–85]. The synthesis
the majority
4,5 or of them corresponding
4,6-disubstituted aminoglycosides to specifically
the pseudo-disaccharide neamine
bind to the A-site [83–85].
with several The synthesis
conserved contacts,of
of hybrid (4,5/4,6-) aminoglycosides, based on the superimposition of crystallographic structures,
hybrid (4,5/4,6-)
the majority of themaminoglycosides,
correspondingbased
to theon the superimposition
pseudo-disaccharide of crystallographic
neamine structures,
[83–85]. The synthesis of
originated a new family of 4,5,6-aminoglycoside derivatives with a better prognosis against AMEs
originated a new aminoglycosides,
hybrid (4,5/4,6-) family of 4,5,6-aminoglycoside
based on thederivatives with a better
superimposition prognosis against
of crystallographic AMEs
structures,
than thethe
than corresponding
originated corresponding natural
a new familynatural
antibiotics
antibiotics[86]
[86] (Figure
of 4,5,6-aminoglycoside (Figure 7). Another
7). Another
derivatives
approachbased
withapproach
basedonon
a better prognosis
structural
structural data
against AMEs
data
showed
showed that
than the thethe
that antibiotic scaffold
antibiotic
corresponding presents
scaffold
natural presents
antibiotics different
different
[86] conformations
(Figure conformations when
whenbound
7). Another approach basedtoto
bound the
on theA-site
A-siteoror
structural totothe
data
AME.
the This
AME.
showed realization
This
that translated
therealization into
translated
antibiotic scaffold the design
into the
presents of a conformationally
design conformations
different of a conformationally locked aminoglycoside
lockedtoaminoglycoside
when bound the A-site or to that
retained
that
the AME.the
retainedantibiotic activity
therealization
This but
antibiotic activity was not
but was
translated susceptible
intonot
thesusceptibleto
design of to enzymatic
a enzymatic modification
modification
conformationally [87,88]
[87,88]
locked (Figure
(Figure 7).
aminoglycoside 7).
that retained the antibiotic activity but was not susceptible to enzymatic modification [87,88] (Figure 7).
Figure 7.7.Structure
Figure Structureofofthe
the4,5,6-Neomycin derivativeand
4,5,6-Neomycin derivative andconstrained
constrainedneomycin.
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 orderCrystallographic studies indicated
to increase the receptor that neither
binding affinity, the 6″-OH group
aminoglycoside dimersinwere
kanamycin
conceivednorand
the
synthesized. Crystallographic studies indicated that neither the 6”-OH group in kanamycin nor the
5-OH group in
synthesized. neamine were essential
Crystallographic for RNA binding;
studies indicated that neitherthus,the
they weregroup
6″-OH selected
in as anchoringnor
kanamycin points
the
5-OH group in neamine were essential for RNA binding; thus, they were selected as anchoring points
for
5-OHthegroup
synthesis of various
in neamine dimers
were equipped
essential for RNA with different
binding; linkers
thus, they[89]
were(Figure 8).as
selected Kanamycin
anchoringdimers
points
for the synthesis of various dimers equipped with different linkers [89] (Figure 8). Kanamycin dimers
(Figure 8) exhibited
for the synthesis the same
of various activity
dimers than the
equipped with parent compound,
different but(Figure
linkers [89] were also inactivated dimers
8). Kanamycin by the
(Figure
same
(Figure
8) exhibited
AMEs the
(ANT(4′),
8) exhibited
same activity
APH(3′),
the
than
and AAC(6′)).
same activity
the parent
than theInparent
the case compound, but
of the neamine
compound,
were
dimers
but were
also
[90]
also
inactivated
(Figure 8),by
inactivated
by the
these
the
same AMEs (ANT(4 0 0 0
presented
same AMEsthe same), biological
(ANT(4′), APH(3
APH(3′),),and
andAAC(6′)).
AAC(6
activity (lower )). In the
In than
the case
of theofneamine
neomycin
case the neamine
dimersdimers
or kanamycin), but
[90] [90]
as
(Figure (Figure
well were 8),
8), these
these presented
inactivated
presented the the
by samesame
the AMEs. biological
However,
biological activity
activity these(lower
(lowercompounds than
than neomycinneomycin or kanamycin),
are interesting probes
or kanamycin), but as well
butforas strains
well were were
not
inactivated
expressing
inactivatedbyAMEs,
the the
by AMEs.
since However,
they However,
AMEs. exhibit these compounds
higher affinity
these forare
compounds theinteresting
receptor probes probes
RNA.
are interesting for strainsfor not expressing
strains not
AMEs, since they exhibit higher affinity for the receptor RNA.
expressing AMEs, since they exhibit higher affinity for the receptor RNA.
Figure 8. Structure of the Neamine and kanamycin dimers.

Figure8.8.Structure
Figure Structureof
of the
the Neamine and kanamycin
Neamine and kanamycindimers.
dimers.
Molecules 2018,
Molecules 2018, 23,
23, 284
x FOR PEER REVIEW 9 of 18
The molecular recognition of aminoglycosides with their receptors, in most cases, is stabilized
The molecular
by a significant recognition
number of aminoglycosides
of salt-bridges and polar with their but
contacts, receptors,
it seemsin most
to becases, is stabilized
promoted by CH/π by
astacking
significant number of salt-bridges and polar contacts, but it seems to be promoted
interactions involving aromatic residues of the protein/RNA with the antibiotic. So, the role by CH/π stacking
interactions
played by CH/π involving aromatic
stacking residues of
interactions in the
theprotein/RNA with the antibiotic.
molecular recognition So, the role played
of aminoglycosides by its
by CH/π stacking interactions in the molecular recognition of aminoglycosides
receptors has been evaluated and proven to be significant [91]. The modification of natural by its receptors has
been evaluated and proven to be significant [91]. The modification of natural
aminoglycosides is a promising direction to search for novel aminoglycosides with potency against aminoglycosides is a
promising direction to search for novel aminoglycosides with potency against resistant strains.
resistant strains.
Recently,
Recently, Crich
Crich etet al.
al. prepared
prepared aa semisynthetic
semisynthetic paromomycin
paromomycin derivative
derivative that
that displays
displays similar
similar
0 ,400 ),
antibacterial
antibacterial activity
activity to
to the
the parent
parent compound
compound againstagainst clinical
clinical strains of E.
strains of E. coli
coli and
and MRSA
MRSA (ANT(4
(ANT(4′,4″),
APH-(3 0 00
,5 ), and
and AAC(6′)).0
AAC(6 )).The The enhanced
enhanced activity
activity on
on the
the ribosome
ribosome hashas been
APH-(3′,5″), been demonstrated
demonstrated to to depend
depend on on
the equatorial hydroxyl group at the 6 0 -position, thereby providing support for the crystallographically
the equatorial hydroxyl group at the 6′-position, thereby providing support for the crystallographically
derived
derived models
models of of aminoglycoside–ribosome
aminoglycoside–ribosome interactions
interactions [92]
[92] (Figure
(Figure 9).
9).
OH
O O
HO NH22
H22N O
O NH22
HO
O OH
OH NH22
O
H22N
OH O OH
Paromomycin derivative
Figure9.9.Structure
Figure Structureof
ofthe
theParomomycin
Paromomycinderivative4. Inhibitors of
derivative4. Inhibitors of Aminoglycoside
AminoglycosideModifying
ModifyingEnzymes.
Enzymes.
Another way
Another waytoto evade
evade aminoglycoside
aminoglycoside resistance
resistance by AMEs
by AMEs is through
is through thespecific
the use of use ofinhibitors
specific
inhibitors
of of these enzymes.
these enzymes. Some attempts
Some attempts have
have been been to
made made to produce
produce inhibitors
inhibitors of oneofor
one or more
more of
of the
the AMEs
AMEs [93–95].
[93–95]. Bi-substrate
Bi-substrate analogs
analogs have
have been
been synthesized
synthesized asasinhibitors
inhibitorsofofAAC
AACandandANT/APH
ANT/APH
enzymes based on the the proposed
proposed kinetic
kinetic mechanism.
mechanism. For instance, the crystallographic
crystallographic structure of
AAC(6 0
AAC(6′)-Ii incomplex
)-Ii in complexwith
withkanamycin
kanamycinB-CoA B-CoA provided
provided a new
a new insight
insight intointo chemical
chemical optimization
optimization [96]
[96] (Figure
(Figure 10). This
10). This adduct
adduct is a good
is a good inhibitor
inhibitor ofAAC
of the the AAC family,
family, but unfortunately
but unfortunately doesdoes not have
not have any
any bacterial activity due to a poor permeability of the cell
bacterial activity due to a poor permeability of the cell wall [97]. wall [97].
AME.
Figure 10. Structure of Kanamycin –CoA inhibitor to AME.
Using the
Using the same
same approach,
approach, aanucleotide–aminoglycoside
nucleotide–aminoglycoside complex
complex for
for the
the inhibition
inhibition of
ofAPHs/ANTs
APHs/ANTs
has been described (Figure 11). Such a tethered bi-substrate design contains a neamine
has been described (Figure 11). Such a tethered bi-substrate design contains a neamine core withcore with the
the
3′-OH linked to adenosine via a non-hydrolyzable linker in place of the triphosphate group
30 -OH linked to adenosine via a non-hydrolyzable linker in place of the triphosphate group [98]. [98].
Molecules 2018, 23, 284 10 of 18
Molecules 2018,
Molecules 2018, 23,
23, x
x FOR
FOR PEER
PEER REVIEW
REVIEW 10 of
10 of 18
18
Figure 11. Structure of nucleotide-neamine complex as inhibitor of APHs and ANTs.

Figure 11.
Figure 11. Structure
Structure of
of nucleotide-neamine
nucleotide-neamine complex
complex as
as inhibitor
inhibitor of
of APHs
APHs and
and ANTs.
ANTs.
Regardingthe
Regarding
Regarding themodification
the modification
modification of of discrete
of discrete
discrete functional
functional
functional groups
groupsgroups
targeted
targetedtargeted by enzymatic
by enzymatic
by enzymatic resistance,
resistance,resistance,
different
different
different aminoglycoside
aminoglycoside
aminoglycoside analogs have
analogs analogs
have beenhave
been been synthesized,
synthesized,
synthesized, which turnwhich
which turn turn into
into suicide
into suicide suicide inactivators
inactivators
inactivators upon
upon enzymatic
upon enzymatic
enzymatic phosphorylation. Such is the case of 2-nitro-2 0 -deaminokanamycin, a good inhibitor of
phosphorylation. Such is the case of 2-nitro-2’-deaminokanamycin,
phosphorylation. Such is the case of 2-nitro-2’-deaminokanamycin, a good inhibitor of APH(3′)-Ia a good inhibitor of APH(3′)-Ia
APH(3 0 )-Ia and APH(30 )-IIa [99] (Figure 12), which generates in situ a nitro-alkene intermediate
and
and APH(3′)-IIa
APH(3′)-IIa [99] (Figure
[99] (Figure 12),
12), which
which generates
generates in in situ
situ aa nitro-alkene
nitro-alkene intermediate
intermediate susceptible
susceptible to to
susceptible
Michael to
addition Michael
by addition
close by close
nucleophilic nucleophilic
residues, thusresidues,
rendering thusa rendering
covalent
Michael addition by close nucleophilic residues, thus rendering a covalent intermediate that blocks a covalent
intermediate intermediate
that blocks
thatactive
the
the blockssite
active theand
site active
and site and
abolishes
abolishes the
theabolishes the activity
activity (Figure
activity (Figure 12). (Figure
12). Another12).
Another Anotherexample
interesting
interesting interesting
example example
is the
is the is the
synthesis
synthesis of
of
0
synthesis of 3 -ketokanamycin, where theis
3′-ketokanamycin,
3′-ketokanamycin, where the
where the keto
keto group
group isketo
known
known group
to is known
to exist
exist to exist inwith
in equilibrium
in equilibrium equilibrium
with ketal with
its ketal
its form,its
form, soketal
so that
that
the phosphorylated ketal can be transformed back into a keto form by eliminating a dibasic phosphate,a
form,
the so that
phosphorylated the phosphorylated
ketal can be ketal
transformed can be
back transformed
into a keto back
form into
by a keto
eliminating form
a by eliminating
dibasic phosphate,
dibasic
and
and then
then phosphate,
it can
it can further and re-regenerate
further then it can further
re-regenerate re-regenerate
the ketal.
the ketal. the ketal.
Interestingly,
Interestingly, Interestingly,
both 3′-ketal-
both 3′-ketal- both 30 -ketal- and
and 2′-nitro-kanamycin
and 2′-nitro-kanamycin
20 -nitro-kanamycin
derivatives
derivatives can derivatives
can inactivate
inactivate the can inactivate
the APH(3′)
APH(3′) in an
in the APH(30manner.
an irreversible
irreversible ) in an irreversible
manner. manner.
Most probably,
Most probably, Most
these
these probably,
compounds
compounds
these
are compounds
inhibitors of are
other inhibitors
kinases
are inhibitors of other kinases too. of
too. other kinases too.
Figure 12.
Figure Structure and
12. Structure and mode
mode of
of action
action of
of the
the 2’-nitro-2’deaminokanamycin
2’-nitro-2’deaminokanamycin B.
B.
Figure 12. Structure and mode of action of the 2’-nitro-2’deaminokanamycin B.
Allen et
Allen et al.
al. reported
reported that
that α-hydroxytropolone
α-hydroxytropolone plusplus the
the appropriate
appropriate aminoglycoside
aminoglycoside substrates
substrates
were active against resistant bacteria
were bacteria possessing
possessing the
the adenylyltransferase
adenylyltransferase phenotype
adenylyltransferase phenotype [100].
phenotype [100]. Recently,
[100]. Recently,
Recently,
α-hydroxytropolone
α-hydroxytropolone derivatives
α-hydroxytropolone derivatives
derivativeshave have also
havealso been
alsobeen described
beendescribed as
asas
described good
good competitive
competitive
good inhibitors
inhibitors
competitive of
of ATP
inhibitors ATP in
in the
of ATP in
the binding
binding site
site site
of ANT(2 00
of ANT(2″)-Ia
ANT(2″)-Ia [101]
)-Ia [101] (Figure
(Figure 13).
13).13).
the binding of [101] (Figure
Figure 13.
Structure of hydroxytropolone
Structure of hydroxytropolone derivatives.
derivatives.
Garneau-Tsodikova
Garneau-Tsodikova
Garneau-Tsodikova et etal.
et al.have
al. havereported
have reportedaaasulfonamide
reported sulfonamidescaffold
sulfonamide scaffoldthat
scaffold thatserved
that served
served as
asas aa pharmacophore
pharmacophore
a pharmacophore to
to
to generate
generate
generate inhibitors
inhibitors
inhibitors of AAC(2″)
of AAC(2″)
of AAC(2 from Mycobacterium
00 ) from Mycobacterium
from Mycobacterium tuberculosis,
tuberculosis,
tuberculosis, whose upregulation
whose upregulation
whose upregulation causes
causes
causes resistance
resistance
resistance to the
to the aminoglycosides
aminoglycosides
to the aminoglycosides [102] (Figure [102]
[102] (Figure 14).
14).(Figure 14).
Molecules 2018, 23, 284 11 of 18

Figure 14. Structure of
of aa sulfonamide
sulfonamide as
as inhibitor
inhibitor of
of the
the AAC(2”).
AAC(2’’).
Given that
Given that AMEs
AMEs share share common
common binding
binding features
featuresand and thatthat many
many of of them
them alsoalso bind
bind peptides
peptides andand
proteins, cationic
proteins, cationic peptides
peptides could
could serve
serve as
as lead
lead molecules
molecules in in the
the development
development of of new
new inhibitors
inhibitors ofof
these enzymes. Therefore,
these Therefore,cationic
cationicpeptides
peptideshave
have beenbeentested
tested as as
inhibitors
inhibitors of APH(30 )-IIIa,
of APH(3′)-IIIa, AAC(60and
AAC(6′)-Ii, )-Ii,
and AAC(60 )-APH(2
AAC(6′)-APH(2″), 00 ), and
and the the
results showed
results showed that thethe
that indolicidin
indolicidinmoietymoietyand andits
its analogs
analogs have an an
inhibitoryeffect
inhibitory effectagainst
againstboth both ACC
ACC and
and APH
APH enzymes,
enzymes, albeitalbeit by different
different mechanisms.
mechanisms. These These peptides
peptides
constitute the
constitute the first
first example
example of of broad-spectrum
broad-spectrum inhibitors
inhibitors of of AMEs,
AMEs, but but unfortunately
unfortunately none none ofof them
them
showedany
showed anyinhibitory
inhibitory effect
effect in vivo.
in vivo. Known
Known inhibitors
inhibitors of eukaryotic
of eukaryotic proteinprotein
kinaseskinases
have been have been
studied
studied
too too to determine
to determine whether they whether they against
were active were active APH(3 0
against APH(3′)-IIIa
)-IIIa and 0
AAC(6 )-APH(2 00
and AAC(6′)-APH(2″)
) because of the
because ofrelation
structural the structural relation found
found between between [95].
these enzymes these enzymes [95].
Some aminoglycoside
Some aminoglycoside dimers have have also
also been
been usedused asas inhibitors
inhibitors of of the
the AMEs.
AMEs. Neamine
Neamine dimersdimers
wereinvestigated
were investigatedforfor theirtheir antibacterial
antibacterial activity
activity and theirandcapability
their capability
to inhibit to theinhibit
action of the action of
bifunctional
AAC(6 0 )-APH(2
bifunctional 00 ), and were proven
AAC(6′)-APH(2″), and were
to beproven to be active
active against against
clinically clinically
isolated isolated
strains strains of
of P. aeruginosa.
P. aeruginosa.
However, the However,
synthesis the synthesis
of one of one
universal universal
inhibitor for inhibitor
all AMEsfor all AMEs
seems seems still unreachable,
still unreachable, since good
since goodrelies
inhibition inhibition
on many relies on many mechanistic
mechanistic and kinetic
and kinetic factors that can factors
varythat can vary
between between
families (AAC, families
ANT,
(AAC, ANT, and APH) and between types
and APH) and between types too (i.e., APH(3 )-I, II). too0 (i.e., APH(3′)-I, II).
4.
4. Combination
Combination Therapy
Therapy
The use of
The use of aacombination
combinationtherapy therapycan can help
help in in solving
solving the the problem
problem of resistance
of resistance by AMEs.by AMEs.This
This approach
approach reliesrelies
on theon the rescue
rescue of original
of original aminoglycosides
aminoglycosides (gentamicin,
(gentamicin, amikacin, amikacin,
or etimicin)or etimicin)
through
through the co-administration
the co-administration of the ofcorresponding
the corresponding inhibitors
inhibitors for for
each each enzyme.Ideally,
enzyme. Ideally, the
the adjuvant
adjuvant
compound
compound (inhibitor) would be targeted preferentially by the resistance enzymes, thus freeing the
(inhibitor) would be targeted preferentially by the resistance enzymes, thus freeing the
antibiotic
antibiotic toto bind
bind the the target
target A-site.
A-site. An An all-purpose
all-purpose inhibitor
inhibitor would
would be be aa compound
compound that that mimics
mimics the the
charge
chargeandandshape
shapeofofananaminoglycoside
aminoglycoside (common
(common forfor
thethethree families
three of AMEs),
families of AMEs),having in mind
having that
in mind
all AMEs have a highly negatively charged surface in their binding
that all AMEs have a highly negatively charged surface in their binding sites such that a must-havesites such that a must-have feature
should
featurebe an overall
should be anpositive
overall charge.
positiveAcharge.proof-of-concept for this strategy
A proof-of-concept for thisis strategy
that the use of streptidine
is that the use of
along
streptidine along with streptomycin in the cell culture restores the activity of the streptomycinANT(6),
with streptomycin in the cell culture restores the activity of the streptomycin against against
because
ANT(6),streptidine competescompetes
because streptidine for the bindingfor thesite with the
binding sitestreptomycin, acting as aacting
with the streptomycin, “decoy asacceptor”
a “decoy
of the enzyme
acceptor” [103]
of the enzyme (Figure 15).(Figure
[103] Thus, 15).
streptidine could be acould
Thus, streptidine good be starting
a good compound for the design
starting compound of
for the
more efficient “decoy acceptors” of AMEs targeting streptomycin,
design of more efficient “decoy acceptors” of AMEs targeting streptomycin, or 2-deoxystreptamine for or 2-deoxystreptamine for those
targeting 4,5- or4,5-
those targeting 4,6-aminoglycosides
or 4,6-aminoglycosides (Figure(Figure
15). 15).
Hybrid
Hybrid antibiotics have also been developedtotobattle
antibiotics have also been developed battlebacterial
bacterial resistance
resistance [104–107].
[104–107]. The ability
The to
ability
slow down the emergence of resistance is probably one of the most
to slow down the emergence of resistance is probably one of the most important advantages of hybrid important advantages of hybrid
drugs
drugs [108,109].
[108,109]. This
This strategy
strategy connects
connects two two antibiotics
antibiotics that that have
have different
differentmodes
modesof of action
action into
into aa single
single
molecule.
molecule. The main difficulty so far has been to find the correct linker that
main difficulty so far has been to find the correct linker that connects the two drugs to connects the two drugs
to provide
provide better
better inhibition
inhibition on both
on both targets.
targets. The synthesis
The synthesis of the Cipro-NeoB/Cipro-KanA,
of the Cipro-NeoB/Cipro-KanA, which waswhich active
was active
against against
a wide range a wide range
of strains, is ofonestrains,
of the mostis onesuccessful
of the most successful
examples of thisexamples
approachof(Figure
this approach
16). The
(Figure 16). The
MIC values MIC values
of these hybrids of these
were hybrids
the same were the same
against against
resistant and resistant and non-resistant
non-resistant strains
strains (AAC(6′),
(AAC(6 0 0 0 00
APH(3′),),andAPH(3 ), and AAC(6 )/APH(2
AAC(6′)/APH(2″) [110,111]. This ) [110,111].
kind ofThis kind of aminoglycoside
aminoglycoside derivative,
derivative, based based
on a hybrid
on a hybrid structure, provides a promising drug with an unusual
structure, provides a promising drug with an unusual dual mechanism of action, a potent profile dual mechanism of action, a potent
profile
againstagainst
AMEs,AMEs,and reducedand reduced
potential potential for generating
for generating bacterial bacterial resistance.
resistance.
Molecules 2018, 23, 284 12 of 18
Molecules
Molecules2018,
2018,23,
23,xxFOR
FORPEER
PEER REVIEW
REVIEW 12
12 of
of18
18
Figure
Figure 15.
15. Structure of
Structure of the
of the streptomycin
the streptomycin
streptomycin and
and streptidine
streptidine as “decoy acceptor”.
as “decoy acceptor”.
Figure
Figure 16.
16. Structure of
Structure of the
of the kanamycin
the kanamycin B-Cipro
kanamycin B-Cipro complex
B-Cipro complex as
complex as hybrid
as hybrid antibiotic.
hybrid antibiotic.
antibiotic.
5.
5. Conclusions
Conclusions
In
Inthis
thisreview,
this review,
review, we have
wewehave aimed
have aimed
aimed to
tocover the
themost
to cover
cover mosttherelevant semi-synthetic
most relevant
relevant aminoglycosides,
semi-synthetic
semi-synthetic inhibitors,
aminoglycosides,
aminoglycosides, inhibitors,
and
anddecoy
decoyacceptors
inhibitors, and decoy
acceptors of
ofthe
theAMEs.
acceptors
AMEs.of By far,
Bythefar,the
themost
AMEs. mostBy successful
far, thechemical
successful approach
most successful
chemical approach to
tomodify
chemical
modify the
thenatural
approach natural to
aminoglycosides
the naturalhas been 00 amino
modify
aminoglycosides been thethe modification
aminoglycosides
has has beenof
modification the
of the N-1/N-3″
N-1/N-3″ amino
modification
the of thegroups
amino N-1/N-3
groups with
with the
the AHBgroups
AHB groupwithor
group the
or aa
guanidino
AHB group substituent
or a that
guanidino has retained
substituent the
that hasparent antibiotic
retained the activity.
parent Alkylation
antibiotic
guanidino substituent that has retained the parent antibiotic activity. Alkylation at the N-6′ position activity.at the N-6′
Alkylation position
at the
of 0 position
of the
N-6 the antibiotic
antibiotic resulted
of the in
in aa decrease
antibiotic
resulted resulted
decreasein of
ofaaffinity
decrease
affinity for
forofresistant
affinity enzymes,
resistant for resistant
enzymes, but
but aa concomitant
enzymes, decrease
decrease in
but a concomitant
concomitant in
bacterial
decrease
bacterial in activity has
bacterial
activity been
been observed
hasactivity has beenas
observed well.
well. Chemical
observed
as as well. deoxygenation
Chemical Chemical
deoxygenation of
of the
deoxygenation of the 30 groups
the 3′/3′,4′-OH
3′/3′,4′-OH /3 0 ,40 -OH
groups of
of
aminoglycosides
groups resulted
resultedin
of aminoglycosides
aminoglycosides incompounds
resulted
compounds with
withhigh
in compounds highaffinitywith for
affinity forthe
high the RNA
RNAand
affinity for resistance
and the RNA against
resistance and MRSA.
resistance
against MRSA.
Comparatively,
against
Comparatively, fewer
fewer inhibitors
MRSA. Comparatively,inhibitors or
or decoy
fewer acceptors
inhibitors
decoy acceptors of
or decoyof the AMEs
AMEs have
theacceptors been
of the
have AMEs
been described.
have been described.
described.
On 0 )-Ia, ANT(2″)-Ia, 00 )-Ia, ANT(3″)(9), 00 )(9), AAC(3)-IIIb, 0 )-IIIa have
Onthetheother
otherhand,
hand,ANT(4′)-Ia,
ANT(4
ANT(4′)-Ia, ANT(2
ANT(2″)-Ia, ANT(3
ANT(3″)(9), AAC(3)-IIIb, and
AAC(3)-IIIb, andAPH(3′)-IIIa
APH(3
APH(3′)-IIIa havebeen
have been
been
described
described as
described as highly
highly dynamic
dynamic enzymes
enzymes and
enzymes and display
display properties
properties of of intrinsically
intrinsically disordered
disordered proteinsproteins in in
the
the absence
absence of of the aminoglycosides
aminoglycosides [112].
the aminoglycosides [112]. The The degree
degree of of promiscuity
promiscuity by by AMEs
AMEs is is governed
governed by
governed by the
the
dynamics
dynamics of
dynamics of the
the protein,
protein, which
which is is strongly
strongly influenced
strongly influenced by by the
the ligand
ligand andand thethe cofactor,
cofactor, as as well
well as as its
its
interaction
interaction with with the
the solvent,
solvent, and and itit should
should be be taken
taken into into consideration
considerationin inthe
thedesign
designof ofnew drugs.
newdrugs.
drugs.
Structural
Structural knowledge
knowledge of of both
both thethe RNA-
RNA- and and AME-aminoglycoside
AME-aminoglycoside complexes complexes has has helped
helped in in the
the
design
design of
design of new
new antibiotics
antibioticsthat
antibiotics thatcan
that canescape
can escapethe
escape theaction
the actionof
action ofofthe
thetheenzymatic
enzymaticmodification
enzymatic modification
modification [113]. In
[113].
[113]. order
In In
orderorderto
to
avoid
to avoid
avoid or
orinhibit the
theactivity
or inhibit
inhibit of
ofAMEs,
the activity
activity AMEs, obtaining
of AMEs,
obtaining structural
obtaining
structural and
andmechanistic
structural information
and mechanistic
mechanistic information isisof
ofparamount
informationparamount is of
importance.
paramount
importance.importance.
Acknowledgments:
Acknowledgments:
Acknowledgments:The The authors
Theauthors thank
authorsthank the
thankthe Spanish
theSpanish Ministeriode
SpanishMinisterio
Ministerio deEconomía
de EconomíayyyCompetitividad
Economía Competitividad(Grant
Competitividad (GrantMAT2015-
(Grant MAT2015-
MAT2015-
65184-C2-2-R and
65184-C2-2-R and CTQ2016-79255-P)
CTQ2016-79255-P) andand the
the CSIC
CSIC project
project iCOOPB20237
iCOOPB20237 for
for their
their support.
support.
65184-C2-2-R and CTQ2016-79255-P) and the CSIC project iCOOPB20237 for their support.
Conflicts of Interest: The authors declare no conflict of interest.
Conflicts
Conflicts of
of Interest:
Interest: The
The authors
authors declare
declare no
no conflict
conflict of
of interest.
interest.
Molecules 2018, 23, 284 13 of 18
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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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Semisynthetic Aminoglycosides

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

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

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

Molecules 2018, 23, x FOR PEER REVIEW 2 of 18

promiscuity seems to be governed by disordered elements that adopt well-defined conformations

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

Enzyme Resistance Profile Bacterial Source Pdb Number

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

Enzyme Resistance Profile Bacterial Source Pdb

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

Enzyme Resistance Profile Bacterial Host Pdb Number

Table 4. Bifunctional modifying enzymes.

Enzyme Resistance Profile Bacterial Source Pdb Number

3. Semi-Synthetic Aminoglycoside Derivatives

Molecules 2018, 23, x FOR PEER REVIEW

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

00 -Guanidino Kanamycin A and Plazomicin.

Figure 8. Structure of the Neamine and kanamycin dimers.

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

Figure 14. Structure

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