Aminoacyl-tRNA synthetase inhibitors as potential antibiotics

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European Journal of Medicinal Chemistry 46 (2011) 5227e5236

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European Journal of Medicinal Chemistry

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Invited review

Aminoacyl-tRNA synthetase inhibitors as potential antibiotics

Gaston H.M. Vondenhoff, Arthur Van Aerschot*

Rega Institute for Medical Research, Laboratory of Medicinal Chemistry, Katholieke Universiteit Leuven, Minderbroedersstraat 10, BE-3000 Leuven, Belgium

a r t i c l e i n f o

Article history:Received 28 June 2011Received in revised form12 August 2011Accepted 15 August 2011Available online 16 September 2011

Keywords:Aminoacyl tRNA synthetase inhibitorsAminoacyl sulfamoyladenosinesAntibioticsDrug design

* Corresponding author. Tel.: þ32 16 337388; fax: þE-mail address: [email protected]

0223-5234/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.ejmech.2011.08.049

a b s t r a c t

Increasing resistance to antibiotics is a major problem worldwide and provides the stimulus for devel-opment of new bacterial inhibitors with preferably different modes of action. In search for new leads,several new bacterial targets are being exploited beside the use of traditional screening methods. Hereto,inhibition of bacterial protein synthesis is a long-standing validated target. Aminoacyl-tRNA synthetases(aaRSs) play an indispensable role in protein synthesis and their structures proved quite conserved inprokaryotes and eukaryotes. However, some divergence has occurred allowing the development ofselective aaRS inhibitors. Following an outline on the action mechanism of aaRSs, an overview will begiven of already existing aaRS inhibitors, which are largely based on mimics of the aminoacyl-adenylates,the natural reaction intermediates. This is followed by a discussion on more recent developments in thefield and the bioavailability problem.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

The major hurdle in treating bacterial infections is the greatadaptability of bacteria to newantibiotics, which leads to resistance.This has posed the scientific community to the challenge of keepingup with (potentially) hazardous pathogens. Examples of bacteriathat are already causing a major threat to public health are methi-cillin resistant Staphylococcus aureus (MRSA), penicillin resistantStreptococcus pneumoniae, vancomycinresistant Enterococcus, andmulti-drug resistant Mycobacterium tuberculosis (MDMT) [1].

Three mechanisms by which resistance to the already existingclasses of antibiotics develops are: (i) modification of the target, (ii)functional bypassing of that target, or (iii) the drug becomingineffective due to bacterial impermeability, efflux or enzymaticinactivation [2,3]. Strategies to overcome resistance involve furtherdevelopment of existing classes of antibiotics and the use ofcombinations of existing antibiotics, as well as searching for newclasses of antibiotics. Indeed, the former strategy seems the mostpromising, since it can build on previous knowledge, and thus isrelatively time-, labor- and cost-saving. However, there is a greaterrisk of rapid reoccurrence of resistance. Therefore new antibioticswith different modes of action need to be developed to preventcross-resistance [4,5].

New antibiotics have to fulfill three criteria: the target should bevital for the cell function of the pathogen, it should be very selectivefor the bacterial target, and it should be difficult for the bacteria to

32 16 337340..be (A. Van Aerschot).

son SAS. All rights reserved.

develop resistance bymutations [6]. One of the more recent targetsfor intervention is the translation of mRNA and thus bacterialprotein synthesis. One way of doing so is by inhibiting the chargingof transfer ribonucleic acid (tRNA) with its cognate amino acid byaminoacyl-tRNA synthetases (aaRSs). These enzymes are found inall living organisms andmost organisms contain at least 20 differentaaRSs, one for each amino acid. Aside from the 20 standard aminoacids, quite a few non-standard amino acids are known, includingselenocysteine and pyrrolysine [7]. In addition, aaRSs are alreadyclinically validated as valuable target for development of antibiotics,e.g. Bactroban� (also known as mupirocin), which is responsible forthe inhibition of isoleucin-tRNA synthetase (IleRS).

Inhibition at this level is interesting for a number of reasons.First, aaRSs have a pivotal role in translation of messenger RNA(mRNA), and thus are of vital importance. Second, strong structuralconservation in the catalytic domains of the synthetase existsthroughout evolution, implying that one type of drug directedagainst a typical active site may inhibit a range of synthetases. Littlestructural variation may also imply that it could be difficult todevelop resistance by mutations in genes coding for the synthe-tases. This also means that if great homology exists betweeneukaryotic and prokaryotic aaRS, selectivity of a potential inhibitorfor the latter may be hard to achieve. Third, depending on whichsynthetase is considered, a full canonical pattern exists, meaningthat there are great sequence differences between prokaryotes andeukaryotes. This is, almost without exception, true for AspRS,GluRS, PheRS, LeuRS, IleRS, HisRS, ProRS, and MetRS. However,structurally, great differences are not always observed. The archi-tecture of the active site is in a structural sense quite conserved

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amongst different species [8e10]. Both in vitro and in vivo, mosteukaryal aaRSs can complement bacterial enzymes [11]. Furthercombined biochemical, bioinformatics, and structural studies areneeded to reveal the exact variations in active sites of aaRSs, whichare the key to rational drug design.

Although several other compounds are found in nature as well,most inhibitors of aaRSs developed to date are non-cleavablemimics of the natural reaction intermediates (i.e.aminoacyl-ade-nylates (aa-AMP)), that act through competitive binding to theaaRSs. Most of these aa-AMP analogues are aminoacyl-sulfamoyl-adenosines (aaSAs), vide infra. Although these compounds arepotent inhibitors of aaRSs, with MIC-values in the nanomolar range[12e15], their in vivo activity is considerably lower and poor uptakehas been suggested to be the main reason [16]. The lack of selec-tivity and poor bioavailability are the most prominent problems forthis new potential class of antibiotics. In this report several solu-tions to these problems are reviewed, with special attention toMicrocin C (McC), which has been the lead-compound for our ownrecent work on aaRS inhibitors.

2. Aminoacyl-tRNA-synthetases: basic mechanisms andactions

2.1. Coupling of amino acids to a cognate tRNA

To create an aminoacyl-tRNA unit, a tRNA-subunit must becovalently attached to a specific amino acid. This reaction is cata-lyzed by aaRSs, which are specific for each amino acid and a cor-responding group of tRNAs (isoacceptors). These enzymes have torecognize two substrates: first, a set of tRNAs which sharea collection of ‘identity elements’ and second, an amino acid thatmay be distinguished by small differences in side-chain properties.

The actual coupling of an amino acid to the corresponding tRNAcomprises two steps. First, the amino acid (aa) is activated bynucleophilic attack on the a-phosphate of adenosinetriphosphate(ATP) giving aminoacyl-adenosine-monophosphate (aaAMP) andpyrophosphate. The second step constitutes the esterification bya nucleophilic attack of the 20- or 30 ribose hydroxyl group at the

Fig. 1. Aminoacylation occurs in two steps, both catalyzed by aaRSs. The aminoacylation reacII enzyme. The 20-hydroxyl is acylated by class I aaRSs.

30-end (A76) of the cognate tRNA to the activated carboxyl group ofthe aaAMP generating the activated aa-tRNA species (Fig. 1). Thecorrect aa-tRNAs interact with elongation factors (EF-1a ineukaryotes and Archaea, EF-Tu in prokaryotes) to translate themRNA within the A site of the ribosome [6]. This process has beenadequately documented many times (see e.g. Refs. [17] and [18]).

2.2. Two distinct classes comprise 21 aminoacyl-tRNA-synthetases

The 21 aaRSs are classified into two distinct classes: 11 in class Iand 10 in class II, with lysRS found in each class. This partition isbased on consensus motifs of the catalytic domains. Class I aaRSscontain two dinucleotide-binding Rossman folds, which is a struc-tural motif that can bind nucleotides. It is composed of three ormore b-strands linked by two a-helices. Since each Rossman foldcan only bind one nucleotide, class I aaRSs contain two pairedRossman folds [19]. The active site of a class II aaRS is a barrel-likestructure of antiparallel b-sheets surrounded by loops and a-helices[20]. This structure forms a template that binds the respectiveamino acid and ATP. Furthermore, class I proteins differ from class IIproteins in the position of esterification at the ribose moiety of the30-adenosine of the tRNA with the amino acid. Class I synthetasesesterify at the 20-hydroxyl group, whereas class II synthetasesesterify at the 30-hydroxyl group of the ribose [1,6,21]. This can beexplained by the fact that class I proteins approach the tRNAacceptor stem from the minor groove side, whereas class IIenzymes approach the tRNA from the major groove [6]. Furthersubdivisions in each class are made, based on sequence homologyand domain architecture [20]. Table 1 shows the classifications ofaaRSs in the six subclasses.

The recognition of the cognate tRNA is for all aaRSs dependenton the discriminator base N73, the acceptor stem and the anti-codon of the tRNA. To maintain high fidelity in the catalyticcoupling of tRNA to amino acids, all aaRSs contain a distinctstructural domain for anticodon recognition. In addition, someaaRSs contain a zinc-binding domain that is involved in therecognition of the acceptor stem [20].

tion depicted here takes place at the 30-hydroxyl moiety and thus is catalyzed by a class

Table 1Classification of the different aaRSs.

Class I Class II

Ia Ib Ic IIa IIb IIc

LeuRSIleRSValRSCysRSMetRS

TyrRSTrpRSLysRSI

ArgRSGlnRSGluRS

HisRSProRSSerRSThrRSGlyRSAlaRS

AspRSAsnRSLysRSII

PheRS

G.H.M. Vondenhoff, A. Van Aerschot / European Journal of Medicinal Chemistry 46 (2011) 5227e5236 5229

Interestingly, LysRS is found in both class I and class II. Mostorganisms contain a class II LysRS, but some bacteria and Archaeaonly possess a structurally distinct class I LysRS instead. As class ILysRS is not found in Eukarya and differs in substrate specificityfrom its class II analogue, this class I LysRS may be an interestingspecies-specific target for antibacterial drug development [22].

2.3. An editing site hydrolyses the misactivated amino acids

Correct aminoacylation depends on the selection of two appro-priate substrates, the tRNAand the amino acid, by the correspondingaaRS. Since the tRNA is a relatively large unit and therefore hasa large number of ‘identity elements’, the selection of tRNA is mucheasier than the selection of the smaller amino acids. Amino acidshave to be selected by the nature of their side chains [22]. Althougheach amino acid has a different structure, some have similarchemical and/or structural properties.

Two different editing mechanisms exist to decrease the numberof incorrectly aminoacylated tRNAs. In pre-transfer editing, themisactivated amino acid is hydrolyzed into the amino acid andAMP, whereas in the post-transfer editing, the incorrectly amino-acylated tRNA is hydrolyzed into the amino acid and tRNA [23]. Thesynthetic site is mostly specific enough so that only the correctamino acid can be activated and transferred, due to the recognitionof specific properties of each amino acid and the steric exclusion ofamino acids with larger side-chains. Also, it has been reported thatthe difference in sugar puckering and the orientation of the C(40)eC(50) bond of the adenosine plays an important role on how aaRSsdiscriminate cognate from non-cognate aminoacyl-AMP [13].

Apart from the synthetic site, an editing site exists to hydrolyzemisactivated amino acids. The presence of two catalytic sites with

Fig. 2. Structures for the amidotransferase inhibitors aspartycin (1a, n¼ 1) and glutamyb-phosphoryl-aspartyl-tRNAAsn, the reaction intermediate in the transamidation reaction, was small molecule inhibitor of the reaction.

different activities led to the proposal of a double-sieve model. Inthis model the synthetic site of the enzyme acts as the first sieve,excluding amino acids that are too large or do not establish theright interactions with the active site. The smaller amino acids thatcan establish sufficient interactions however, may slip through thisfirst sieve and may be incorrectly activated. The editing site, whichis too small to fit the cognate amino acids, is capable of hydrolyzingmisactivated amino acids. This double sieving mechanism raisesthe accuracy to about one mistake in 40,000 aminoacylation reac-tions [24]. The interplay between pre- and post-transfer editing intRNA synthetases has been reviewed recently by Martinis andBoniecki [25].

2.4. Indirect biosynthesis of tRNAAsn and tRNAGln by transamidation

Beside the sievingmechanism in the aaRSs, Archaea and bacteriahave an additional system involved in the editing of non-cognateaminoacylated tRNA. The aminoacyl-tRNA amidotransferase (AdT)canmodify the coupled amino acid to obtain the cognate aminoacyl-tRNA. This is not only an error reducing mechanism, but alsoan indirect pathway for the biosynthesis of Asn-tRNAAsn andGln-tRNAGln, which is for some bacteria the only source to obtainthese charged tRNAs, e.g. for Helicobacter pylori [26]. The mis-acylated tRNAs are synthesized by non-discriminating GluRS andAspRS, which also aminoacylate Glu onto tRNAGln and Asp ontotRNAAsn [27]. Since thismechanism is not present in eukaryotic cells,these enzymes are interesting targets for drugdevelopment. Hereto,some analogues bearing resemblance to the 30-end of amino-acylated tRNA like aspartycin and glutamycin (1a,b; Fig. 2) or to thereaction intermediates of the transamidation reaction like 3, havebeen synthesized and tested for antibacterial activity [27]. Morerecently a series of chloramphenicol analogues was synthesized,uncovering compound 4 within their series as the most activeinhibitor of the transamidase activity with respect to Asp-tRNAAsn

with a Ki value of 27 mM [28].

2.5. Non-ribosomal peptide synthetases fulfill a similar role asaaRSs

Many important peptides, produced by bacteria and fungi, aresynthesized by non-ribosomal peptide synthetases (NRPSs) with-cyclosporin A, gramicidin S and bleomicyn A2 being examples ofsuch peptides. NRPSs can be seen as a series of modules, in whicheach module incorporates a polypeptide in the NRPS. Each module

cin (1b, n¼ 2), and for the chloramphenicol analogue 4. While structure 2 depictsith R indicating the remainder of the tRNA, compound 3 is a stable phosphonate mimic


is responsible for the incorporation of an amino acid into thegrowing polypeptide chain. In contrast to aaRSs, NRPSs are bothtemplate and peptide producing enzymes. Each module can besubdivided into three domains, which are similar to the ribosomalpeptide machinery. Hence, both systems use aminoacyl-adenylatesas their building blocks. As a consequence, also the NRPSs can beinhibited by 50-O-(N-aminoacyl)-sulfamoyladenosines (aaSAs, videinfra) at concentrations in the low nanomolar range [29]. However,both systems bind these inhibitors in different conformations.

Peptide synthesis by the ribosomal machinery is extraordinaryefficient and occurs with high fidelity, but is restrained to a set of20e22 amino acids. In contrast, peptide synthesis by NRPS lacksproofreading activity, but can use a variety of substrates, includingD-amino acids, fatty acids, and aryl acids [30]. This makes NRPSideal targets for potential antibiotics.

3. Existing aminoacyl-tRNA-synthetase inhibitors

Most inhibitors of aaRSs act by competitive binding at the activesite where normally the cognate amino acid would bind. Manyinhibitors known to date are natural products or derivatives ofthem. Only few compounds have reached the stage of clinicaldevelopment. A selection of the most important compounds (alldepicted in Fig. 3) will be discussed in the next section.

3.1. Mupirocin is the only approved aaRS inhibitor

Of thenumerous aaRS inhibiting compoundsmupirocin (4; Fig. 3),marketed as Bactroban� by GSK, is the only aaRS inhibitor approvedby the FDA [17,31]. Originally it was isolated from Pseudomonas flu-orescens. It is targeted against IleRS, and functions as a competitiveinhibitor at the synthetic active site (Ki: 2.5 nM for Escherichia coli

Fig. 3. Structures of some well-kno

IleRS). The tetrahydropyran ring binds at the place where ribosewould normally bind, the epoxy-group binds in the amino acidpocket and the fatty acid binds inside the adenine pocket. Mupirocinis primarily active against Gram-positive bacteria, e.g. methicillinresistant S. aureus (MRSA) [1] (MIC: 0.25e0.5 mg/mL) showing an8000-fold selectivity for pathogenic aaRS over human aaRS [21]. It isless active against Gram-negative bacteria (MIC: 128 mg/mL forE. coli). Because it is used as topical ointment, high local concentra-tions can be achieved, making it sometimes suitable for Gram-negative bacteria as well.

Unfortunately, it appears that resistance is developing againstthis antibiotic as well. Low-level resistance has been reported bymutation of its target, IleRS, whereas high-level resistance is founddue to the presence of a second IleRS with many similarities toeukaryotic enzymes, due to acquisition of the mupA gene [1,32].Another drawback of mupirocin is its poor bioavailability; since itsesterfunction is highly unstable, it is rapidly hydrolyzed in bloodand tissue. Hence, its use is limited to topical treatment. Therefore,many analogues of mupirocin have been created, although none ofthese have reached the clinic. Themost successful analoguewas SB-234764 (5), which combined structural features of IleSA andmupirocin. However, with the aim of gaining selectivity, furthermodifications especially to the base moiety have been envisaged,culminating in substitution of a phenyltetrazole moiety for theadenine (6, CB-432, vide infra).

3.2. Clinical development candidates

Indolmycin (7) is an inhibitor of tryptophanyl-tRNA synthetase(TrpRS) [33]. Indolmycin is a biosynthetic derivative of Trp,which hasa number of other intracellular functions that affect viability [17].Probablydue to its hydrophobicity,whichmay impair cellularuptake,

wn synthetic aaRS inhibitors.


the overall in vivo inhibition of TrpRS is limited. Pfizer filed a patentalready in 1965, although further development was discontinued,since it appeared that indolmycin was not sufficiently active againstthe majority of commonly occurring pathogenic bacteria like Strep-tococci, Enterococci and Enterobacteriaceae [20]. More recently,indolmycinhas been shown to exert a bacteriostatic activity againstS. aureus. However, certain strains have been isolated that haveadopted resistance via a point mutation (H43N) in TrpRS [34]. AlsoStreptomyces griseus was shown to adopt resistance to indolmycin.H.pylorion theotherhand, showed tobeunable to develop resistance[35].

REP8839 (8) is a fluorvinylthiophene linked via a 1,3-diaminopropane with a quinolone. It is a very potent analogue ofthe original quinoline derivative 9 found via a high throughputscreening effort by Jarvest et al. [36]. REP8839 is a fully syntheticinhibitor of MetRS, currently in phase I clinical trials for the treat-ment of skin and wound infections of S. aureus. Aside frommupirocin or oxacillin resistant S. aureus strains (MIC90: 0.5 mg/mL),this compound also showed good activity against Streptococcuspyogenes (MIC: 0.03e0.5 mg/mL) as well as against a number ofother Staphylococci and Enterococci. Interestingly, the Ki for S. aureusMetRS was found to be w10 pM, while this compound showedsignificantly weaker inhibition for E. coli MetRS with a Ki value of300 nM.Moreover, no inhibition of mammalian rat liver MetRS wasfound, while humanmitochondrial and cytoplasmic MetRS showedKi values three- to six-fold higher than S. aureus MetRS. ThusREP8839 is a relatively selective compound with high potential[37]. The compound also proved to be well tolerated when appliedfor intranasal ointments [38].

AN-2690 (10) is a fluorinated benzoxaborole with activityagainst dermatophytes, yeasts, and molds. It is perfectly capable topenetrate nail tissue. For this feature it is being pursued to treatonychomycosis (infection of the nail). In contrast to most otheraaRS inhibitors, it is a non-competitive inhibitor of LeuRS, and bindsin the editing site of the enzyme [12]. Here it traps tRNALeu throughthe formation of two covalent bonds of boron with the 20,30-hydroxyl groups of the 30-terminal adenosine of tRNALeu withformation of acyclic borate structure. Phase I and II clinical trialshave shown efficacy and safety [20], with phase III trials ongoing,carried out by its developer Anacor. Very recently analogues ofAN2690 were found to strongly inhibit LeuRS of Trypanosomabrucei, paving the way likewise for anti-parasitic drug development[39].

Icofungipen (11) is an antifungal that inhibits IleRS. Throughactive transport by permeases this compound accumulates in yeastcells up to 200-fold of the extracellular concentration [40]. It wasdiscovered through a program directed toward a more potentderivative of cispentacin (12). The 1R, 2S-configuration was foundto be essential, and, aside from the methylene addition at the4-position, no other additions or substitutions were allowed, inorder to retain high activity [41]. Good clinical efficacy and safetywere observed in phase I and II clinical trials, although low myco-logic eradication rates were observed in HIV-positive patients. Tothis end, higher dosage may be desirable [20].

3.3. Other natural aaRS inhibiting products

Cispentacin (12) (PLD-118; Fig. 4) is a cyclic b-amino acid thathas been isolated from two species: Bacillus cereus and Strepto-myces setonii. It is effective against Candida albicans infection inmice. Although icofungipen is a follow up of this compound andinhibits IleRS [1, 20], cispentacin itself is a millimolar inhibitor ofProRS.

Chuangxinmycin (13) bears some resemblance to indolmycin,and as a consequence it also inhibits TrpRS. Initially it was reported

for its activity against a range of Gram-positive and Gram-negativebacteria. It showed good efficacy against Shigella dysenteria andE. coli in infected mice. Despite its apparent potency, there are noreports on further development of this compound [1].

In contrast to most other aaRS inhibitors, borrelidin (14) is anallosteric inhibitor. By binding to a hydrophobic patch of ThrRS, itimpairs catalytic conformational changes necessary for Thr and ATPbinding [42]. Apart from its ThrRS inhibition, borrelidin also acti-vates caspases 3 and 8, hence inducing apoptosis. As of theseeffects, borrelidin is evaluated in further studies for its potency forangiogenesis inhibition. The compound showed good absorptionand membrane permeability, and proved to be non-mutagenic inthe Ames test, but an inhibitor of CYP3A [43,44].

Non-hydrolyzable analogues of the aminoacyl-AMP form thelargest class of potentially active compounds against aaRSs [31].Agrocin 84 (15, Fig. 4) is such a well-known aaRS-inhibitor and isused to inhibit the formation of plant tumors caused by Agro-bacterium tumefaciens. Agrocin 84 is a derivative of leucyl-adenylate, but contains a D-glucofuranosyloxyphosphoryl moietywhich is important for uptake by the pathogen. This moiety iscleaved off intracellularly following uptake [17]. A similar mode ofaction is presented by Microcin C (McC, 20), which will be furtherdiscussed in the next sections. The toxic moiety of agrocin 84inhibits cellular leucyl-tRNA synthetases, but the agrocin 84producing strain K84 carries a second, self-protective copy of thesynthetase, termed AgnB2 and providing immunity to the antibi-otic [45]. The genetic basis for the production and self-protection toagrocin 84 has been discussed by Kim et al. [46]. The full synthesisof Agrocin 84 was already described by Moriguchi et al. [47].

Another interesting natural antibiotic, which is also an aa-AMPanalogue, is ascamycin (16), produced by Streptomyces and carryinga 2-chloroadenine moiety. Ascamycin inhibits incorporation ofphenylalanine in Xanthomonas citri and Xanthomonas oryzae. Noactivity was observed against E. coli., which was blamed on thecompound being charged, hampering permeation of the bacterialmembrane [48]. X. citri and X. oryzae possess an Xc-aminopeptidaseat their cell surfaces that metabolizes ascamycin into its dealanylderivative. The dealanyl analogue showed activity against a range ofGram-negative and Gram-positive bacteria [49].

Phosmidosine (17) is a proline-AMP analogue and according toits structure, most likely also targets the corresponding ProRS,although this has never been reported. The natural compound wasfirst described in 1991 as an antifungal nucleotide antibioticinhibiting spore formation of Botrytis cinerea at the concentrationof 0.25 mg/mL [50]. In view of its rare O-methylated phosphor-amidate structure, the compound is rather base unstable. Sekineet al. [51] tried to circumvent this instability in synthesizingdifferent analogues and studied the structure-activity relationshipof this potent antitumoral compound with its unique property ofarresting cell growth at the G1 phase in the cell cycle. While bothphosphoramidate isomers proved equally active, presence of the L-proline part was mandatory for the activity.

Albomycin (18) is another interesting natural seryl tRNAsynthetase repressor which is actually working as a Trojan horseinhibitor. The active moiety is attached to a specific hydroxamate-derived siderophore uptake part which is hydrolyzed followingactive transport. The intracellular released inhibitor is an adenosineanalogue with a thioxylofuranosyl moiety substituting for theribofuranosyl, and carrying a glycine at its 50-carbon via a CeC bond.The awkward 50-part apparently mimics a phosphate and a serinemoiety is further acylating the a-amine, generating the aminoacyl-AMP analogue. The structure for the different albomycin congeners(d1, d2 and 3)was already revealed in 1982 [52] and the strong in vitroinhibitory activity of 8 nM for the intracellular released nucleosideanalogue (19) (coined SB-217452) versus seryl tRNA synthetases has

Fig. 4. Chemical structures for some well-known natural aaRS inhibitors.


been documented by Stefanska et al. [53]. Remarkably, a syntheticderivative with oxygen substituting for the sulfur (thus based ona xylofuranosyl derived compound) annihilated the activity for thed1 compound [54]. A practical synthetic method for the hydrox-amate partwas nicelyworkedout by Lin andMiller, opening thewayfor other siderophore carrying compounds [55]. More recently, itwas shown that albomycin producing strains at the same timeencode for a second seryl-tRNA synthetase to avoid self-poisoning,a situation as described with agrocin 84 [56].

Microcin C (McC, 20) exerts a bacteriostatic activity againsta wide range of Gram-negative bacteria including Escherichia,Klebsiella, Salmonella, Shigella, and Proteus species [57], as well asagainst some Gram-positives [58,59]. McC is a potent antibacterialcompound produced by some E. coli strains and functions througha Trojan-Horse mechanism: it is actively taken up inside a sensitivecell through the function of the YejABEF-transporter and thenprocessed by cellular aminopeptidases. Processed McC (21) isa non-hydrolyzable aspartyl-adenylate analogue that inhibitsaspartyl-tRNA synthetase (AspRS).

3.4. More recent synthetic aaRS inhibitors

The search for new and further improved RS inhibitors is stillongoing. For instance, mutations in S. aureus MetRS have beenfound that conferred resistance to REP8839. However, thesemutations also severely reduced bacterial fitness [20]. Therefore,many new quinolinone congeners have been prepared but

unfortunately with reduced activity. Some however displayedsignificant inhibitory properties against Enterococci [60]. Likewise,a series of 3-aryl-4-alkylaminofuran-2(5H)-ones were preparedand proved strongly inhibitory to Gram-positive organismsculminating in compound 22 (Fig. 5) endowed with MIC50 of0.42 mg/ml against S. aureus, but lacking activity versus Gram-negative bacteria [61]. Enzymatic tests indicated Tyr RS to be thetarget and molecular docking proved a nice fit of the inhibitor withthe Tyr RS active site.

In addition, protozoal tRNA synthetases recently have been tar-geted. Benzoxaborole (see also 10) was used as the lead structure fordevelopment of T. brucei LeuRS inhibitors, resulting in compound 23with an IC50 of 1.6 mM. All stronger LeuRS inhibitors also affordedexcellent T. bruceiparasite growth inhibition activity,with the betterresults obtained for themore lipophilic congener 24with an EC50 of0.37 mM[62]. Several research groups are using in silico strategies touncover new leads for different tRNA synthetases, but confirmedresults using such strategies are largely missing. For instance,a series of imidazolidin-2-ones as depicted in the general structure25, were believed to be inhibitory for IleRS following in silicoscreening, but unfortunately no activity could be detected followingsynthesis of the proposed structures [63]. Hoffmann and Torchalalikewise proposed a series of potential LeuRS inhibitors which canbe easily obtained via click chemistry. However, confirmation of theactivity profile so far is lacking [64]. However, a last example provedthe strategy can be relatively fruitful as three moderately activeinhibitors of Staphylococcus epidermidis TrpRS were uncovered

Fig. 5. Some more recent developments lead to a series of furan-2(5H)-ones (22) and to benzoxaborole derivatives (23,24) and in addition to many in silico active inhibitorsrepresented by structure 25.


using a structure-based virtual screening effort [65]. While all threecompounds displayed some common characteristics like a benzoicacid terminal end, overall structures proved quite diverse. Effectivebinding to the bacterial TrpRS was demonstrated by surface plas-mon resonance, and low cytotoxicity to mammalian cells was re-ported. Bacterial growth inhibition assays however, showed onlymoderate activity with MIC50values of 6.25, 25 and 100 mMrespectively.

4. Recent developments in aminoacyl-AMP type inhibitors

A good aaRS inhibitor has tomeet several criteria. First, it shouldbe able to interact with great affinity in the active site of the aaRS,relative to the normal substrates, i.e. ATP and the cognate aminoacid. Second, the activation energy for hydrolysis of the analogueinto AMP and the amino acid should be sufficiently high. Aside fromthe natural aaRS inhibitors, many analogues of aminoacyl-AMPhave been created that are currently under study.

Fig. 6. Possible linkages between adenosine and the amino acid as isosteres of theacyl-phosphate bond.

4.1. Aminoacyl sulfamoyl-adenosines are the most potent inhibitorsof aaRSs

Initially it was hypothesized that in general replacement of thelabile aminoacyl-phosphate (26) (a mixed anhydride) by a non-hydrolyzable bio-isoster, such as an aminoalkyl adenylate [31,66,67](27), an aminoacylsulfamoyl adenosine [13, 68] (28) linkage or aneven more simplified linkage like an amide [69] would lead toa similar interaction with the enzyme (Fig. 6). It was observed thatthe phosphoramidate linkage, as found in McC and Agrocin 84, isunstable in acidic environment [70] and physiological conditions.However Moriguchi et al. [47] reported an unstable phosphor-amidate linkage in Agrocin 84 in basic conditions. Also, the phos-phoramidate linkage in phosmidosine was found to be base labile[71], but the latter two findings refer to lability of the additionalester linkages.

Most analogues consist of an adenosine coupled to an aminoacid (analogue) via a stable sulfamate/ester/phosphonate linkage

instead of a labile phosphoanhydride linkage. In addition, thesynthesis of b-ketophosphonates (29) has been reported [72].

The most potent analogues are those bearing a sulfamoyllinkage as found in structures 16 and 28. By X-ray analysis it wasshown that the stable sulfamoyl (sulfamate) linkage in 50-O-[N-(L-seryl)-sulfamoyl]adenosine, a SerRS inhibitor, establishes similarhydrogen bonds inside the hydrophilic cleft of the enzyme [68].Therefore it may be generally true that although the linkage ismuchmore stable, the electron distributions closely resemble thoseof aa-AMP and the number of hydrogen bonds remains the same.

Forrest et al. [66] reported the presence of the carbonyl group,present in a sulfamoyl linkage but not in an aminoalkyl adenylatelinkage, to be crucial for the recognition by a class II synthetase.Both acyl-phosphate mimics are negatively charged in solution dueto acidity of either the phosphodiester or the NH-function in thesulfamoyl moiety, respectively. Only the acyl sulfamate, containinga carbonyl is able to delocalize this negative charge, seemingly ofgreat importance in stabilization of the transition state.

An X-ray crystallographic conformation study of AlaSA showedthe compound to be in a zwitterionic state in the crystal. Themolecule was found to be in a stretched conformation, with thealanyl- and adeninyl-moieties at different sides of the molecules.

Fig. 7. General structure for the dipeptidyl-sulfamoyladenosine analogues 30, and the recently developed Microcin C analogues 31, where the phosphoramidate is replaced bya sulfamoyl linker. For both structures R1 and R2 can be any amino acid side chain.


Ribose puckering was in the expected C(30)-endo or North confor-mation, and the adenine base was in anti-position with respect tothe sugar. This work also confirmed that sulfamate and phosphateare indeed close isosteres [13]. As a consequence, aaRSs bind theircognate aaSAwith great affinity, but the carboxyl-sulfamate bond isconsiderably stronger than the carboxyl-phosphate bond in aa-AMP. This way the aaRS catalytic site becomes blocked, providingthe sulfamate containing analogues the best potential towardfurther drug development. In general, all reported Ki values are inthe low nanomolar range up to 50 and 60 nM for GlySA [29] andPheSA [30].

4.2. Aminoacyl sulfamoyl-adenosines are not selective and displaypoor bioavailability

All compounds, discussed in the previous section, are reactionintermediate mimics, implying that these compounds differ onlylittle from the natural reaction intermediates, and thus selectivityfor either eukaryotic or prokaryotic aaRSs is low. As was discussedin Section 2.2, during evolution extensive divergence occurred inthe amino acid sequences of prokaryotic and eukaryotic aaRSs,however this did not lead to great structural difference. Asa consequence, both prokaryotic and eukaryotic aaRSs use identicalreaction intermediates.

By modifications at the adenine base, researchers have tried toincrease the selectivity for bacterial aaRSs. The most successfulexample is probably CB-432 (6), an IleSA analogue whereby theadenine is replaced by a large apolar substituent. CB-432 showed570 fold more affinity for the E. coli IleRS relative to the corre-sponding human enzyme [10]. Unfortunately, clinical developmentof this compound was discontinued as a consequence of lowbioavailability, due to binding to serum albumin. Also otherheterocycles like thiazole [73], and tetrazole [74] have been eval-uated for activity and selectivity. Some of the thiazole derivativeshave shown inhibitory activity against Gram-positive and Gram-negative bacteria as well as some selectivity over human aaRS [74].

Although aaSAs are potent inhibitors of aaRSs, their whole cell(i.e. in vivo) activity is rather low, probably due to poor uptake [16].Clues to this observation may come from the structure of any aaSA,since under physiological conditions, apart from the amino acidside chain, all aaSAs contain one negative charge at the sulfamatelinkage and a positive charge at the amino terminal end. Thus, theseare highly polar compounds that will not diffuse easily through thehydrophobic cellmembrane. Ubukata et al. [48] found that L-prolyl-L-prolyl-sulfamoyl-2-Chloroadenosine had an increased in vivoactivity against both Gram-negative and Gram-positive bacteria,when compared to L-prolyl-SA. It was therefore suggested thatpeptide transporters are involved in increased uptake of thedipeptidyl-SA compound, leading to an increased activity, althoughthis was not further investigated [48].

Following this example, Van de Vijver et al. [75] synthesizeda diverse set of dipeptidyl-SAs (Fig. 7, 30) that differed in

physicochemical parameters such as hydrophobicity, net chargeand size. These compounds were evaluated against several Gram-negative and Gram-positive bacteria. In general, whole cellactivity was shown to be relatively low. The reason as to why the L-Pro-L-Pro-sulfamoyl-2-Chloroadenosine inhibitor did show a niceactivity against a range of bacteria may be the presence of a proline-rich peptide transporter, such as SbmA, which is found in E. coli[76]. However, Van de Vijver et al. [75] found that such compounds,where proline was used as the C-terminal amino acid suffered fromextensive decomposition. Several other dipeptide transporters havebeen described already before by different authors [77,78]

Likewise, the natural compounds Agrocin 84 (16) and McC (20)circumvent the problem of poor uptake using peptide transporters.These compounds are so-called Trojan horse antibiotics, by theirmode of action. Once taken up by a peptide transporter [79], theseprodrugs are at first processed, thereby liberating the activecompound [80,81]. This principle has been demonstrated topromote the active uptake of some aaSAs [82] and many morestudies by the authors of this review are ongoing to implement thisTrojan horse principle. Hereto also a new synthesis has beenelaborated allowing for the production of a wide variety of McCanalogues that can target virtually any aaRS [83]. These analoguesretain theMcCmode of action, and thus are actively taken up by theYejABEF transporter, followed by intracellular processing by thedifferent peptidases, after which the respective aaRSs are targeted.

5. Conclusions

Aminoacyl-tRNA synthetases are structurally quite conserved inprokaryotes and eukaryotes. As a consequence, all aaRSs use thesame reaction intermediates in the aminoacylation of tRNA.However, some divergence has occurred throughout evolution,making it possible to develop selective aaRS inhibitors. Manynatural compounds have been found by means of high throughputscreening, although few have reached the level of clinical trials.Mupirocin is the only drug that is used in the clinic, although high-level resistance to this drug has restricted its use. Hereto, newcompounds that limit resistance should be developed. To addressthis resistance issue, one might also try to look for a multi-synthetase inhibitor. The concept still needs to be borne out, butone could think of an inhibitor targeting simultaneously ValRS,IleRS and LeuRS, as these recognize isosteric amino acids. Likewise,a single inhibitor could be envisaged for inhibition of AspRS andGluRS, or for AsnRS and GlnRS.

AaSAs for a long time have been evaluated as high-potentialantibiotics. However, these compounds lack bioavailability andselectivity, and therefore cannot be used as such. Most importantfinding however is that the Trojan horse concept, as presented byMcC and its newly developed analogues, could offer some solutionsto address the selectivity and bioavailability issues, opening thedoors for a new plethora of antibacterial agents. However, insuffi-cient research so far has been carried out on the possible cytotoxic


effects on mammalian cells. A recent report by Messmer et al. [84]confirmed Asp-SA to be an efficient inhibitor of many differentbacterial AspRSs, but for the first time likewise a strong inhibitionwas found on the human mitochondrial synthetase. However, it isnot clear yet whether such possible side effects on the host mito-chondrial enzymes will be translated to an in vivo setting.

Finally, the benzoxaborole type structures seem to be the mostadvanced class of compounds, endowed with low toxicitycombined with strong inhibitory activity on dermatophytes, moldsand more recently T. brucei, with the additional advantage of beingsmall molecules. Overall, it should be clear from this review thatsome of the here discussed prototype compounds are bound toreach the market in the near future, which will further establishaaRSs as a valuable target for development of antibiotics and morein general antimicrobials.

Acknowledgements

We are indebted to C. Biernaux for generous assistance in finaltypesetting.

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