Phenyltriazole-functionalized sulfamate inhibitors targeting tyrosyl- or isoleucyl-tRNA synthetase

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Highlights

  • Eight new CB 168 analogues targeting isoleucyl- or tyrosyl-tRNA synthetase.

  • New IleRS-targeting compounds with 3-carbon linker showed good inhibitory activity.

  • Docking approach for 11 revealed a different binding mode compared to CB 168.

  • Activity decrease was due to loss of crucial pyran hydroxyl group interactions.

  • Enhanced lipophilic/hydrophilic balance suggested an improved bacterial uptake.

Abstract

Antimicrobial resistance is considered as one of the major threats for the near future as the lack of effective treatments for various infections would cause more deaths than cancer by 2050. The development of new antibacterial drugs is considered as one of the cornerstones to tackle this problem. Aminoacyl-tRNA synthetases (aaRSs) are regarded as good targets to establish new therapies. Apart from being essential for cell viability, they are clinically validated. Indeed, mupirocin, an isoleucyl-tRNA synthetase (IleRS) inhibitor, is already commercially available as a topical treatment for MRSA infections. Unfortunately, resistance developed soon after its introduction on the market, hampering its clinical use. Therefore, there is an urgent need for new cellular targets or improved therapies. Follow-up research by Cubist Pharmaceuticals led to a series of selective and in vivo active aminoacyl-sulfamoyl aryltetrazole inhibitors targeting IleRS (e.g. CB 168).

Here, we describe the synthesis of new IleRS and TyrRS inhibitors based on the Cubist Pharmaceuticals compounds, whereby the central ribose was substituted for a tetrahydropyran ring. Various linkers were evaluated connecting the six-membered ring with the base-mimicking part of the synthesized analogues. Out of eight novel molecules, a three-atom spacer to the phenyltriazole moiety, which was established using azide-alkyne click chemistry, appeared to be the optimized linker to inhibit IleRS. However, 11 (Ki,app = 88 ± 5.3 nM) and 36a (Ki,app = 114 ± 13.5 nM) did not reach the same level of inhibitory activity as for the known high-affinity natural adenylate-intermediate analogue isoleucyl-sulfamoyl adenosine (IleSA, CB 138; Ki,app = 1.9 ± 4.0 nM) and CB 168, which exhibit a comparable inhibitory activity as the native ligand. Therefore, 11 was docked into the active site of IleRS using a known crystal structure of T. thermophilus in complex with mupirocin. Here, we observed the loss of the crucial 3′- and 4′- hydroxyl group interactions with the target enzyme compared to CB 168 and mupirocin, which we suggest to be the reason for the limited decrease in enzyme affinity. Despite the lack of antibacterial activity, we believe that structurally optimizing these novel analogues via a structure-based approach could ultimately result in aaRS inhibitors which would help to tackle the antibiotic resistance problem.

Introduction

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes for protein synthesis as they are required for a correct attachment of amino acids to their corresponding tRNA molecule.1 These enzymes are divided in two classes based on their different overall fold.2 Class I aaRSs contain a Rossmann binding fold, which is characterized by the conserved KMSKS and HIGH sequence motifs.3 On the contrary, the active site of Class II aaRS synthetases have a unique seven-stranded β-sheet flanked by α-helices. Despite these conserved features, it was shown that selective targeting of bacterial isoleucyl-tRNA synthetase (IleRS) is feasible. Polyketide IleRS inhibitor mupirocin (Fig. 1A)4 was approximately 8000 times more active against Gram-positive bacteria versus human cells.1 Unfortunately, its activity spectrum is in practice restricted to Gram-positive bacteria due to poor penetration through the outer membrane of Gram-negative variants.5 Nevertheless, mupirocin exhibits a remarkable activity towards the ESKAPE pathogen MRSA in particular. The ESKAPE pathogens are bacteria that are often resistant to many of the available antibiotics, and mupirocin therefore is included on the WHO list of essential medicines in 2019.6

However, low- and high-level resistance in Gram-positive bacteria against mupirocin appeared quickly after it was clinically introduced in 1985 because of decolonization failure and its widespread use led to an increase in resistance.7 Low-level resistance (Minimal Inhibitory Concentration (MIC) < 64 µg/mL) arises when point mutations emerge during bacterial proliferation affecting the Rossmann fold in the synthetase.8 However, the main concern is about the high-level resistance (MIC > 512 µg/mL), originating from bacteria expressing a novel IleRS9 of which the plasmid encoding for the resistant enzyme (mupA gene) can be passed on via conjugal mating.10 Nonetheless, the emergence of cross-resistance is quite unlikely due to the rather unexplored target. Halofuginone and tavaborole (Fig. 1A) are the only other two commercially available aaRS inhibitors. Halofuginone is a competitive inhibitor competing with proline in the active site of ProRS and is solely used in cattle breeding.11 It is a non-toxic derivative of the plant alkaloid febrifugine, which was historically recognized for its antiprotozoal activity. Tavaborole on the other hand is a topical antifungal drug for the treatment of onychomycosis which targets the editing site of LeuRS.12 Its boron atom makes a covalently bound complex with the ribose hydroxyl groups of the 3′-terminal adenosine of a tRNALeu molecule, prohibiting the charging with leucine. Also, resistance to tavaborole after repeated exposure has not been demonstrated,13 unlike mupirocin.

Therefore, research started at the end of the 1990s in order to find new IleRS inhibitors. SmithKline Beecham Pharmaceuticals (now GSK) synthesized analogues of mupirocin whereby they replaced the labile ester functional group in the long aliphatic chain of mupirocin with various heterocyclic groups in order to avoid systemic breakdown to monic acid A.14, 15 Most of these new compounds could not reach the same antibacterial activity due to poor cell wall penetration, but increased polarity in subsequent research resulted in a compound which had potential to be systemically used in vivo.16 Despite these promising results, no further follow-up was reported. Cubist Pharmaceuticals (since 2015 acquired by Merck & Co.) used the high-affinity aminoacyl-sulfamate adenosines (aaSAs, Fig. 1B) as a starting point to synthesize IleRS inhibitors with improved chemical stability and bacterial selectivity. Substituted thiazoles were directly attached to the ribose ring as well, replacing the adenine group.17 No antibacterial activity was reported for these selective molecules in contrast to another compound series in which the sugar ring was connected with aryl-substituted tetrazoles via a two-carbon atom linker (Fig. 1B).18 This approach gave rise to selective IleRS inhibitors of which the phenoxyphenyltetrazole (CB 432, Fig. 1B) in particular had antibacterial activity. However, research was halted due to its low bioavailability in vivo.

A recent report by the WHO stated that 10 million people would decease every year by 2050 due to antibacterial resistance.19 Therefore, antibiotic research awakened with several global action plans as a result (e.g. Joint programming initiative on AntiMicrobial Resistance (AMR), Transatlantic task force on AMR, Global action plan on AMR, New drugs for bad bugs and only very recently the AWaRe campaign) and triggered academic groups to find new cellular targets or improve existing therapies. For example, Oxford Drug Design synthesized N-leucinyl benzenesulfonamides, based on previously reported benzenesulfonamide aaRS inhibitors,20, 21 which selectively and strongly inhibited E. coli LeuRS.22 More specifically for mupirocin, it recently was shown by Lounsbury et al. that a combination therapy could resensitize mupirocin-resistant bacteria.23

Following this trend, we envisioned to design novel inhibitors based on the tetrazole approach of Cubist Pharmaceuticals (Fig. 1C). The ribose of the latter compounds was replaced by a tetrahydropyran ring as in mupirocin, however with a different implantation of both side chains on this ring. The base-mimicking chain was moved to the C2 position in analogy with the base connection in 1,5-anhydrohexitol nucleosides (HNA), which have been extensively studied in our research group for other purposes.24, 25, 26 The sulfamate-containing chain was consequently shifted to C5 next to the ring oxygen, preserving a 1,4-cis relationship of both substituents on the tetrahydropyran ring. The sulfamate was further coupled to isoleucine or tyrosine to target IleRS and Class I TyrRS, respectively. Additionally, the importance of the epoxide functional group in mupirocin was investigated by introducing an epoxide-mimicking chain in our compounds because in the mupirocin-IleRS complex structure (PDB entry 1JZS) no significant interactions with this part of the molecule are noted. The optimal distance between the tetrahydropyran ring and the heterocycle needed evaluation as well because our six-membered ring is less flexible than the ribose ring used by Cubist Pharmaceuticals.

Following synthesis of all compounds, the enzymatic and antibacterial activity was evaluated. We here show that using this altered scaffold it is feasible to ultimately develop new aaRS inhibitors which can contribute in the fight against the antibiotic resistance problem.

Section snippets

Synthesis of phenyltriazoles/tetrazoles with different linkers

The new implantation of both side chains on the pyran ring provoked some uncertainty regarding how the molecules would bind inside the active site of the synthetase. Consequently, the optimal aliphatic linker between the six-membered ring and the heterocycle for obtaining the best ligand-enzyme interactions needed to be determined. We started out synthesizing a C3 spacer linking the pyran ring to a 1,4-phenyl-substituted 1,2,3-triazole (Scheme 1). This heterocyclic scaffold was chosen because

Discussion and conclusion

Mupirocin has become the most prescribed topical antibiotic and is used as a last resort for treating MRSA infections of open wounds owing to its rather unexplored mechanism of action, the inhibition of isoleucyl-tRNA synthetase (IleRS).33 Unfortunately, the emergence of high-level resistance against mupirocin appeared soon after it was clinically introduced.7 Therefore, pharmaceutical companies have tried to develop analogues of mupirocin based on monic acid A or bisubstrate inhibitors

Reagents and analytical procedures

Reagents and solvents were purchased from commercial suppliers (Acros, Sigma-Aldrich) and used as provided, unless indicated otherwise. DMF, THF, DCM and MeOH were of analytical grade and were stored over 4 Å molecular sieves. All other solvents used for reactions were analytical grade and used as provided. Reactions were carried out in oven-dried glassware under a nitrogen atmosphere with stirring at room temperature, unless indicated otherwise. 14C-radiolabeled amino acids and scintillation

Funding

This work was supported by the Research Foundation - Flanders [Fonds voor Wetenschappelijk Onderzoek, 1109117N to D.D.R., 1S23217N to C.M., G077814N to S.S. and A.V., G0A4616N to S.W. and A.V., 1S53516N to S.D.G.]; the KU Leuven Research Fund [3 M14022 to S.W. and A.V.]; and the Chinese Scholarship Council [to L.P.].

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors thank the Belgian FWO for providing a PhD Fellowship of the Research Foundation - Flanders to Dries De Ruysscher and SB Fellowships to Charles-Alexandre Mattelaer and Steff De Graef; the China Scholarship Council for providing a scholarship to Luping Pang and KU Leuven for financial support. Mass spectrometry was made possible by the support of the Hercules Foundation of the Flemish Government [20100225E7]. We are indebted to Davie Cappoen and Paul Cos of the Laboratory of

References (37)

  • J.J. Perona et al.

    Structural diversity and protein engineering of the Aminoacyl-tRNA synthetases

    Biochemistry

    (2012)
  • C.M. Thomas et al.

    Resistance to and synthesis of the antibiotic mupirocin

    Nat Rev Microbiol

    (2010)
  • J.E. Hodgson et al.

    Molecular characterization of the gene encoding high-level mupirocin resistance in staphylococcus aureus J2870

    Antimicrob Agents Chemother

    (1994)
  • Essential medicines and health products. WHO model lists of essential medicines....
  • J.B. Patel et al.

    Mupirocin resistance

    Clin Infect Dis

    (2009)
  • M. Antonio et al.

    Mutations affecting the Rossman fold of isoleucyl-tRNA synthetase are correlated with low-level mupirocin resistance in Staphylococcus aureus

    Antimicrob Agents Chemother

    (2002)
  • E.E. Udo et al.

    Genetic analysis of methicillin-resistant Staphylococcus aureus expressing high- and low-level mupirocin resistance

    J Med Microbiol

    (2001)
  • J.G. Hurdle et al.

    In vivo transfer of high-level mupirocin resistance from Staphylococcus epidermidis to methicillin-resistant Staphylococcus aureus associated with failure of mupirocin prophylaxis

    J Antimicrob Chemother

    (2005)
  • Cited by (6)

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