Discovery and optimisation of potent, selective, ethanolamine inhibitors of bacterial phenylalanyl tRNA synthetase

doi:10.1016/j.bmcl.2005.03.003

Bioorganic & Medicinal Chemistry Letters

Volume 15, Issue 9, 2 May 2005, Pages 2305-2309

https://doi.org/10.1016/j.bmcl.2005.03.003 Get rights and content

Abstract

High throughput screening of Staphylococcus aureus phenylalanyl tRNA synthetase (FRS) identified ethanolamine 1 as a sub-micromolar hit. Optimisation studies led to the enantiospecific lead 64, a single-figure nanomolar inhibitor. The inhibitor series shows selectivity with respect to the mammalian enzyme and the potential for broad spectrum bacterial FRS inhibition.

Graphical abstract

High throughput screening of Staphylococcus aureus phenylalanyl tRNA synthetase (FRS) identified an ethanolamine as a sub-micromolar hit. Optimisation studies led to the enantiospecific lead 64, a single-figure nanomolar inhibitor. The inhibitor series shows selectivity with respect to the mammalian enzyme and the potential for broad spectrum bacterial FRS inhibition.

Section snippets

Acknowledgments

We thank Margarita Rodriguez-Lens and David McNair for assistance with preparation of compounds 39/40 and 64/65, respectively, Greg Jonas for the chiral preparative chromatography to resolve 62/63; Doug Minick for assignment of absolute configuration; Stephen Rittenhouse and his team for antibacterial testing; and Nicola Wallis and Christine Richardson for useful discussions.

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Exploring the binding sites of Staphylococcus aureus phenylalanine tRNA synthetase: A homology model approach
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Validation data from the template (2RHQ) was used as the baseline to evaluate the model. Phenylalanine as the natural substrate, phenylalanyl-adenylate [21] and known inhibitors with reported microbiological activity were built as ligands using MOE-Builder [22,23], and the energy was minimised for each ligand. These ligands were docked by running MOE-Dock with default setting: Placement: Triangular Matcher, Rescoring 1: London ΔG, 30 poses were constructed for each compound and the best scoring model-ligand complexes were selected.
Increased resistance of MRSA (multidrug resistance Staphylococcus aureus) to anti-infective drugs is a threat to global health necessitating the development of anti-infectives with novel mechanisms of action. Phenylalanine tRNA synthetase (PheRS) is a unique enzyme of the aminoacyl-tRNA synthetases (aaRSs), which are essential enzymes for protein biosynthesis. PheRS is an (αb)₂ tetrameric enzyme composed of two alpha subunits (PheS) and two larger beta subunits (PheT). Our potential target in the drug development for the treatment of MRSA infections is the phenylalanine tRNA synthetase alpha subunit that contains the binding site for the natural substrate. There is no crystal structure available for S. aureus PheRS, therefore comparative structure modeling is required to establish a putative 3D structure for the required enzyme enabling development of new inhibitors with greater selectivity. The S. aureus PheRS alpha subunit homology model was constructed using Molecular Operating Environment (MOE) software. Staphylococcus haemolyticus PheRS was the main template while Thermus thermophilus PheRS was utilised to predict the enzyme binding with tRNA^phe. The model has been evaluated and compared with the main template through Ramachandran plots, Verify 3D and Protein Statistical Analysis (ProSA). The query protein active site was predicted from its sequence using a conservation analysis tool. Docking suitable ligands using MOE into the constructed model were used to assess the predicted active sites. The docked ligands involved the PheRS natural substrate (phenylalanine), phenylalanyl-adenylate and several described S. aureus PheRS inhibitors.
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Catalytic hydrogenation of nitro derivatives 12–14 gave corresponding amines 15–17 in good yields [43]. Different classes of amidine based minor groove binders exhibited activity against Gram-positive bacteria while the related QSAR studies revealed benzoxazole, benzimidazole and benzothiazole as necessary functionalities for adequate antibacterial activity [44–47]. For that reason, we decided to use these pharmacophores in the design and synthesis of here reported antibacterial compounds.
Current antibacterial chemotherapeutics are facing an alarming increase in bacterial resistance pressuring the search for novel agents that would expand the available therapeutic arsenal against resistant bacterial pathogens. In line with these efforts, a series of 9 amidine derivatives of 3,4-ethylenedioxythiophene were synthesized and, together with 18 previously synthesized analogs, evaluated for their relative DNA binding affinity, in vitro antibacterial activities and preliminary in vitro safety profile. Encouraging antibacterial activity of several subclasses of tested amidine derivatives against Gram-positive (including resistant MRSA, MRSE, VRE strains) and Gram-negative bacterial strains was observed. The bis-phenyl derivatives were the most antibacterially active, while compound 19 from bis-benzimidazole class exhibited the widest spectrum of activity (with MIC of 4, 2, 0.5 and ≤0.25 μg/ml against laboratory strains of Staphyloccocus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, respectively and 4–32 μg/ml against clinical isolates of sensitive and resistant S. aureus, Staphylococcus epidermidis and Enterococcus faecium) and also demonstrated the strongest DNA binding affinity (ΔT_m of 15.4 °C). Asymmetrically designed compounds and carboxamide-amidines were, in general, less active. Molecular docking indicated that the shape of the 3,4-ethylenedioxythiophene derivatives and their ability to form multiple electrostatic and hydrogen bonds with DNA, corresponds to the binding modes of other minor-groove binders. Herein reported results encourage further investigation of this class of compounds as novel antibacterial DNA binding agents.
The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability
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Citation Excerpt :
Mitochondrial PheRS is a monomeric protein that appears to have evolved from an αβ chimeric protein (14). These structural differences hold promise for the development of selective inhibitors, as demonstrated by the discovery of a wide array of bacterial PheRS inhibitors with broad biochemical spectrum and selectivity against their human counterparts, such as spirocyclic furans and pyrrolidines (15, 16), ethanolamines (17), and benzyl phenyl ethers (18). Unfortunately, the antimicrobial activity of these compounds was marginal.
The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.
Preparation, antibacterial evaluation and preliminary structure-activity relationship (SAR) study of benzothiazol- and benzoxazol-2-amine derivatives
2012, Bioorganic and Medicinal Chemistry Letters
In this study, a novel benzothiazol- and benzooxazol-2-amine scaffold with antibacterial activity was designed and synthesized. Preliminary structure–activity relationship analysis displayed that compound 8t with a 5,6-difluorosubstituted benzothiazole was found to be a potent inhibitor of Gram-positive pathogens, and exhibited some potential against drug-resistant bacteria and without cytotoxicity in therapeutic concentrations. In addition, molecular docking studies indicated that Staphylococcus aureus methionyl-tRNA synthetase might be the possible target of these compounds. Taken together, the present study provides an effective entry to the synthesis of a good lead for subsequent optimization and a new small molecule candidate drug for antibacterial therapeutics.
Discovery and SAR of benzyl phenyl ethers as inhibitors of bacterial phenylalanyl-tRNA synthetase
2009, Bioorganic and Medicinal Chemistry Letters
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2009, European Journal of Medicinal Chemistry
Citation Excerpt :
A very potent analogue of 2 has been reported and is in clinical trials [12–14]. Recently, ethanolamine derivatives, a new class of inhibitors of bacterial phenylalanyl-tRNA synthetase [15], have been discovered by high throughput screening. Herein, we report the synthesis and the methionyl-tRNA synthetase inhibitory and antibacterial activities of a new series of 2-substituted-4-quinolones (26a, b, 39a–e, 32, 33, 36, 38).
New analogues of 2-[2-substituted-3-(3,4-dichlorobenzylamino)propylamino]quinolin-4-ones, 26a, 26b, 31a–e, 34, 35, 38 and 40, have been synthesized and evaluated against Staphylococcus aureus methionyl-tRNA synthetase. All of the synthesized compounds were less active than the reference compound 2. The compounds were also screened against various strains of S. aureus and Enterococci for their antibacterial activities. Among the compounds, 26b, 31c and 31e displayed significant inhibitory properties against various strains of Enterococci compared to compound 2.

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Discovery and optimisation of potent, selective, ethanolamine inhibitors of bacterial phenylalanyl tRNA synthetase

Abstract

Graphical abstract

Section snippets

Acknowledgments

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Antimicrob. Agents Chemother.

J. Med. Chem.

Biochemistry

Biochemistry

Biochemistry