Discovery and structure–activity relationship studies of irreversible benzisothiazolinone-based inhibitors against Staphylococcus aureus sortase A transpeptidase

Zhulenkovs, Dmitrijs; Rudevica, Zhanna; Jaudzems, Kristaps; Turks, Māris; Leonchiks, Ainars

doi:10.1016/j.bmc.2014.09.011

Cited by 58 publications

(91 citation statements)

References 49 publications

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“…The inhibitor used here ( Figure S4B) is known to bind irreversibly by formation of a covalent bond to the active-site cysteine. 18 As expected for a system with slow or no chemical exchange, the tert-butyl resonances of the inhibitor bound and unbound states of the protein appeared simultaneously at half-saturation ( Figure 2B). A control experiment confirmed that the dimethyl sulfoxide (DMSO) of the inhibitor stock solution caused much smaller chemical shift changes than the ligand ( Figure S3B).…”

Section: ■ Resultssupporting

confidence: 73%

O-tert-Butyltyrosine, an NMR Tag for High-Molecular-Weight Systems and Measurements of Submicromolar Ligand Binding Affinities

Chen

Kuppan

Lee³

et al. 2015

J. Am. Chem. Soc.

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O-tert-Butyltyrosine (Tby) is an unnatural amino acid that can be site-specifically incorporated into proteins using established orthogonal aminoacyl-tRNA synthetase/tRNA systems. Here we show that the tert-butyl group presents an outstanding NMR tag that can readily be observed in one-dimensional 1 H NMR spectra without any isotope labeling. Owing to rapid bond rotations and the chemical equivalence of the protons of a solvent-exposed tert-butyl group from Tby, the singlet resonance from the tert-butyl group generates an easily detectable narrow signal in a spectral region with limited overlap with other methyl resonances. The potential of the tert-butyl 1 H NMR signal in protein research is illustrated by the observation and assignment of two resonances in the Bacillus stearothermophilus DnaB hexamer (320 kDa), demonstrating that this protein preferentially assumes a 3-fold rather than 6-fold symmetry in solution, and by the quantitative measurement of the submicromolar dissociation constant K d (0.2 μM) of the complex between glutamate and the Escherichia coli aspartate/ glutamate binding protein (DEBP, 32 kDa). The outstanding signal height of the 1 H NMR signal of the Tby tert-butyl group allows K d measurements using less concentrated protein solutions than usual, providing access to K d values 1 order of magnitude lower than established NMR methods that employ direct protein detection for K d measurements.

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Section: ■ Resultssupporting

confidence: 73%

O-tert-Butyltyrosine, an NMR Tag for High-Molecular-Weight Systems and Measurements of Submicromolar Ligand Binding Affinities

Chen

Kuppan

Lee³

et al. 2015

J. Am. Chem. Soc.

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“…39, 40, 41 Although there is high amino acid sequence homology (29%) between the SrtA of S. aureus and S. mutans , 14 there is no evidence that homologous proteins have the same inhibitors. Moreover, compared with S. mutans , the major pathogen of dental caries, S. aureus is a leading cause of hospital- and community-acquired infections ranging from minor skin infections to osteomyelitis, meningitis, endocarditis, septicaemia and toxic shock syndrome.…”

Section: Discussionmentioning

confidence: 99%

In silico identification of potential inhibitors targeting Streptococcus mutans sortase A

et al. 2017

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Dental caries is one of the most common chronic diseases and is caused by acid fermentation of bacteria adhered to the teeth. Streptococcus mutans (S. mutans) utilizes sortase A (SrtA) to anchor surface proteins to the cell wall and forms a biofilm to facilitate its adhesion to the tooth surface. Some plant natural products, especially several flavonoids, are effective inhibitors of SrtA. However, given the limited number of inhibitors and the development of drug resistance, the discovery of new inhibitors is urgent. Here, the high-throughput virtual screening approach was performed to identify new potential inhibitors of S. mutans SrtA. Two libraries were used for screening, and nine compounds that had the lowest scores were chosen for further molecular dynamics simulation, binding free energy analysis and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties analysis. The results revealed that several similar compounds composed of benzofuran, thiadiazole and pyrrole, which exhibited good affinities and appropriate pharmacokinetic parameters, were potential inhibitors to impede the catalysis of SrtA. In addition, the carbonyl of these compounds can have a key role in the inhibition mechanism. These findings can provide a new strategy for microbial infection disease therapy.

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“…The structure of the SrtA: 2‐salt complex is the first high‐resolution structure of a SrtA‐type enzyme bound to a small‐molecule inhibitor. Previously, Zhulenkovs and colleagues solved the NMR structure of S. aureus SrtA in complex with a benzisothiazolinone‐based inhibitor . However, the co‐ordinates of the inhibitor were not well defined, as only nine intermolecular enzyme–inhibitor NOEs defined its positioning.…”

Section: Discussionmentioning

confidence: 99%

“…This is because the active site of SrtA is structurally disordered in its apo‐state making it difficult to model drug–enzyme interactions computationally and potentially hindering the application of X‐ray crystallography that require crystallization of the SrtA–inhibitor complex. At present, only Zhulenkov and colleagues have used NMR spectroscopy to visualize how SrtA binds to an inhibitor, a benzisothiazolinone‐based small molecule that irreversibly modifies the enzyme . However, the structure of the inhibitor–SrtA complex was determined at low resolution.…”

Section: Introductionmentioning

confidence: 99%

“…At present, only Zhulenkov and colleagues have used NMR spectroscopy to visualize how SrtA binds to an inhibitor, a benzisothiazolinone-based small molecule that irreversibly modifies the enzyme. [16] However, the structure of the inhibitor-SrtA complex was determined at low resolution. Maresso, et al have also structurally characterized covalent inhibitors of the aryl (β-amino)ethyl ketone class by co-crystallizing the inhibitors with the sortase B from Bacillus anthracis (Ba-SrtB).…”

Section: Introductionmentioning

confidence: 99%

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NMR structure‐based optimization of Staphylococcus aureus sortase A pyridazinone inhibitors

Chan

Weiner

et al. 2017

Chem Biol Drug Des

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Staphylococcus aureus is a leading cause of hospital-acquired infections in the United States and is a major health concern as methicillin-resistant S. aureus (MRSA) and other antibiotic resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti-infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure-based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone-based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second generation analogs that are ~70-fold more potent than the lead molecule. These optimized molecules exhibit broad-spectrum activity against other types of class A sortases, have reduced cytotoxicity and impair SrtA-mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogs are attractive candidates for further development into anti-infective agents, and highlights the utility of employing NMR spectroscopy and solubility-optimized small molecules in structure-based drug discovery.

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Discovery and structure–activity relationship studies of irreversible benzisothiazolinone-based inhibitors against Staphylococcus aureus sortase A transpeptidase

Cited by 58 publications

References 49 publications

O-tert-Butyltyrosine, an NMR Tag for High-Molecular-Weight Systems and Measurements of Submicromolar Ligand Binding Affinities

O-tert-Butyltyrosine, an NMR Tag for High-Molecular-Weight Systems and Measurements of Submicromolar Ligand Binding Affinities

In silico identification of potential inhibitors targeting Streptococcus mutans sortase A

NMR structure‐based optimization of Staphylococcus aureus sortase A pyridazinone inhibitors

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