Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination

Woods, Lucy A.; Dolezal, Olan; Ren, Bin; Ryan, John H.; Peat, Thomas S.; Poulsen, Sally‐Ann

doi:10.1021/acs.jmedchem.5b01940

Cited by 53 publications

(82 citation statements)

References 70 publications

(108 reference statements)

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“…Automated nESI‐MS screenings of a fragment library have been reported by several groups, and are typically achieved with the aid of a liquid handling and sample infusion robot . However, without access to such automation, a smaller library of 80 fragments was employed for the proof of concept of the use of native MS for screening fragments against a protein–DNA interaction.…”

Section: Figuresupporting

confidence: 53%

“…Validation and characterization of hits is usually performed by a combination of isothermal calorimetry (ITC), protein‐observed NMR spectroscopy, and X‐ray crystallography . Whereas the use of nanoelectrospray ionization mass spectrometry (nESI‐MS) to study biomolecular assemblies has been well‐documented, native MS is a relatively underutilized technique, especially in fragment screening for drug discovery …”

Section: Figurementioning

confidence: 99%

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Fragment Screening against the EthR–DNA Interaction by Native Mass Spectrometry

et al. 2017

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Native nanoelectrospray ionization mass spectrometry is an underutilized technique for fragment screening. In this study, the first demonstration is provided of the use of native mass spectrometry for screening fragments against a protein–DNA interaction. EthR is a transcriptional repressor of EthA expression in Mycobacterium tuberculosis (Mtb) that reduces the efficacy of ethionamide, a second‐line antitubercular drug used to combat multidrug‐resistant Mtb strains. A small‐scale fragment screening campaign was conducted against the EthR–DNA interaction using native mass spectrometry, and the results were compared with those from differential scanning fluorimetry, a commonly used primary screening technique. Hits were validated by surface plasmon resonance and X‐ray crystallography. The screening campaign identified two new fragments that disrupt the EthR–DNA interaction in vitro (IC50=460–610 μm) and bind to the hydrophobic channel of the EthR dimer.

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Section: Figuresupporting

confidence: 53%

Section: Figurementioning

confidence: 99%

Fragment Screening against the EthR–DNA Interaction by Native Mass Spectrometry

et al. 2017

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“…Native MS has recently attracted further interest in the field of FBS and has been employed in a few studies for screening and additional support in hit validation . In a recent work, the Poulsen group screened a library of 70 fragments independently with native MS and SPR against human carbonic anhydrase II with a good agreement of 80 % for the two screening methods . This study illustrates that native MS is able to fit in the fragment‐screening cascade.…”

Section: Introductionmentioning

confidence: 76%

“…A further question that arises from our experiments is how to rank hits according to their apparent affinity. In previous native MS studies, fragments were ranked either by their dissociation constant or classified in different binding categories (A+, A, B) based on their fraction of complex formation . Of course, the most accurate way of affinity ranking would be to determine a dissociation constant.…”

Section: Discussionmentioning

confidence: 99%

Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment‐Based Screening

et al. 2017

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Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention in recent years. So far, only a few studies have discussed mass spectrometry as a screening technology for fragments. Herein, we report the application of native electrospray ionization mass spectrometry (MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screening conducted with a thermal shift assay (TSA) and represented different binding categories. Our data indicated that, beside specific complex formation, many fragments show extensive multiple binding and also charge-state shifts. Both of these factors complicate automated data analysis and decrease the attractiveness of native MS as a primary screening tool for fragments. A comparison of the hits identified by native MS and TSA showed good agreement for two of the proteins. Furthermore, we discuss general challenges, including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS to be a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening.

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“…We evaluated PAL probe–protein binding, protein crosslinking efficiency, and CuAAC efficiency of probes 1 – 11 by use of stepwise analysis by native state mass spectrometry and in‐gel fluorescence. Native state nanoelectrospray ionisation mass spectrometry (nanoESI‐MS) can be used for direct observation of native state proteins and protein complexes (covalent or noncovalent) and was employed in this study to evaluate the efficiency of the probes for binding and subsequent crosslinking to CA II. The nanoESI‐MS source was interfaced with a Bruker SolariX XR 12.0 Tesla Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) fitted with a ParaCell, and samples were introduced by direct infusion.…”

Section: Resultsmentioning

confidence: 99%

Characterisation of Photoaffinity‐Based Chemical Probes by Fluorescence Imaging and Native‐State Mass Spectrometry

et al. 2017

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Chemical probes are small-molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer-associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug-resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein-probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein-probe binding, and covalent protein-probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure-activity relationships to support the development of optimum chemical probes.

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Native State Mass Spectrometry, Surface Plasmon Resonance, and X-ray Crystallography Correlate Strongly as a Fragment Screening Combination

Cited by 53 publications

References 70 publications

Fragment Screening against the EthR–DNA Interaction by Native Mass Spectrometry

Fragment Screening against the EthR–DNA Interaction by Native Mass Spectrometry

Critical Evaluation of Native Electrospray Ionization Mass Spectrometry for Fragment‐Based Screening

Characterisation of Photoaffinity‐Based Chemical Probes by Fluorescence Imaging and Native‐State Mass Spectrometry

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