eSPC: an online data-analysis platform for molecular biophysics

Burastero, Osvaldo; Niebling, Stephan; Defelipe, Lucas A.; Günther, Christian; Struve, Angelica; Alai, Maria Garcia

doi:10.1107/s2059798321008998

Cited by 34 publications

(28 citation statements)

References 35 publications

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“…Data were analyzed and visualized with self-written python scripts using the Python modules Numpy ( Oliphant, 2006 ; van der Walt et al, 2011 ), Matplotlib ( Hunter, 2007 ), Scipy ( Virtanen et al, 2020 ) and Pandas ( McKinney, 2010 ) and the publicly available eSPC data analysis platform ( Burastero et al, 2021 ). The fluorescence titration of T5 was fitted with a simple 1:1 binding model.…”

Section: Methodsmentioning

confidence: 99%

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease

et al. 2022

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The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.

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Section: Methodsmentioning

confidence: 99%

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease

et al. 2022

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“…The fluorescence signal was fitted to a one-site binding model using the ThermoAffinity online tool. 56 Asymmetric confidence intervals were calculated as suggested by Paketurytė et al , 2021. 39 …”

Section: Methodsmentioning

confidence: 99%

“… 42 , 57 Data analysis was performed using the FoldAffinity online tool. 40 , 56 For the isothermal analysis, we used 35 to 50 °C as the temperature window and the “local” option to fit the melting curves. Asymmetric confidence intervals were calculated as previously mentioned.…”

Section: Methodsmentioning

confidence: 99%

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Cosolvent Sites-Based Discovery ofMycobacterium TuberculosisProtein Kinase G Inhibitors

et al. 2022

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Computer-aided drug discovery methods play a major role in the development of therapeutically important small molecules, but their performance needs to be improved. Molecular dynamics simulations in mixed solvents are useful in understanding protein–ligand recognition and improving molecular docking predictions. In this work, we used ethanol as a cosolvent to find relevant interactions for ligands toward protein kinase G, an essential protein of Mycobacterium tuberculosis ( Mtb ). We validated the hot spots by screening a database of fragment-like compounds and another one of known kinase inhibitors. Next, we performed a pharmacophore-guided docking simulation and found three low micromolar inhibitors, including one with a novel chemical scaffold that we expanded to four derivative compounds. Binding affinities were characterized by intrinsic fluorescence quenching assays, isothermal titration calorimetry, and the analysis of melting curves. The predicted binding mode was confirmed by X-ray crystallography. Finally, the compounds significantly inhibited the viability of Mtb in infected THP-1 macrophages.

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“…Mass photometry data acquired on a Refeyn instrument can be analyzed using the proprietary software that requires a license by Refeyn DiscoverMP. Here, we are releasing a new user-friendly software module for MP analysis and publicly available at the eSPC data analysis platform: spc.embl-hamburg.de ( Burastero et al, 2021 ). This is particularly important when the proprietary software is not accessible.…”

Section: Stepwise Proceduresmentioning

confidence: 99%

Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins

Niebling

Veith

Vollmer

et al. 2022

Front. Mol. Biosci.

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Successful sample preparation is the foundation to any structural biology technique. Membrane proteins are of particular interest as these are important targets for drug design, but also notoriously difficult to work with. For electron cryo-microscopy (cryo-EM), the biophysical characterization of sample purity, homogeneity, and integrity as well as biochemical activity is the prerequisite for the preparation of good quality cryo-EM grids as these factors impact the result of the computational reconstruction. Here, we present a quality control pipeline prior to single particle cryo-EM grid preparation using a combination of biophysical techniques to address the integrity, purity, and oligomeric states of membrane proteins and its complexes to enable reproducible conditions for sample vitrification. Differential scanning fluorimetry following the intrinsic protein fluorescence (nDSF) is used for optimizing buffer and detergent conditions, whereas mass photometry and dynamic light scattering are used to assess aggregation behavior, reconstitution efficiency, and oligomerization. The data collected on nDSF and mass photometry instruments can be analyzed with web servers publicly available at spc.embl-hamburg.de. Case studies to optimize conditions prior to cryo-EM sample preparation of membrane proteins present an example quality assessment to corroborate the usefulness of our pipeline.

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eSPC: an online data-analysis platform for molecular biophysics

Cited by 34 publications

References 35 publications

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease

Hydrazones and Thiosemicarbazones Targeting Protein-Protein-Interactions of SARS-CoV-2 Papain-like Protease

Cosolvent Sites-Based Discovery ofMycobacterium TuberculosisProtein Kinase G Inhibitors

Biophysical Screening Pipeline for Cryo-EM Grid Preparation of Membrane Proteins

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