Fragment-based screening by protein-detected NMR spectroscopy
Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information ab...
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| Vydané v: | Methods in enzymology Ročník 690; s. 285 |
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| Hlavní autori: | , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | English |
| Vydavateľské údaje: |
United States
2023
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| ISSN: | 1557-7988, 1557-7988 |
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| Abstract | Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and K
values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins. |
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| AbstractList | Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins.Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins. Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and K values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins. |
| Author | Peterson, Francis C Jensen, Davin R Nuñez, Raymundo Volkman, Brian F Smith, Brian C Zhou, Angela L Hill, R Blake Kerber, Paul J |
| Author_xml | – sequence: 1 givenname: Paul J surname: Kerber fullname: Kerber, Paul J organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States – sequence: 2 givenname: Raymundo surname: Nuñez fullname: Nuñez, Raymundo organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States – sequence: 3 givenname: Davin R surname: Jensen fullname: Jensen, Davin R organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States – sequence: 4 givenname: Angela L surname: Zhou fullname: Zhou, Angela L organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States – sequence: 5 givenname: Francis C surname: Peterson fullname: Peterson, Francis C organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States – sequence: 6 givenname: R Blake surname: Hill fullname: Hill, R Blake organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States – sequence: 7 givenname: Brian F surname: Volkman fullname: Volkman, Brian F email: bvolkman@mcw.edu organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States. Electronic address: bvolkman@mcw.edu – sequence: 8 givenname: Brian C surname: Smith fullname: Smith, Brian C email: brismith@mcw.edu organization: Department of Biochemistry, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States; Program in Chemical Biology, Medical College of Wisconsin, Watertown Plank Road, Milwaukee, WI, United States. Electronic address: brismith@mcw.edu |
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| Keywords | Thermostability Chemical shift perturbations Fragment-based drug discovery Difference intensity analysis Python scripts Nuclear magnetic resonance Biophysical techniques Structure–activity relationships K-means clustering Principal component analysis |
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| Title | Fragment-based screening by protein-detected NMR spectroscopy |
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