A general assay platform to study protein pharmacology using ligand-dependent structural dynamics
Abstract Drug design strategies represent a fundamental challenge in chemical biology that could benefit from the development of next-generation high-throughput assays. Here we demonstrate that structural dynamic changes induced by ligand binding can be transmitted to a sensor protein fused to a tar...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-59658-6 |
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| author | Daniel A. Ciulla Patricia K. Dranchak Mahesh Aitha Renier H. P. van Neer Divia Shah Ravi Tharakan Kelli M. Wilson Yuhong Wang John C. Braisted James Inglese |
| author_facet | Daniel A. Ciulla Patricia K. Dranchak Mahesh Aitha Renier H. P. van Neer Divia Shah Ravi Tharakan Kelli M. Wilson Yuhong Wang John C. Braisted James Inglese |
| author_sort | Daniel A. Ciulla |
| collection | DOAJ |
| description | Abstract Drug design strategies represent a fundamental challenge in chemical biology that could benefit from the development of next-generation high-throughput assays. Here we demonstrate that structural dynamic changes induced by ligand binding can be transmitted to a sensor protein fused to a target protein terminus. Here, NanoLuc luciferase, used as the intact protein or its α-complementation peptide, was fused to seven proteins from distinct enzyme superfamilies resulting in sensitive ligand-dependent bioluminescent outputs. This finding allows a general non-competitive, function-independent, quantitative, isothermal gain-of-signal ligand binding readout. As applied to chemical library high throughput screening, we can observe multivariate pharmacologic outputs including cofactor-induced synergy in ligand binding, as well as an example of allosteric site binding. The structural dynamics response assay format described here can enable the investigation of proteins precluded from study due to cost-prohibitive, insensitive, or technically challenging assays, including from cell lysates containing endogenously expressed gene edited proteins. |
| format | Article |
| id | doaj-art-dfa9a22d696a4f59b6f2f9530d24a300 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-dfa9a22d696a4f59b6f2f9530d24a3002025-08-20T03:52:53ZengNature PortfolioNature Communications2041-17232025-05-0116111610.1038/s41467-025-59658-6A general assay platform to study protein pharmacology using ligand-dependent structural dynamicsDaniel A. Ciulla0Patricia K. Dranchak1Mahesh Aitha2Renier H. P. van Neer3Divia Shah4Ravi Tharakan5Kelli M. Wilson6Yuhong Wang7John C. Braisted8James Inglese9Division of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, NIHAbstract Drug design strategies represent a fundamental challenge in chemical biology that could benefit from the development of next-generation high-throughput assays. Here we demonstrate that structural dynamic changes induced by ligand binding can be transmitted to a sensor protein fused to a target protein terminus. Here, NanoLuc luciferase, used as the intact protein or its α-complementation peptide, was fused to seven proteins from distinct enzyme superfamilies resulting in sensitive ligand-dependent bioluminescent outputs. This finding allows a general non-competitive, function-independent, quantitative, isothermal gain-of-signal ligand binding readout. As applied to chemical library high throughput screening, we can observe multivariate pharmacologic outputs including cofactor-induced synergy in ligand binding, as well as an example of allosteric site binding. The structural dynamics response assay format described here can enable the investigation of proteins precluded from study due to cost-prohibitive, insensitive, or technically challenging assays, including from cell lysates containing endogenously expressed gene edited proteins.https://doi.org/10.1038/s41467-025-59658-6 |
| spellingShingle | Daniel A. Ciulla Patricia K. Dranchak Mahesh Aitha Renier H. P. van Neer Divia Shah Ravi Tharakan Kelli M. Wilson Yuhong Wang John C. Braisted James Inglese A general assay platform to study protein pharmacology using ligand-dependent structural dynamics Nature Communications |
| title | A general assay platform to study protein pharmacology using ligand-dependent structural dynamics |
| title_full | A general assay platform to study protein pharmacology using ligand-dependent structural dynamics |
| title_fullStr | A general assay platform to study protein pharmacology using ligand-dependent structural dynamics |
| title_full_unstemmed | A general assay platform to study protein pharmacology using ligand-dependent structural dynamics |
| title_short | A general assay platform to study protein pharmacology using ligand-dependent structural dynamics |
| title_sort | general assay platform to study protein pharmacology using ligand dependent structural dynamics |
| url | https://doi.org/10.1038/s41467-025-59658-6 |
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