A general assay platform to study protein pharmacology using ligand-dependent structural dynamics

Daniel A. Ciulla, Patricia K. Dranchak, Mahesh Aitha, Renier H. P. Neer, Divia Shah, Ravi Tharakan, Kelli M. Wilson, Yuhong Wang, John C. Braisted and James Inglese ()
Additional contact information
Daniel A. Ciulla: NIH
Patricia K. Dranchak: NIH
Mahesh Aitha: NIH
Renier H. P. Neer: NIH
Divia Shah: NIH
Ravi Tharakan: NIH
Kelli M. Wilson: NIH
Yuhong Wang: NIH
John C. Braisted: NIH
James Inglese: NIH

Nature Communications, 2025, vol. 16, issue 1, 1-16

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-59658-6

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