Development of NanoBRET Cellular Target Engagement Assays in Primary Neurons for Activating Mutants of p21-Activated Kinase 1
The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimer’s disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in cerebral cells.
Authors
- Abdiel Badillo-Martinez (ORCID: https://orcid.org/0000-0002-6854-6136)
- Jacob L. Capener (ORCID: https://orcid.org/0000-0001-5051-2010)
- Bassel Awada (ORCID: https://orcid.org/0000-0003-4451-8533)
- Alison Donnelly Axtman (ORCID: https://orcid.org/0000-0003-4779-9932)
- Frances M. Bashore (ORCID: https://orcid.org/0000-0003-4241-9873)
- Zachary W. Davis‐Gilbert (ORCID: https://orcid.org/0000-0002-4070-0832)
- Thomas Krämer (ORCID: https://orcid.org/0000-0003-0507-7843)
- Hassan Al‐Ali (ORCID: https://orcid.org/0000-0002-8899-3443)
- Cameron S. Blair
Institutions
- University of North Carolina at Chapel Hill (US)
Publication Details
- Journal
- UNC Libraries
- Published
- 2026-10-30
- DOI
- https://doi.org/10.17615/pesw-wg70
- Primary Topic
- Protein Kinase Regulation and GTPase Signaling
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Howard Hughes Medical Institute
- U.S. Department of Energy
- Alfred P. Sloan Foundation
- March of Dimes Foundation
- Welch Foundation
- Gordon and Betty Moore Foundation
- American Heart Association
- Camille and Henry Dreyfus Foundation
- William K. Warren Foundation
- Pharmaceutical Research and Manufacturers of America Foundation
- Alexander von Humboldt-Stiftung
- Texas A and M University
- Cancer Research Society
- Lotte and John Hecht Memorial Foundation
- Robertson Foundation
- UK Research and Innovation
- Gaussian
- Ministerio de Ciencia, Innovación y Universidades
- Heinz Family Foundation
- Georgia Tech Foundation
- Canadian Lung Association
- National Institute for Health and Care Research
- British Heart Foundation
- Royal Society
- European Commission
- Commonwealth Scholarship Commission
- Deutsche Forschungsgemeinschaft
- Ministry of Education, Culture, Sports, Science and Technology
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- National Natural Science Foundation of China
- Bundesministerium für Bildung und Forschung
- Chinese Academy of Sciences
- China Postdoctoral Science Foundation
- Fonds Wetenschappelijk Onderzoek
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek
- Cambridge Trust
- Generalitat Valenciana
- Ministero dell’Istruzione, dell’Università e della Ricerca
- Karolinska Institutet
- Max-Planck-Gesellschaft
- Helse Vest
- Terry Fox Research Institute
- Riksbankens Jubileumsfond
- Fondazione Umberto Veronesi
- Otsuka Pharmaceutical
- Svenska Läkaresällskapet
- Yunnan Provincial Department of Education
- Xunta de Galicia
- Shanghai Key Laboratory of Peripheral Nerve and Microsurgery
- Maurice Wilkins Centre for Molecular Biodiscovery
- Lung Health Foundation
- European Academy of Dermatology and Venereology
- Ministero dell'Università e della Ricerca
- National Institutes of Health
- Directorate for Biological Sciences
- National Institute of Food and Agriculture
- American Chemical Society Petroleum Research Fund
- Horizon 2020 Framework Programme
- Leonard M. Miller School of Medicine, University of Miami
- Cornell Atkinson Center for Sustainability, Cornell University
- Kavli Institute at Cornell, Cornell University
- Canadian Institutes of Health Research
- Natural Sciences and Engineering Research Council of Canada
- Engineering and Physical Sciences Research Council
- Health Research Council of New Zealand
- Japan Science and Technology Agency
- Instituto de Salud Carlos III
- Estonian Research Competency Council
- Chinese Universities Scientific Fund
- European Regional Development Fund
- Faculty of Medicine, Chiang Mai University
- Agencia Estatal de Investigación
- National Key Research and Development Program of China
- Fundamental Research Funds for the Central Universities
- Research England
- National Research, Development and Innovation Office
- NIHR Nottingham Biomedical Research Centre
- National Institute on Aging
- National Heart, Lung, and Blood Institute
- National Institute of General Medical Sciences
- National Institute of Diabetes and Digestive and Kidney Diseases
- National Institute of Neurological Disorders and Stroke
- Division of Chemistry
- Division of Integrative Organismal Systems
- Basic Energy Sciences
- National Institute on Minority Health and Health Disparities
- Stanford Maternal and Child Health Research Institute
- Core Research for Evolutional Science and Technology
- Air Force Office of Scientific Research
- Army Research Office
- H2020 Marie Skłodowska-Curie Actions
- Energy Frontier Research Centers
- HORIZON EUROPE Marie Sklodowska-Curie Actions
- HORIZON EUROPE Health
- HORIZON EUROPE European Research Council