Development of NanoBRET cellular target engagement assays in primary neurons for activating mutants of p21-activated kinase 1

Abstract 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 primary neurons. Competing Interest Statement The authors have declared no competing interest. Abbreviation - ACN - acetonitrile - AD - Alzheimer’s disease - AID - autoinhibitory domain - ATP - adenosine 5′-triphosphate - Boc - tert-butyloxycarbonyl - BRET - bioluminescence resonance energy transfer - CDC42 - cell division cycle 42 - CDI - 1,1′-carbonyldiimidazole - cd3od - methanol-d4 - DCM - dichloromethane - DIPEA - N,N-diisopropylethylamine - DMEM - Dulbecco’s Modified Eagle Medium - DMF - N,N-dimethylformamide - DMSO - dimethyl sulfoxide - DMSO-d6 - dimethyl sulfoxide-d6 - DNA - deoxyribonucleic acid - EC50 - half-maximal effective concentration - FBS - fetal bovine serum - FL - full-length - HATU - 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate - HEK293 - human embryonic kidney 293 - HPLC - high-performance liquid chromatography - IC50 - half-maximal inhibitory concentration - Ki,app - apparent inhibition constant - Km - Michaelis constant - LCMS - liquid chromatography–mass spectrometry - MAPK - mitogen-activated protein kinase - mBU - milliBRET units - MeI - methyl iodide - MOI - multiplicity of infection - NanoBRET - nanoluciferase-based bioluminescence resonance energy transfer - NB - NanoBRET - NLuc - Nanoluciferase - NMR - nuclear magnetic resonance - PAK - p21-activated kinase - PEG - polyethylene glycol - PROTAC - proteolysis-targeting chimera - Rac1 - Ras-related C3 botulinum toxin substrate 1 - SEM - standard error of the mean - SGC - Structural Genomics Consortium - SIK - salt-inducible kinase - SYK - spleen tyrosine kinase - TEA - triethylamine - TFA - trifluoroacetic acid - WNT - wingless-related integration site - WT - wild-type.