Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
Year: 2016
Type: article
Abstract: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows 'hit' compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that 'hit' compounds are not artifactual.
DOI: 10.3791/54540
Institution: Northwestern University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
Funders: Brain Research Foundation, Chicago Community Trust, Northwestern University, Chicago Biomedical Consortium, National Institutes of Health, Robert H. Lurie Comprehensive Cancer Center, National Institute of Mental Health, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke, National Center for Advancing Translational Sciences
Awards: G1164661550, G1284175734, G1382277591, G1624640492, Discovery of novel small molecule antidepressants, Medical Scientist Training Program, G317886719, G4159268500, Multidisciplinary Clinical and Translational Science (MCTS) Program (UL1), G6040778250, Evaluation of antidepressant-like effects of hippocampal HCN channel modulation, G6758896395, HCN channel trafficking in epilepsy, G9000046704, Training Program in Neurobiology of Information Storage, The Robert H. Lurie Comprehensive Cancer Center
OpenAlex ID: W2553188030