title: Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b.
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.
authors.affiliation: Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University; ye-han@northwestern.edu.
authors.forename: Ye
authors.initials: Y
authors.lastname: Han
authors.affiliation: Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University.
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Lyman
authors.affiliation: Center for Molecular Innovation and Drug Discovery, Northwestern University.
authors.forename: Matt
authors.initials: M
authors.lastname: Clutter
authors.affiliation: Department of Pharmacology, Feinberg School of Medicine, Northwestern University.
authors.forename: Gary E
authors.initials: GE
authors.lastname: Schiltz
authors.affiliation: Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University.
authors.forename: Quratul-Ain
authors.initials: QA
authors.lastname: Ismail
authors.affiliation: Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University.
authors.forename: Xiangying
authors.initials: X
authors.lastname: Cheng
authors.affiliation: High Throughput Analysis Laboratory, Department of Molecular Biosciences, Northwestern University.
authors.forename: Chi-Hao
authors.initials: CH
authors.lastname: Luan
authors.affiliation: Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University; Department of Physiology, Feinberg School of Medicine, Northwestern University.
authors.forename: Dane M
authors.initials: DM
authors.lastname: Chetkovich
mesh_terms: D003865:Major Depressive Disorder / Q000378:metabolism / Q000628:therapy; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000378:metabolism*; D008565:Membrane Proteins; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D054852:Small Molecule Libraries
chemical_list: D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D008565:Membrane Proteins; C529762:PEX5L protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D054852:Small Molecule Libraries