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Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Published: November 11th, 2016



1Davee Department of Neurology and Clinical Neurosciences, Feinberg School of Medicine, Northwestern University, 2Center for Molecular Innovation and Drug Discovery, Northwestern University, 3Department of Pharmacology, Feinberg School of Medicine, Northwestern University, 4High Throughput Analysis Laboratory, Department of Molecular Biosciences, Northwestern University, 5Department of Physiology, Feinberg School of Medicine, Northwestern University
* These authors contributed equally

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

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.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the heart and central nervous system where they play an important role in regulating membrane excitability1. HCN channels have been implicated in the pathogenesis of Major Depressive Disorder (MDD)2, which has led several groups to propose that limiting HCN channel function pharmacologically may be effective as a novel treatment for MDD3. However, directly targeting HCN channels is not viable because of their important role in the cardiac action potential4. Ivabradine, the only FDA approved HCN channel antagonist, is used for the treatment of....

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1. Purification of TRIP8b (241-602) Protein

  1. Transform the plasmid containing TRIP8b (241-602) in the bacterial protein expression vector pGS213 into competent E. coli for protein expression according to manufacturer's instructions. Plate 300 µl of the culture on Luria Broth (LB)-agar with 5 µg/ml of Chloramphenicol and Ampicillin. Incubate the plate at 37 °C for 16 h.
  2. The next day, pick a single colony to inoculate 50 ml LB with 50 µg/ml of Chloramphenicol.......

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To avoid copyright issues with our previous publication, a TAMRA-tagged probe HCN1TAMRA was used to generate Figures 2 and 3. Note that this substitution did not make an appreciable difference in the results, and the protocols are identical to those outlined above with HCN1FITC. To assess the interaction with HCN1TAMRA, TRIP8b (241-602) was titrated into a fixed concentration of HCN1TAMRA using the protocol outl.......

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Because of its potential as a therapeutic target in MDD24, there has been considerable interest in pharmacological approaches that antagonize HCN channel function in the central nervous system4. However, these efforts have been stalled by the important role of HCN channels in cardiac pacemaking and the risk of arrhythmia25. We reasoned that disrupting the interaction between HCN and its brain specific auxiliary subunit, TRIP8b8, might be sufficient to produce antidepressant-lik.......

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This work was supported by National Institutes of Health Grant R21MH104471 and R01MH106511 (D.M.C.), Brain Research Foundation SG 2012-01 (D.M.C.), Northwestern University Clinical and Translational Sciences Institute 8UL1TR000150 (Y.H.), Chicago Biomedical Consortium HTS-004 (Y.H. and D.M.C.), and National Institutes of Health Grant 2T32MH067564 (K.L.). A part of this work was performed by the Northwestern University Medicinal and Synthetic Chemistry Core (ChemCore) at the Center for Molecular Innovation and Drug Discovery (CMIDD), which is funded by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust and Cancer Center ....

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Name Company Catalog Number Comments
Ni-NTA agarose  Qiagen 30210
Dialysis cassette  ThermoFisher 66456
Isopropyl b-D-1-thiogalactopyranoside  Sigma-Aldrich I5502-1G
384 Well Black plate  Corning 3820
Proxiplate  Perkin-Elmer  6008289
Anti-GST Acceptor beads  Perkin-Elmer  6760603C
NiChelate Perkin-Elmer  AS101D
pGS21-a Genscript SD0121
PMSF Sigma-Aldrich 10837091001
Coomassie Kit ThermoFisher 23200
Protein concentrator ThermoFisher 88527
Perkin Elmer Enspire Multimode Plate reader Perkin-Elmer  #2300-001M
BL21 (DE3) Competent Cells Agilent 200131

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