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

Summary

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.

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.

Introduction

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....

Protocol

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 and Ampicillin. Incubate the culture at 37 °C for 16 hr (with shaking).
3. Add 50 ml culture to 1 L of LB with 50 µg/ml of Ampicilli....

Results

To avoid copyright issues with our previous publication, a TAMRA-tagged probe HCN1_TAMRA 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 HCN1_FITC. To assess the interaction with HCN1_TAMRA, TRIP8b (241-602) was titrated into a fixed concentration of HCN1_TAMRA using the protocol outl.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ni-NTA agarose	Qiagen	30210
Dialysis cassette	ThermoFisher	66456
Isopropyl b-D-1-thiogalactopyranoside	Sigma-Aldrich	I5502 - 1 gr
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