Name: method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b
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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 Support Grant P30 CA060553 from the National Cancer Institute awarded to the Robert H. Lurie Comprehensive Cancer Center. The high throughput screen work was performed in the High Throughput Analysis Laboratory which is also a core facility of the Robert H. Lurie Comprehensive Cancer Center.

1. Purification of TRIP8b (241-602) Protein
<ol>
	<li>Transform the plasmid containing TRIP8b (241-602) in the bacterial protein expression vector pGS213 into competent E. coli for protein expression according to manufacturer&#39;s instructions. Plate 300 &#181;l of the culture on Luria Broth (LB)-agar with 5 &#181;g/ml of Chloramphenicol and Ampicillin. Incubate the plate at 37 &#176;C for 16 h.</li>
	<li>The next day, pick a single colony to inoculate 50 ml LB with 50 &#181;g/ml of Chloramphenicol...

<ol>
	<li>Biel, M., Wahl-Schott, C., Michalakis, S., Zong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarization-activated+cation+channels:+from+genes+to+function.">Hyperpolarization-activated cation channels: from genes to function.</a> Physiol Rev. 89 (3), 847-885 (2009).</li><li>Shah, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN1+channels:+a+new+therapeutic+target+for+depressive+disorders?.">HCN1 channels: a new therapeutic target for depressive disorders?.</a> Sci Signal. 5 (244), pe44 (2012).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Small-Molecule+Inhibitors+of+Hyperpolarization-Activated+Cyclic+Nucleotide-Gated+Channels.">Identification of Small-Molecule Inhibitors of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels.</a> J Biomol Screen. 20 (9), 1124-1131 (2015).</li><li>Postea, O., Biel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+HCN+channels+as+novel+drug+targets.">Exploring HCN channels as novel drug targets.</a> Nat Rev Drug Discov. 10 (12), (2011).</li><li>Stieber, J., Wieland, K., St&#246;ckl, G., Ludwig, A., Hofmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bradycardic+and+proarrhythmic+properties+of+sinus+node+inhibitors.">Bradycardic and proarrhythmic properties of sinus node inhibitors.</a> Mol Pharmacol. 69 (4), 1328-1337 (2006).</li><li>Santoro, B., Wainger, B. J., Siegelbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+HCN+channel+surface+expression+by+a+novel+C-terminal+protein-protein+interaction.">Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction.</a> J Neurosci. 24 (47), 10750-10762 (2004).</li><li>Lewis, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+hyperpolarization-activated+cyclic+nucleotide-gated+channel+auxiliary+subunit+TRIP8b+impairs+hippocampal+Ih+localization+and+function+and+promotes+antidepressant+behavior+in+mice.">Deletion of the hyperpolarization-activated cyclic nucleotide-gated channel auxiliary subunit TRIP8b impairs hippocampal Ih localization and function and promotes antidepressant behavior in mice.</a> J Neurosci. 31 (20), 7424-7440 (2011).</li><li>Heuermann, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+thalamic+and+cortical+Ih+by+deletion+of+TRIP8b+produces+a+mouse+model+of+human+absence+epilepsy.">Reduction of thalamic and cortical Ih by deletion of TRIP8b produces a mouse model of human absence epilepsy.</a> Neurobiol Dis. 85, 81-92 (2016).</li><li>Steru, L., Chermat, R., Thierry, B., Simon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tail+suspension+test:+A+new+method+for+screening+antidepressants+in+mice.">The tail suspension test: A new method for screening antidepressants in mice.</a> Psychopharmacology. 85 (3), 367-370 (1985).</li><li>Petit-Demouliere, B., Chenu, F., Bourin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+swimming+test+in+mice:+a+review+of+antidepressant+activity.">Forced swimming test in mice: a review of antidepressant activity.</a> Psychopharmacology. 177 (3), 245-255 (2004).</li><li>Cryan, J. F., Mombereau, C., Vassout, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tail+suspension+test+as+a+model+for+assessing+antidepressant+activity:+Review+of+pharmacological+and+genetic+studies+in+mice.">The tail suspension test as a model for assessing antidepressant activity: Review of pharmacological and genetic studies in mice.</a> Neurosci Biobehav Rev. 29 (4-5), 571-625 (2005).</li><li>Han, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+and+gating+of+hyperpolarization-activated+cyclic+nucleotide-gated+channels+are+regulated+by+interaction+with+tetratricopeptide+repeat-containing+Rab8b-interacting+protein+(TRIP8b)+and+cyclic+AMP+at+distinct+sites.">Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites.</a> J Biol Chem. 286 (23), 20823-20834 (2011).</li><li>Hu, L., Santoro, B., Saponaro, A., Liu, H., Moroni, A., Siegelbaum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+the+auxiliary+subunit+TRIP8b+to+HCN+channels+shifts+the+mode+of+action+of+cAMP.">Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP.</a> J Gen Physiol. 142 (6), 599-612 (2013).</li><li>Saponaro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+mutual+antagonism+of+cAMP+and+TRIP8b+in+regulating+HCN+channel+function.">Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function.</a> Proc Natl Acad Sci U S A. 111 (40), 14577-14582 (2014).</li><li>Bankston, J. R., Camp, S. S., DiMaio, F., Lewis, A. S., Chetkovich, D. M., Zagotta, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+stoichiometry+of+an+accessory+subunit+TRIP8b+interaction+with+hyperpolarization-activated+cyclic+nucleotide-gated+channels.">Structure and stoichiometry of an accessory subunit TRIP8b interaction with hyperpolarization-activated cyclic nucleotide-gated channels.</a> Proc Natl Acad Sci U S A. 109 (20), 7899-7904 (2012).</li><li>Gatto, G. J., Geisbrecht, B. V., Gould, S. J., Berg, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+targeting+signal-1+recognition+by+the+TPR+domains+of+human+PEX5.">Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.</a> Nat Struct Biol. 7 (12), 1091-1095 (2000).</li><li>Owicki, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+polarization+and+anisotropy+in+high+throughput+screening:+perspectives+and+primer.">Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer.</a> J Biomol Screen. 5 (5), 297-306 (2000).</li><li>Smith, D. S., Eremin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+polarization+immunoassays+and+related+methods+for+simple,+high-throughput+screening+of+small+molecules.">Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules.</a> Anal Bioanal Chem. 391 (5), 1499-1507 (2008).</li><li>Roehrl, M. H. A., Wang, J. Y., Wagner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+framework+for+development+and+data+analysis+of+competitive+high-throughput+screens+for+small-molecule+inhibitors+of+protein-protein+interactions+by+fluorescence+polarization.">A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization.</a> Biochemistry. 43 (51), 16056-16066 (2004).</li><li>Arkin, M. R., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+protein-protein+interactions:+progressing+towards+the+dream.">Small-molecule inhibitors of protein-protein interactions: progressing towards the dream.</a> Nat Rev Drug Discov. 3 (4), 301-317 (2004).</li><li>Zhang, J., Chung, T., Oldenburg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4 (2), 67-73 (1999).</li><li>Eglen, R. 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Y., Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+dorsal+hippocampal+activity+by+knockdown+of+HCN1+channels+leads+to+anxiolytic-+and+antidepressant-like+behaviors.">Enhancement of dorsal hippocampal activity by knockdown of HCN1 channels leads to anxiolytic- and antidepressant-like behaviors.</a> Neuron. 75 (3), 503-516 (2012).</li><li>Wahl-Schott, C., Biel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN+channels:+structure,+cellular+regulation+and+physiological+function.">HCN channels: structure, cellular regulation and physiological function.</a> Cell Mol Life Sci. 66 (3), 470-494 (2009).</li><li>DeBerg, H. A., Bankston, J. R., Rosenbaum, J. C., Brzovic, P. S., Zagotta, W. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hill+equation+revisited:+uses+and+misuses.">The Hill equation revisited: uses and misuses.</a> FASEB J. 11 (11), 835-841 (1997).</li><li>Prinz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hill+coefficients,+dose-response+curves+and+allosteric+mechanisms.">Hill coefficients, dose-response curves and allosteric mechanisms.</a> J Chem Biol. 3 (1), 37-44 (2009).</li><li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Guide+to+Simple+and+Informative+Binding+Assays.">A Guide to Simple and Informative Binding Assays.</a> Mol Biol Cell. 21 (23), 4061-4067 (2010).</li><li>Rossi, A. M., Taylor, C. 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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...

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 heart failure to produce a bradycardic effect5. As such, there is a need for pharmacologic agents that limit HCN channel function exclusively in the central nervous system.
Tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b) is a brain specific auxiliary subunit of HCN channels that controls the surface expression and localization of HCN channels6,7. Genetic knockout of TRIP8b causes a reduction of brain HCN channels7 without affecting HCN expression in the heart8. Interestingly, TRIP8b knockout mice spend less time immobile on the forced swim task and tail suspension task7, two commonly used screening tests for antidepressant efficacy9-11. These results suggest that rather than directly targeting HCN channels with a small molecule antagonist of HCN channel function, disrupting the interaction between TRIP8b and HCN may be sufficient to produce antidepressant-like behavior.
TRIP8b binds to HCN at two distinct binding sites. The cyclic nucleotide binding domain (CNBD) of HCN interacts with a conserved domain of TRIP8b located N terminal to the TPR domains of TRIP8b12,13. Although the residues of the CNBD that are involved in this interaction have been mapped14, the region of TRIP8b that is involved has not been narrowed down beyond an-80-amino acid fragment13. A second interaction occurs between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tripeptide of HCN (&#39;SNL&#39; in HCN1, HCN2, and HCN4, but &#39;ANM&#39; in HCN3)3,12. The recently solved crystal structure15 of this C tail interaction revealed substantial structural similarity to the interaction between the peroxisomal import receptor, peroxin 5 (PEX5), and its interacting partners, containing type 1 peroxisomal targeting sequences (PTS1)16.
Although both interaction sites are required for HCN channel function, the interaction between the TPR domains of TRIP8b and the C terminal tripeptide of HCN1 serves as the dominant binding site and regulates HCN surface expression. Therefore, this interaction was chosen as the targeting site in this study. For the remainder of the manuscript, when a reference is made to the interaction between TRIP8b and HCN, it is this interaction that is being referred to. This interaction is recapitulated by a highly soluble fragment of TRIP8b corresponding to its conserved C terminal&#160;containing the TPR domains required for binding the C terminal tail of HCN (residues 241-602 of the 1a-4 isoforms of TRIP8b)3.
In order to develop a high throughput screen to identify small molecules capable of disrupting this interaction, a fluorescence polarization (FP)-based assay was employed17. Fluorescence polarization is based on the excitation of a fluorophore-tagged ligand with polarized light, and measuring the degree of polarization of the emitted fluorescence18. In the presence of a binding partner, the rotational motion of the fluorescent ligand is constrained and polarized light is emitted19. In the absence of a binding partner, the rotational motion of the ligand leads to the emission of depolarized light.
In the enclosed protocol, a method for the purification of N terminal-tagged (6xHis) TRIP8b (241-602) using Nickel-Nitrilotriacetic acid (Ni-NTA) beads is presented. A similar protocol was employed to purify the Glutathione-S-Transferase (GST)-tagged C terminal 40 amino acids of HCN1 (HCN1c40) used in step 7 of the protocol. For space considerations, a detailed description of that procedure was omitted.
In steps 2 through 7 of the protocol, a high throughput screening workflow is presented (see Figure 1). Protein-protein interactions are a notoriously difficult target for high throughput screening and readers are advised to seek out additional resources on the topic20.
Steps 2 and 3 of the procedure characterize the in vitro affinity of the purified TRIP8b (241-602) construct for a fluorescein isothiocyanate (FITC)-tagged eleven amino acid peptide corresponding to the C terminal tail of HCN1 (HCN1FITC). Based on the crystal structure of the TRIP8b-HCN complex15, this eleven amino acid segment is sufficient to produce binding with TRIP8b (241-602). In step 2, the Kd of the interaction is measured by titrating TRIP8b (241-602) into a fixed concentration of HCN1FITC. In step 3, an unlabeled version of the HCN peptide used in step 2 is titrated into a fixed concentration of both TRIP8b (241-602) and HCN1FITC to examine if the FITC tag interferes with binding. These experiments are essential to selecting the appropriate concentrations of TRIP8b (241-602) and HCN1FITC used in the high throughput screen.
The premise of the high throughput screen is that a small molecule capable of disrupting the interaction between TRIP8b (241-602) and HCN1FITC will produce a decrease in polarized light. In step 4, the Z factor of the assay is calculated21 for a given concentration of TRIP8b (241-602) and HCN1FITC to ensure that the assay is appropriate for high throughput screening (step 5). Steps 6 and 7 are validation assays to confirm that the hits identified in the primary high throughput screen are acting by disrupting the interaction between TRIP8b (241-602) and HCN1FITC rather than through a nonspecific mechanism. In step 6,&#160;carboxytetramethylrhodamine (TAMRA)-labeled HCN1 peptide (HCN1TAMRA) is used in an otherwise identical fluorescence polarization assay to filter fluorescent compounds that compromise the FP assay using the FITC tag. Step 7 utilizes a larger HCN1 C terminal fragment (HCN1C40) and employs a bead-based proximity assay, which is based on the &#39;tunneling&#39; of a singlet oxygen from a donor bead to an acceptor bead brought close to one another by interacting proteins22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ni-NTA agarose&#160;</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis cassette&#160;</td>
			<td>ThermoFisher</td>
			<td>66456</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl b-D-1-thiogalactopyranoside&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I5502-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Black plate&#160;</td>
			<td>Corning</td>
			<td>3820</td>
			<td></td>
		</tr>
		<tr>
			<td>Proxiplate&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6008289</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GST Acceptor beads&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6760603C</td>
			<td></td>
		</tr>
		<tr>
			<td>NiChelate</td>
			<td>Perkin-Elmer&#160;</td>
			<td>AS101D</td>
			<td></td>
		</tr>
		<tr>
			<td>pGS21-a</td>
			<td>Genscript</td>
			<td>SD0121</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>10837091001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Kit</td>
			<td>ThermoFisher</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein concentrator</td>
			<td>ThermoFisher</td>
			<td>88527</td>
			<td></td>
		</tr>
		<tr>
			<td>Perkin Elmer Enspire Multimode Plate reader</td>
			<td>Perkin-Elmer&#160;</td>
			<td>#2300-001M</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent Cells</td>
			<td>Agilent</td>
			<td>200131</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b

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 &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

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

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 ...

The overall goal of this procedure is to identify small molecules that are capable of disrupting the interaction between TRIP8b and HCN channels. This method identifies candidate molecules that could be developed into new treatments for depression. The main advantage of this technique is that it can completed in a high throughput fashion.This technique has implications for the development of a novel therapy for major depressive disorder because our lab has shown that disrupting TRIP8b binding to HCN may be of therapeutic benefit. Though this method was developed with depression in mind disrupting the interaction between TRIP8b and HCN may be of therapeutic benefit in other disorders such as chronic pain. Visual demonstration of this protocol is important because it illustrates the benefits of using automated high throughput instruments to advance basic and translational research at open facilities like Northwestern University's high throughput analysis lab.After purifying the TRIP8b 241-602 protein fragment and examining protein protein interactions with a positive control according to the text protocol thaw aliquots of TRIP8b and HCN1-FITC and keep them on ice. Use FP Buffer to prepare 20 milliliters of 2 micromolar TRIP8b and 50 nanomolar HCN1-FITC. Then dispense 25 microliters of the mixture into each well of a low binding 384 well black microtiter plate.For library screening, use an acoustic liquid handler to dispense 100 nanoliters of compounds into each well of columns 3 to 22 for a final concentration of 40 micromoles. Dispense 100 nanoliters of DMSO into each well of columns 1 and 23 as negative controls. Dispense 100 nanoliters of unlabeled HCN1 peptide into each well of columns 2 and 24 as positive controls with a final concentration of 10 micromoles.Incubate the plates at room temperature for two hours. Using a plate reader, measure the fluorescence polarization. Alternatively plates may be stored for 16 hours at 4 degrees Celsius before reading.Using the following equation calculate the percent inhibition where XN is the normalized percent inhibition corresponding to the signal X.X positive is the average signal from the positive control wells and X negative is the average signal from the negative control wells. To validate Hits with over 50%inhibition prepare a master mix of 20 milliliters of 2 micromolar TRIP8b and 50 nanomolar HCN1 TAMRA in FP buffer. Dispense 25 microliters of the master mix into each well of a black 384 well microtiter plate.Then add 100 nanoliters of compounds from a plate containing two-fold serial dilutions of each active compound into the respective wells. For the negative control wells add 100 nanoliters of DMSO. For a positive control, add 100 nanoliters of serially diluted unlabeled HCN1 peptide with final concentrations ranging from 200 micromoles to 0.1 micromoles.Incubate the plate at room temperature for two hours, or at 4 degrees Celsius for 16 hours. Using a plate reader measure the fluorescence polarization. Calculate the IC50 values by fitting the concentration dependent FP data of each compound using a 4 parameter nonlinear regression model.Thaw one aliquot each HIS tagged TRIP8b and GST tagged HCN1 fusion proteins and keep them on ice. Prepare 1 milliliter of 200 nanomolar HIS TRIP8b and 20 nanomoles of GST-HCN1C40 in assay buffer for each compound to be tested. Use a 16 channel pipette to add 7 microliters of the HIS-TRIP8b GST-HCN1C40 mixture to three wells per row of rows A through N to test each compound in triplicate.Briefly centrifuge the plate to ensure that the liquids are at the bottom of each well, and to remove any bubbles. Next, dispense the Hit compounds to be tested into the plate to achieve a final concentration of 200 micromoles in row A and finishing with 0.1 micromoles in row L.Add the same volume of DMSO into row M.Then dispense unlabeled HCN1 peptide to a final concentration of 10 micromoles into row N.Shake the plate for two minutes and incubate for 2.5 hours at room temperature. Using assay buffer, dilute the Anti-GST acceptor beads 1 to 50.With a 16 channel pipette, add 3.5 microliters of the diluted acceptor beads to each well of the plate and mix by gently pipetting to avoid creating bubbles. Incubate the plate in the dark at room temperature for one hour. Dilute the Nickel Chelate donor beads 1 to 50 with assay buffer while not exposing the mixture to light.Add 3.5 microliters of the diluted donor beads to each well of the plate and gently mix, avoiding air bubbles. Seal the plate and incubate it in the dark at room temperature for 1.5 hours. Then use a plate reader to read the plate with the measurement parameters listed here.Finally, calculate the IC50 values by fitting data for each compound using a 4 parameter nonlinear regression model. To assess the interaction with HCN1 TAMRA TRIP8b was titrated into a fixed concentration of HCN1 TAMRA. This graph shows that as the concentration of TRIP8b increases, more HCN1 TAMRA molecules become bound and polarization increases.In this experiment as unlabeled HCN1 was titrated into a fixed concentration of TRIP8b and HCN1 TAMRA, the labeled peptide was displaced and the signal decreased. A high throughput screening shows each compound's percent inhibition represented by single points on the graph. The results of this screen are plotted with positive and negative controls, and each each compound's average percent inhibition across two runs of the assay.Compounds showing greater than 50%inhibition were then validated in this experiment. The X and Y coordinates were determined by the percent inhibition in two runs of the assay. In this experiment, HCN1C40 contains an end terminal GST tag that binds acceptor beads.The interaction of the TPR domains of TRIP8b and the C terminal tripeptide of HCN1 excite the donor bead with 680 nanometer wavelength light to produce Singlet oxygen. Finally, as show in this graph, titration of increasing concentrations of the hit compound NUCC-5953 into a fixed concentration of TRIP8b and HCN1C40 increased the inhibition of the TRIP8b HCN1 interaction. Before starting high throughput screening validate the protein preparation and the fluorescence polarization in bead-based proximity assays.Ensure the protein is of high purity. Aliquot the protein to avoid multiple freeze thaw cycles. It is important to use high quality reagents, including fresh DMSO and Triton.Don't forget that working with the DMSO can be dangerous, and the precautions such as wearing appropriate personal protective equipment should always be taken while performing this procedure. Following this procedure, other methods like electrophysiology and flow cytometry can be performed in order to determine if the hit compounds have activity in cells. After watching this video, you should have a good understanding of how to perform a high throughput screen with a fluorescence polarization based assay.

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