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.