GSH Conjugates

Stability: Stability was determined for ML204 at 23°C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) and is shown in the table presented below. The solubility of ML204 in PBS (>700 μM) suggests that the stability measurements may not be affected by precipitation, although direct determinations of compound decomposition were not made.

ML204: CID 230710, SID 97362164 (assay data) and CID 49786978, SID 103061324 (deposited Probe)

Compounds added to the SMR collection (MLS#s): 003227299 (ML204, 26.5 mg), 003227300 (SID103061325, CID16800920, 22.2mg), 003227301 (SID103061326, CID 49786979, 20.1mg), 003227302 (SID 103061323, CID 49786977, 21.6mg), 003227303 (SID 103061328, CID 49786981, 20mg), 003227304 (SID 103061327, CID 49786980, 20.2mg).

Screening Summary

1. 305k compounds initially screened → 1189 hits (AID 2247)
2. 856 compounds retested → 735 compounds validate as TRPC4 inhibitors using assay from primary screen (AID 2636, 2637)
3. 856 compounds → 541 compounds show TRPC4 dependent inhibition using an alternative TRPC4 expressing cell line (AID 434978)
4. 506 compounds tested against TRPC6 → 421 show no activity against TRPC6 expressing cells (AID 434948)
5. 421 tested for dose dependent inhibition of TRPC4 → 393 compounds generated CRC curves (AID 434942)

After the screening hits were finalized, there were approximately 393 confirmed hits with dose dependent effects (see table for activity breakdown).

3.2.1. Fluorescent assay

The primary HTS assay used to identify TRPC4 inhibitors measured calcium influx in a HEK293 cell line (AID 2247). TRPC4 channel activation was triggered by GPCR activation in HEK293 cells stably expressing TRPC4 channels along with μ-opioid receptors. Addition of 300 nM DAMGO activates the μ-opioid receptors and G_i/o signaling pathways that cause TRPC4 channels to open. Calcium influx is measured by a calcium-sensitive fluorescent dye (Fluo-4 AM, Invitrogen) on an FDSS instrument. Figure 1 shows full block of TRPC4-mediated calcium influx by ML204 with an IC50 value of 0.99 μM. In replicate experiments (n=5), the mean ± SD IC50 values were 0.96 ± 0.26 μM.

Figure 1

Inhibition of calcium influx in TRPC4- and μ-opioid receptor-expressing cells by ML204 after activation by DAMGO. Left panel (A) displays fluorescent signals in individual wells in the presence of different concentrations of ML204. Dose-response (more...)

3.2.2. Electrophysiological assay

In order to more directly monitor block of TRPC4 channels, an electrophysiological assay was developed and implemented on the QPatch16 automated electrophysiology instrument (AID 492991, 504589). Whole cell voltage clamp recordings were made from the HEK293 TRPC4 and μ-opioid receptor expressing cells that were used in the primary, confirmatory and SAR assays. Cells were held at 0 mV, stepped to −100 mV for 50 ms, and voltage ramps from −100 mV to +120 mV were applied every 15 s to initiate low levels of voltage-dependent channel opening to monitor TRPC4 channel activity. In Figure 2A, the voltage ramp occurred between 250 ms and 370 ms. Addition of 50 nM DAMGO to the bath solution produced large increases in TRPC4 channel activity that can best be seen at +120 mV at the end of the voltage ramps (Fig. 2A). DAMGO-activated TRPC4 currents were reversibly blocked by ML204 (Fig. 2A and Fig. 2B). Figure 2B shows a diary plot of the current amplitudes recorded from one cell at +120 mV over time. After an initial stabilization period, DAMGO, ML204 or buffer were added with a double addition protocol to ensure more complete fluid exchange. 50 nM DAMGO added to the bath solution produced a large increase in TRPC4 currents, which were blocked by more than 50% by 3.3 μM ML204. Subsequent washout of the compound and DAMGO returned TRPC4 currents to control levels. Subsequent reapplication of 50 nM DAMGO produced a robust activation of TRPC4 currents to levels similar to the initial DAMGO activation levels. This protocol enabled reliable determination of TRPC4 inhibition and was used to produce concentration response curves for ML204. ML204 blocked TRPC4 with an IC value of 2.9 μM using this protocol (n=7 cells). A similar IC50 value of 2.6 μM was obtained using this protocol, but with a modification allowing application of multiple concentrations of this compound for each cell (n=5 cells). The modified protocol was used to evaluate selected TRPC4 inhibitors and data are shown in the SAR tables.

Figure 2

Block of TRPC4 currents by ML204 in automated electrophysiology (QPatch) experiments. (A) shows effects of DAMGO and ML204 on membrane currents elicited by voltage ramps from −100 mV to +120mV applied every 15 s from a holding potential of 0 mV. (more...)

3.6.1. Profiling Panels

ML204 was tested in 397 assays listed in PubChem and was active in only one assay (Cycloheximide Counterscreen for Small Molecule Inhibitors of Shiga Toxin) that is not TRPC4 based. ML204 was tested at Ricerca’s (formerly MDS Pharma’s) Lead Profiling Screen (binding assay panel of 68 GPCRs, ion channels and transporters screened at 10 μM), and was found to not significantly interact with 61 out of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM). ML204 did have activity against several targets (see Table 3 below); however, it should be pointed out that these are only single-point values and that functional selectivity may be significantly better than suggested by these “% activities.”

Table 3

Ricerca profiling of ML204 (SID 97362164/VU0024172-3).

3.6.2. Metabolic and Physical Properties

ML204 and other analogs were also evaluated in our tier 1 in vitro DMPK battery to further establish its utility as a small molecule probe. In CYP₄₅₀ assay, ML204 was clean against three of the CYP enzymes (>30 μM, 2C9, 2D6, 3A4), but was a potent inhibitor of CYP1A2 (310 nM). This trend held true for most of the analogs as well, with CYP1A2 the only CYP with significant inhibition (<1 μM). Due to the structural features of ML204 and analogs (small, planar aromatic structures) – this is an expected result as these compounds has similar structural features as other known CYP1A2 inhibitors.

Table 4CYP₄₅₀ inhibition for ML204 and analogs

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Entry	Cmpd	Structure	CYP450 (μM)
			1A2	2C9	2D6	3A4
1	97362164/VU0024172-3		0.31	>30	>30	>30
2	97362171/VU0180678-3		0.96	>30	26.4	>30
3	97362165/VU0418904-1		0.50	>30	>30	>30
4	97362170/VU0324728-2		0.81	>30	11.1	>30
5	97362178/VU0418913-1		0.24	9.7	4.6	16.6
6	97362175/VU0418910-1		0.74	>30	>30	>30
7	99344394/VU0419898-1		0.56	>30	2.85	>30
8	99344398/VU0419902-1		0.37	>30	>30	>30
9	99344396/VU0419900-1		<0.1	>30	<0.1	>30

The metabolic stability of ML204 and analogs were evaluated in assays which can predict rat and human hepatic clearance from in vitro microsomal clearance values (Table 5). This allows for a rank ordering of compounds that would be predicted to have poor stability in our in vivo testing protocols (oral dosing). Unfortunately, all of the compounds evaluated showed high (near hepatic blood flow) clearance in human and rat liver microsomes. This result is not surprising as all the compounds tested contain an unsubstituted benzylmethyl and unsubstituted cycloalkyl groups which is known to undergo oxidation by liver microsomes. In addition, these compounds all showed high protein binding as well (>99% bound), except for SID 97362170, Entry 4 which showed a favorable free fraction (%fu, human = 2.7%; rat = 3.5%).

Table 5

Intrinsic clearance and protein binding data of ML204 and analogs.

3.6.3. Selectivity Studies

The selectivity of ML204 for blocking TRPC4 channels compared with block of other TRP channels was determined using a combination of electrophysiological and fluorescent assays that measured activity of recombinantly expressed TRP channels.

3.6.3.1. Selectivity in Electrophysiological Studies

Whole cell voltage clamp recordings were made from HEK293 cells expressing TRPC6, TRPC5 and TRPC3 channels using the following protocols.

Voltage ramp protocol: Vh=0 mV. The protocol started at 0 mV for 5 ms, then at +100 mV for 20 mV, followed by a ramp from +100 mV to −100 mV within 500 ms, finally at −100 mV for 5 ms. Each protocol cycle was 2 s and the sampling rate was 5 kHz. Extracellular solution (ECS) contained (in mM): 145 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 Glucose, 10 HEPES, pH7.4. Recording pipettes were pulled from micropipette glass (A-M Systems, Inc., Carlsborg, WA) to 3–5 MΩ when filled with an intracellular solution containing (in mM): CsCl₂ 110, MgCl₂ 1, CaCl₂ 4.77, BAPTA 10, HEPES 10, pH 7.2. The concentrations of CaCl₂ and BAPTA in the internal solution were calculated using MAXCHELATOR to give a free calcium concentration of 200 nM in the internal solution. ML204 was dissolved in DMSO and stored in an amber glass bottle at room temperature. Before each recording, 100 mM ML204 stock solution was diluted in ECS and delivered via a gravity-driven perfusion system (an array of 8 parallel tubes). Drugs were applied by manually moving the tubing array so that the target cell was exposed to the direct stream from one tube. Stimulation protocol: after whole cell configuration is formed for 1 min, the recorded cell was first exposed to receptor agonist alone (100 nM DAMGO or 10 μM carbachol plus 100 nM DAMGO for TRPC5 and TRPC4 expressing cells, 100 μM carbachol for TRPC3 and TRPC6 expressing cells and 10 μM OAG for TRPC6 expressing cells), followed by the mixture of agonist plus 10 μM ML204, and then again with the agonist alone. Due to the quick desensitization of the channels, the second agonist stimulation may not be as effective as the first one, but it should be possible to observe the inhibitory effect induced by ML204. All recordings were made at room temperature (22–24°C).

ML204 exhibited modest inhibitory effects on TRPC6 channels activated by bath application of 100 μM carbachol to cells stably expressing TRPC6 channels and transiently expressing M5AchR receptors as shown in Figure 3A. In an additional set of experiments, 10 μM ML204 produced no appreciable block of TRPC6 currents activated by bath application of OAG.

Figure 3

Whole cell voltage clamp recordings from cells expressing TRPC6 and M5 AchR (A) and TRPC5 and μ-opoid receptors (B). Effects of ML204 on currents recorded during voltage ramps following exposure to carbachol (A) or DAMGO (B) are shown in the lower (more...)

ML204 exhibited modest block of TRPC5 channels activated by μ-opioid and muscarinic acetylcholine receptor co-stimulation as shown in Figure 3B. At 10 μM, ML204 blocked less than half of the TRPC5 current, although quantitation of the magnitude of the block is complicated by the steadily increasing current magnitude in the presence of carbachol and DAMGO.

ML204 at 10 μM blocked TRPC3 channels activated by carbachol in HEK293 cells expressing TRPC3 channels and endogenous muscarinic acetylcholine receptors as shown in figure 4A.

Figure 4

Effect of ML204 on whole cell voltage clamp recording from a cell expressing TRPC3 channels following activation by carbachol is shown in (A). Currents recorded during voltage ramps are shown in the lower panel and the time course of changes in currents (more...)

A summary of ML204 effects on TRPC channels in manual electrophysiological experiments is shown in figure 4B. At 10 μM, ML204 produced nearly complete block of TRPC4 channels activated by muscarinic or μ-opioid receptor signaling pathways. Lower levels of block of TRPC3 and TRPC5 channels were observed indicating moderate selectivity against these channels and TRPC6 was weakly blocked by ML204 indicating a higher level of selectivity against this channel.

ML204 at 10 μM caused no significant inhibition (<20%) of TRPV1 channels activated by addition of 30 nM capsaicin in automated electrophysiological experiments performed on the QPatch instrument. In addition, ML204 produced no significant inhibition (10% change at 30 μM) of KCNQ2 potassium channels in electrophysiological experiments using the IonWorks Quattro instrument in population patch clamp mode (protocol similar to AID 2603 except that inhibition is calculated).

3.6.3.2. Selectivity in Fluorescent Experiments

ML204 was evaluated for block of a closely related channel, TRPC6, using a HEK cell line stably expressing TRPC6 channels (AID 492987 and 492981). TRPC6 activation was initiated by muscarinic receptor activation following addition of acetylcholine to the cells. Cation influx was measured using a membrane potential sensitive dye (blue dye, Molecular Devices, R8034) and changes in fluorescent intensity were determined on a FDSS instrument (Hamamatsu). ML204 afforded weak block of TRPC6 activity in this assay with an IC50 of 17.9 ± 2.3 μM (n=3).

The effects of ML204 on a panel of TRP channels were evaluated using a Flex Station (Molecular Devices, Inc.) measuring changes in fluorescent intensity due to changes in intracellular calcium measured with Fluo-4 or to changes in membrane potential measured with a membrane potential sensing dye (FMP). Cells seeded in wells of 96-well plates were loaded with either Fluo-4 or FMP dye and assayed using Flex Station. Results are shown in Figure 5. ML204 caused no inhibition of TRPA1, TRPM8, TRPV3 or TRPV1. High concentrations of ML204 caused weak and slow activation of TRPA1. ML204 inhibited TRPC5-mediated Ca²⁺ influx in a dose-dependent manner with an IC₅₀ value of 9.2 μM. The effects of high concentrations of ML204 on TRPC3 are difficult to interpret because high concentrations of ML204 caused a partial inhibition of Fluo-4 calcium influx signals triggered by carbachol in HEK293 cells not expressing TRPC channels and it produced a concentration dependent decrease in the fluorescence intensity of the membrane potential sensing dye. Overall, ML204 afforded a favorable selectivity profile for inhibition of TRPC4 versus five other TRP channels, with significant block only observed for the closely related TRPC5 (9.2 μM).

Figure 5

Selectivity panel for effects of ML204 on TRP channels in fluorescent assays. Cells expressing different TRP channels were loaded with Fluo-4 to measure calcium influx or with FMP to measure membrane potential changes. TRP channel opening was triggered (more...)

4.2.1. Manual Electrophysiological Assay

Whole cell voltage clamp recordings (AID 492983) were made from a HEK293 cell line stably expressing TRPC4 channels and μ-opioid receptors, which was also used for the fluorescent assay and QPatch assay presented in Figures 1 and 2. For manual voltage clamp recordings, the internal solution had ~400 nM free Ca and was composed of (in mM): 110 CsCl, 10 HEPES, 10 BAPTA 1 MgCl₂, 6.46 CaCl₂, pH adjusted to 7.2 with HCl. The external solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES, pH 7.4 adjusted with NaOH. The pipette resistances were 2–4 MΩ and recordings were performed at the room temperature (22°C). Cells were held at 0 mV and repeated voltage commands stepping to −100 mV for 20 ms followed by a voltage ramp to 100 mV in 100 ms were given at a 0.5 sec interval. Drugs were applied as indicated though perfusion. Typically, ML204 was applied before the establishment of the whole-cell configuration and treated cells were compared with control, untreated cells.

HEK293 cells stably expressing TRPC4β + μ-opioid receptor were used. Cells were pretreated with or without 1 μM ML204 (SID 97362164) for > 2 min before the addition of 10 μM carbachol + 0.1 μM DAMGO. This condition was used to maximally activate TRPC4 whole-cell currents. Figure 6A shows representative traces of current development at −100 and +100 mV for a cell not treated with ML204. The I–V curve at the peak of current development is shown below. Figure 6B is similar to figure 6A but the cell was treated with ML204. Figure 6C shows summary data (mean ± SEM, n = 13 for both) of current densities at −100 and +100 mV for untreated (cntl) and ML204-treated cells.

Figure 6

Whole-cell recordings of TRPC4 currents activated by carbachol plus DAMGO. (A) and (B), representative traces for voltage ramps in the absence (A) or presence of 1μM ML204 (B) are shown in the lower panel and the time course of changes in currents (more...)

In addition, acute effect on TRPC4 currents by ML204 was determined in cells expressing mTRPC4 channels along with μ-opioid receptors. TRPC4 currents were activated by co-application of 0.1 μM DAMGO and 10 μM carbachol. ML204 (10 μM) inhibited >80% of the TRPC4 current amplitude at +100 mV; averaged responses are plotted in figure 4B.

4.2.2. Electrophysiological Assays Using Different Activation Mechanisms

ML204 inhibited TRPC4-mediated cation influx in both fluorescent (AID 492996) and electrophysiological (AID 492991) assays, in which TRPC4 channels were activated by the μ-opioid receptor agonist, DAMGO alone or in combination with carbachol, which activates muscarinic receptors. Inhibitory effects of ML204 on TRPC4 channels are difficult to distinguish from possible effects of ML204 on the GPCR signaling pathways used to activate TRPC4 channels in these assays. A number of alternative assays for TRPC4 function were employed using both electrophysiological and fluorescent measurements to evaluate effects of compounds on TRPC4 activity following channel activation by alternative signaling mechanisms.

In manual electrophysiological experiments, TRPC4 currents were recorded from HEK293 cells expressing guinea pig TRPC4 channels along with M2 muscarinic receptors. 10 μM ML204 nearly completely blocked currents activated by 10 μM carbachol (figure 4B). This level of inhibition is similar the inhibition observed at 10 μM ML204 after TRPC4 activation by μ-opioid receptor activation in QPatch experiments.

An additional protocol was employed to confirm direct inhibition of TRPC4 channels by ML204. In automated electrophysiological experiments (QPatch: protocol similar to AID 492991), GTPγS was included in the intracellular solution to produce constitutive activation of G-proteins. In the absence of μ-opioid receptor activation, the amplitude of TRPC4 currents slowly increased above initial levels (figure 7B). Addition of 50 nM DAMGO produced large and sustained increases in TRPC4 currents that were reversibly inhibited by ML204. After washout of both ML204 and DAMGO, TRPC4 currents were maintained at high levels indicating that TRPC4 currents were no longer dependent on GPCR activation. ML204 inhibited TRPC4 currents in this assay format with a potency (IC50=3.6 ±1.3 μM, n=4) similar to TRPC4 inhibition in other electrophysiological and fluorescent assays in which TRPC4 activation was dependent on GPCR activation.

Figure 7

Block of TRPC4 currents by ML204 in automated electrophysiology (QPatch) experiments using alternative activation mechanism. GTPγS was included in the intracellular solution to produce sustained channel activation by G-proteins following μ-OR (more...)

TRPC channels can be partially activated by increases in intracellular calcium [6]. TRPC4 currents were recorded with a QPatch automated electrophysiology instrument with a protocol similar to AID 492991 with an alteration in the intracellular solution to give a free calcium concentration of 1.8 μM. Small (<1 nA) outwardly rectifying currents were observed that were similar to the currents activated by GPCR activation in the same cells, but were smaller in amplitude. At 10 μM, ML204 inhibited most of this current, but quantification of the degree of block was not performed due to the small amplitude of the current and difficulties in subtracting small amplitude background currents. These qualitative data further confirm a direct inhibitory effect of ML204 on TRPC4 channels.

4.2.3. Fluorescent Assays Utilizing Different Activation Mechanism

A HEK cell line stably expressing TRPC4 channels along with serotonin (5HT1a) receptors was used to evaluate cation influx through TRPC4 channels triggered by serotonin. Cation influx was measured using either a membrane potential sensitive dye (blue dye, Molecular Devices, R8034) or by a calcium sensitive dye (Fluo-4 AM, Invitrogen) as in AID 492995 and 434978 respectively. ML204 inhibited membrane depolarization following 5HT1a activation with an IC50 value of 1.68 ± 0.79 μM (n=2), similar to effects observed in assays with μ-opioid receptor activation, suggesting that ML204 inhibitory effects observed in figures 1 and 2 were not due to modulation of μ-opioid receptors or effects on intracellular calcium signaling. These data provide support for a mechanism in which ML204 acts directly on TRPC4 channels rather than the signaling pathways that activate these channels.

Protocol

1. Cell culture: TRPC4β + μ−Opioid Receptor-expressing HEK293 Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 μg/ml streptomycin, 500 μg/ml G418 and 40 μg/ml hygromycin
2. Cell plating: Add 50 μl/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS
3. Incubate overnight at 37°C and 5% CO₂
4. Remove medium and add 20 μl/well of 1x Fluo-4 solution to cells
5. Incubate 45 minutes at room temperature (RT) in the dark
6. Prepare 7.5x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), and ECmax of DAMGO
7. Remove Fluo-4 dye solution and add 40 μl/well of assay buffer to cells
8. Remove 40 μl solution and add 20 μl/well of assay buffer to cells
9. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 5 seconds at 1 Hz to establish baseline
11. Add 4 μl of 7.5x compound stock into the cell plates
12. Incubate plates for 110 seconds
13. Add submaximal concentration of DAMGO and incubate for 110 seconds
14. Add maximally activating concentration of DAMGO (DAMGO ECmax) and read for another 110 seconds
15. Calculate ratio readout as F(max-min)/F₀ and integrated ratio readout
16. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z′ factors
17. Calculate B scores for test compounds using integrated ratios calculated in Step 15
18. Outcome assignment: If the B score of the test compound is less than the mean minus 3 times the standard deviation (SD) of the B scores of integrated ratios of all library compounds (B score Inhibitor Ratio <-3*SD), the ratio of initial fluorescence intensity is within 3 times the standard deviation plus the mean of the ratios of the complete library AND having a positive integrated ratio, the compound is designated in the Outcome as active (value=2) as an inhibitor of the TRPC4 channel. Otherwise, it is designated as inactive (value=1).
19. Score assignment: An active test compound is assigned a score between 5 and 100 by calculation of Integer ((Log10(abs(B score Inhibitor Ratio))-0.7)*250); they are normalized to the smallest and largest LOG10(B score Inhibitor Ratio), B score Inhibitor Ratio, as in the result definition. The inactive test compounds are assigned a score of 0.

Protocol

1. Cell culture: TRPC6 expressing cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 μg/ml streptomycin, and 400 μg/ml G418
2. Cell plating: Add 50 μl/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS, 50 IU/ml penicillin, and 50 μg/ml streptomycin
3. Incubate overnight at 37°C and 5% CO₂
4. Remove medium and add 20 μl/well of Membrane Potential Dye
5. Incubate 45 minutes at room temperature (RT) in the dark
6. Prepare 7.5x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), ECmax of Acetylcholine
7. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
8. Measure fluorescence for 5 seconds at 1 Hz to establish baseline
9. Add 4 μl of 7.5x compound stock into the cell plates
10. Incubate plates for 110 seconds
11. Add 6 μl maximally activating concentration of Acetylcholine (Acetylcholine ECmax) and read for another 110 seconds
12. Calculate ratio readout as F(max-min)/F₀ and integrated ratio readout
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z′ factors
14. Outcome assignment: If the compound caused a decrease of the membrane potential signal greater than 3*SD of the positive control and retained a positive fluorescent signal, it is termed active. If the compound is active in both duplicates and in the primary screen (pubchem AID: 2247), the compound is considered a non-specific modulator of membrane potential in HEK293 cells expressing TRPC6 channels (Value=2). Otherwise, it is designated as inactive (Value=1).
15. Score assignment: An inactive test compound is assigned the score of 0. An active test compound is assigned a score between 5 and 100 by calculating Integer(24.3*log(AvPercentage - 0.27))

Protocol

1. Cell culture: TRPC4β+ 5HT1A receptor expressing cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 μg/ml streptomycin, 500 μg/ml G418 and 40 μg/ml hygromycin
2. Cell plating: Add 50 μl/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS
3. Incubate overnight at 37°C and 5% CO₂
4. Remove medium and add 20 μl/well of 1x Fluo-4 solution to cells
5. Incubate 45 minutes at room temperature (RT) in the dark
6. Prepare 7.5x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), and ECmax of serotonin
7. Remove Fluo-4 dye solution and add 40 μl/well of assay buffer to cells
8. Remove 40 μl solution and add 20 μl/well of assay buffer to cells
9. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 5 seconds at 1 Hz to establish baseline
11. Add 4 μl of 7.5x compound stock into the cell plates.
12. Incubate plates for 110 seconds
13. Add submaximal concentration of serotonin and incubate for 110 seconds
14. Add maximally activating concentration of serotonin (serotonin ECmax) and read for another 110 seconds.
15. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
16. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z′ factors
17. Outcome assignment: If the compound causes a decrease of the normalized ratio greater than the mean normalized ratio of the ECMax control plus 3*SD, the compound is considered active. If the compound is active in both duplicates and in the primary screen, the compound is considered an inhibitor of TRPC4 channels. Otherwise, it is designated as inactive.
18. Score assignment: An active compound is assigned to a score between 5 and 100 by calculating Integer(25.0 * log(AvPercentage + 7.50)); while an inactive compound is assigned to a score of 0.

Protocol

1. Cell culture: TRPC4β +μ−Opioid Receptor-expressing HEK293 Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 μg/ml streptomycin, 500 μg/ml G418 and 40 μg/ml hygromycin
2. Cell plating: Add 50 μl/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS.
3. Incubate overnight at 37°C and 5% CO₂
4. Remove medium and add 20 μl/well of 1x Fluo-4 solution to cells
5. Incubate 45 minutes at room temperature (RT) in the dark
6. Prepare 7.5x compound plates and control plates on Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (EC0), and ECmax of AChCl
7. Remove Fluo-4 dye solution and add 40 μl/well of assay buffer to cells
8. Remove 40 μl solution and add 20 μl/well of assay buffer to cells
9. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 5 seconds at 1 Hz to establish baseline
11. Add 4 μl of 7.5x compound stock into the cell plates.
12. Incubate plates for 110 seconds
13. Add maximally activating concentration of AChCl (AChCl ECmax) and read for another 110 seconds.
14. Calculate ratio readout as F(max-min)/F₀ and integrated ratio readout
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z′ factors
16. IC50 and Hill Constant calculation from replicates was generated using Microcol Origin 6.0
17. Outcome assignment: If the test compound causes a maximum inhibition of TRPC4 greater than 30% in any concentration tested and a dose response curve is generated the compound is considered to be active (outcome=2). If the test compound does not cause inhibition of TRPC4 at any concentration tested or a dose response is not generated, the compound is designated as inactive (outcome=1).
18. Score assignment: Compounds with an IC50 less than 1 μM are given a score of 100, 1 μM-5 μM a score of 75, 5 μM-10 μM a score of 50, 10 μM- 20μM a score of 25 and any compound with an IC50 greater than 20 μM or those that are designated inactive in the outcome are given a score of 0

Protocol

1. Cell culture: Cells are routinely cultured as described above
2. Electrophysiological recording: For whole cell patch clamp recordings, the internal solution has ~400 nM free Ca and is composed of (in mM): 110 CsCl, 10 HEPES, 10 BAPTA 1 MgCl₂, 6.46 CaCl₂, pH adjusted to 7.2 with HCl. The osmolarity is 274. The external solution contains (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH7.4 adjusted with NaOH. The pipette resistance is 2–4 Mohms and recording is performed at the room temperature (22°C). Cells were held at 0 mV and repeated voltage commands stepping to −100 mV for 20 ms followed by a voltage ramp to 100 mV in 100 ms were given at a 0.5 sec interval.
3. Test compound application: Cells were pretreated with or without 1 μM of drug for > 2 min before the addition of stimulus compound. This condition was used to maximally activate TRPC4 whole-cell currents
4. Current amplitudes were measured at −100 mV and +100 mV for each cell and normalized to cell capacitance. Current densities were compared between treated and untreated cells.
5. Outcome assignment: If the average current density for treated cells was decreased by ≥40 % at either voltage, the compound was considered active (Outcome=2). Otherwise, it is designated as inactive (Outcome=1).
6. Score assignment: an inactive test compound is assigned the score of 0. An active test compound is assigned a score between 1 and 100 according to the maximal percent block at either voltage.

Protocol

1. Cell culture: Cells were routinely cultured as previously described
2. Cells were grown in to 80–90% confluency and dissociated with trypsin immediately prior to use. Cells were washed, resuspended in external buffer solution at 5–7 million cells per ml and loaded in the QPatch16 instrument. Cells were pipetted to QPlate wells and whole cell recordings established using the instrument’s integrated fluidics and pressure controls and following the manufacturer’s recommended protocols. Currents were sampled at 5 KHz and filtered at 1 KHz. 80% series resistance compensation was employed.
3. The TRPC4-μOR expressing HEK293 cells were voltage clamped at 0 mV and brief voltage ramps were applied every 5 s. During the voltage ramp segments, cells were held at 0 mV for 200 ms, then at −100 mV for 50 ms, followed by a ramp from −100 mV to +120 mV in 110 ms, held at +120 mV for 8 ms, and then back to 0 mV for 180 ms. TRPC4 currents were measured at +120 mV at the end of depolarizing ramp voltage commands. Cells with stable current amplitudes in baseline conditions and following addition of 50 nM DAMGO were used to evaluate effects of test compounds.
4. In order to evaluate effects of test compounds as inhibitors of TRPC4 channels, the channels were first activated by addition of a μ-opioid agonist (50 nM DAMGO) to the extracellular bath solution. Two compound addition protocols were used.
   1. Cumulative addition of three doses on individual cells.
      1. Cells were stabilized in saline for 5 min with two saline additions. After two additions of 50 nM DAMGO, cells were recorded for 3 min. Then 1.11 μM of compound with the presence of 50 nM DAMGO was added and recorded for 50 sec; second addition of 1.11 μM of compound with the presence of 50 nM DAMGO was recorded for another 100 sec. Similar additions and recordings were done with 3.33 and 10 μM of test compounds (with the presence of 50 nM DAMGO) sequentially. Cells are then washed with 2 saline additions for 2.5 min followed by addition of 50 nM DAMGO.
   2. Titration experiments using a single dose on individual cells:
      1. Cells were stabilized in saline for 5 min with two saline additions. After two addition of 50 nM DAMGO, cells were recorded for 3 min, followed by another addition of DAMGO for 3 min. One concentration of a compound with 50 nM DAMGO was added twice and recorded for 3 min, followed by another addition for 3 min. Test compound and DAMGO were washed off for 5 min with 3 saline additions. Cells were tested again for their response to 50 nM DAMGO with 2 additions and two 100 sec recording periods. DAMGO was then washed off by two saline additions in 2.5 min.
5. Percent inhibition of TRPC4 currents was calculated at the end of each test period as 100×(1−(I_test−I_baseline/I_DAMGO−I_baseline)).
6. Percent inhibition values calculated at each test compound concentration for a number of cells were combined and fitted with a Hill equation to provide IC50 values and Hill slope values.
7. Outcome assignment: IC50 values were calculated for each compound using a number of cells in each test plate. If the calculated IC50 values was less that 100 μM, the compound was considered active (Outcome=2). Otherwise, it is designated as inactive (Outcome=1).
8. Score assignment: an inactive test compound is assigned the score of 0. An active test compound is assigned a score between 1 and 100 according to the following criteria: 25 for compounds with 10 μM≤IC50<100 μM; 50 for compounds with 3 μM≤IC50<10 μM: 75 for compounds with 1 μM≤IC50<3 μM; 100 for compounds with IC50<1 μM.