A. Probe Chemical Name, Structure and Physiochemical Data

The IUPAC name of the probe ML365 is 2-methoxy-N-(3-(3-methylbenzamido)phenyl)benzamide. The batch prepared and submitted to the MLSMR is archived as SID 104221970, corresponding to CID 46785920.

B. Structure Verification and Purity: ¹H NMR, ¹³C NMR, LCMS, and HRMS Data

Proton and carbon NMR data for ML365/ SID 104221970/ CID 46785920: Detailed analytical methods and instrumentation are described in Section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental proton and carbon data are represented below, and the experimental proton and carbon spectra are included for reference (Appendix 6, Figures A6A and A6B, respectively).

Proton NMR Data for ML365/ SID 104221970/ CID 46785920:¹H NMR (400 MHz, CDCl₃) δ 9.86 (s, 1H), 8.25 (dd, J₁ = 7.8 Hz, J₂ = 1.9 Hz, 1H), 8.14 (br. s, 1H), 8.12 (br. t, J = 2.0 Hz, 1H), 7.67 (br. s, 1H), 7.66 – 7.59 (m, 2H), 7.51 – 7.46 (m, 1H), 7.39 – 7.30 (m, 4H), 7.14 – 7.09 (m, 1H), 7.03 (br. d, J = 8.4 Hz, 1H), 4.05 (s, 3H), 2.59 (s, 3H).

Carbon NMR Data for ML365/ SID 104221970/ CID 46785920:¹³C NMR (126 MHz, CDCl₃) δ 166.00, 163.37, 157.19, 138.89, 138.74, 138.56, 134.86, 133.31, 132.50, 132.42, 129.50, 128.56, 127.70, 124.06, 121.59, 121.56, 116.16, 115.76, 111.89, 111.49, 56.23, 21.32.

LCMS and HRMS Data for ML365/ SID 104221970/ CID 46785920: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental LCMS and HRMS data are represented as follows: LCMS retention time: 3.159 min. LCMS purity at 214 nm: 100%. HRMS: m/z calcd for C₂₂H₂₀N₂O₃ (M + H⁺) 361.1474, found 361.1557. The experimental HRMS and LCMS spectra are included for reference (Appendix, Figure A6C and A6D, respectively).

If available from a vendor, please provide details. This probe is not yet commercially available, as it was designed and prepared as an original analog for this project. A sample of ML365, synthesized at the KU SCC, has been deposited in the MLSMR; however, the compound is also available free of charge from the KU SCC[10].

C. Solubility

Aqueous solubility was measured in phosphate buffered saline (PBS) at room temperature (23°C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4. Probe ML365 (SID 104221970/ CID 46785920) was found to have a solubility measurement of 0.010 μg/mL, or 0.028 μM, under these conditions. Solubility was also assessed in thallium flux (FDSS) assay medium (1× HBSS pH 7.4), as well as in 140K QPatch assay medium (14 mM NaCl, 140 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 10 mM HEPES, pH 7.4). Under the FDSS assay medium conditions, solubility was determined to be 0.47 μg/mL, or 1.30 μM. Under the QPatch assay medium conditions, solubility was determined to be 0.17 μg/mL, or 0.47 μM[11]. These measurements can be related to the potency values of the assays used in the following ways:

The team recognized the overall modest solubility of ML365; however, the probe criteria (> 10-fold the IC₅₀ value) were met given the potency of the compound. Additional refinements to address this issue will be pursued in the next phase of funding for this project.

D. Stability

Aqueous and Chemical Stability: ML365 was evaluated for stability in PBS, for susceptibility to nucleophilic addition and formation of conjugates when treated with thiol-bearing dithiothreitol (DTT). Figures 3 and 4 represent the time course experiment with ML365 under various conditions. ML365 was dissolved at 10 μM in 1:1 PBS/acetonitrile at pH 7.4 (1% DMSO) and independently incubated at room temperature with no nucleophile present or 50 μM dithiothreitol (DTT). The test reactions were sampled every hour for eight hours (or up to 48 hours, for PBS stability) and analyzed by RP HPLC/UV/HRMS. The analytical RP HPLCUV/HRMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity BEH C18 column (2.1 × 50mm, 1.8um) and elution with a linear gradient of 99% water to 100% CH₃CN at 0.6 mL/min flow rate. Peaks on the 214 nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent compound remaining. All samples were prepared in duplicate and the average plotted[12].

Figure 3

Synthetic scheme for preparation of ML365.

Figure 4

Inhibition of thallium signal in TASK1/KCNK3-expressing cells by ML365. Left panel (A) displays fluorescent signals in individual wells in the presence of different concentrations of ML365. Concentration response curve normalized to controls showing inhibition (more...)

For the aqueous stability experiment, 100% of ML365 was detected remaining after 48 hr in 1:1 PBS/acetonitrile (Figure 1).

Figure 1

Aqueous stability of ML365 over 48 h in 1:1 PBS/acetonitrile.

For the chemical stability experiment, ethacrynic acid, a known Michael acceptor, was used as a positive control and was tested in 1:1 PBS/acetonitrile (Figure 2, panel A). After exposing ML365 to 5-fold concentrations of DTT for 8 h in 1:1 PBS/acetonitrile, 100% of ML365 was remaining (Figure 2, panel B). The masses of potential adducts and dimers of ML365 were searched for in the final samples to determine if any detectable adduct formed or dimerization had occurred. In the case of ML365, no adducts were detected at any time point using LCMS detection[12].

Figure 2

Control experiment with ethacrynic acid (panel A) and chemical stability of ML365 over 8 h in the presence of a 5-fold concentration of dithiothreitol (panel B).

A. Fluorescent Assay

The primary HTS assay utilized a thallium sensitive dye to measure potassium channel activity in a CHO cell line expressing TASK1/KCNK3 (AID 602410). An extracellular solution containing both thallium and potassium was added triggering thallium influx into the cells. The electrochemical gradient drives the net inflow of thallium down its concentration gradient and the accumulation of intracellular thallium increases the fluorescence of the FluxOR™ dye (Invitrogen). The reported IC₅₀ values of ML365 were determined by averaging multiple independent experiments. Figure 4 illustrates the typical titratable inhibition of TASK1/KCNK3-mediated thallium influx by ML365 in one experiment. After normalization to buffer controls (0% inhibition) and TASK1/KCNK3-independent thallium signal (100% inhibition), the resulting dose-dependent curve was fit with an IC₅₀ value of 4 nM. In replicate experiments (n=4), the mean ± SD of the determined IC₅₀ values were 4.3 ± 0.4 nM.

B. Automated Electrophysiological Assay

To further validate the compound effect on TASK1/KCNK3 channels, an electrophysiological assay was developed on the QPatch 16X automated electrophysiology instrument (Sophion) (AID 652209). Whole cell voltage clamp recordings were made using the same TASK1/KCNK3-expressing CHO cells used in the primary screen. A complete description of the protocol is included in this report within the appendix (Appendix 6.3). Concentration response curves were generated from data collected at −30 mV and normalized to 100% inhibition determined through the application of 2 mM barium in 140 mM potassium solution at pH 5.8. This protocol was used to evaluate select compounds and the data has been collected in the SAR tables. ML365 effect on the TASK1/KCNK3 channel was evaluated in multiple independent experiments using this method. Figure 5A illustrates dose-dependent inhibition of ML365 on whole cell currents in one experiment. Figure 5B summarizes the inhibition of TASK1/KCNK3 currents at −30 mV and shows that ML365 inhibited TASK1/KCNK3 with an IC₅₀ of 12 ± 1 nM (n=4).

Figure 5

Inhibition of TASK1/KCNK3 currents in automated electrophysiology (QPatch) experiments. Left panel (A) shows ML365 caused dose-dependent inhibition in TASK1 currents. After ramping from −100 mV to +50 mV, the cells were held at +50 mV for 5 ms, (more...)

A. Profiling Against Host Targets

Probe ML365 was submitted to Eurofins PanLabs (formerly Ricerca Biosciences) to evaluate it in radioligand binding assays against a panel of 68 GPCRs, ion channels and transporters at a single concentration of 10 μM, each in duplicate. The following activities were determined as > 50% inhibition at 10 μM (Table 3). The entire data set is included in the Appendix, section 6.4.

Table 3

Targets that were inhibited by ML365 with > 50 percent inhibition at 10 μM.

B. Channel Selectivity Studies

The selectivity of ML365 for inhibiting the TASK1 potassium channel as compared to block of other related and non-related potassium channels was determined using fluorescent and electrophysiological assays.

C. Selectivity in Fluorescent Experiments

ML365 was evaluated for its ability to block a closely related potassium channel, TASK3 (KCNK9), using HEK293 cell line stably expressing the channel under a tetracycline inducible promoter (AID 652211). TASK3 activity was monitored using a fluorescence assay similar to that described for TASK1. Changes in fluorescence intensity due to modulation of channel activity were recorded on an FDSS instrument (Hamamatsu). Based on this assay, ML365 inhibited TASK3 activity with an IC₅₀ of 0.26 ± 0.02 μM (n=4, Figure 6) The effect of all SAR compounds on a panel of potassium channels was also evaluated using an FDSS instrument (Hamamatsu) measuring changes in fluorescence intensity and the data for all compounds has been collected in the SAR tables including their respective AIDs. ML365 activity against all cell lines tested is summarized in Figure 6 indicating the average percent inhibition of thallium signal in cells treated with 1 μM compound. ML365 showed modest effect against KCNQ2, inhibiting less than 20% of the signal. Little to no inhibition was observed in hERG- and Kir2.1-expressing cells.

Figure 6

Selectivity of ML365 effect against various potassium channels using fluorescent-based assays. Left panel (A) shows cells expressing various potassium channels loaded with FluxOR™ to measure changes in thallium influx due to application of ML365. (more...)

D. Selectivity in Electrophysiological Studies

Whole cell voltage clamp recordings were made from HEK293 cells expressing the closely related K2P channel, TASK3, using the QPatch 16X automated electrophysiology instrument (Sophion) (AID 652210). A voltage protocol similar to that used for monitoring TASK1 function was utilized and inhibition of TASK3 current determined at −30 mV. ML365 exhibited modest inhibition of TASK3 with an IC₅₀ 40 times that observed for TASK1 (0.012 μM and 0.48 μM, respectively). Figure 7 compares the concentration response curves generated for ML365 against both cell types tested in automated electrophysiology experiments demonstrating the selectivity of ML365 for TASK1 over TASK3.

Figure 7

Selectivity of ML365 effect for TASK1 as compared to TASK3 using QPatch 16X. Percent inhibition of channel current elicited at −30 mV after application of various concentrations of ML365 for both TASK1 and TASK3 channels. ML365 shows a 40-fold (more...)

HTS for identification of compounds that inhibit the two-pore domain potassium channel TASK1

1.

Cell culture: Cells are routinely cultured in Hams F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 μg/ml streptomycin, 400 μg/ml hygromycin and 10 μg/mL Blasticidin S

2.

Cell plating: Add 50 μl/well of 120,000 cells/ml re-suspended in Hams F12 medium with 10% FBS and 1μg/μl Tetracycline

4.

Remove medium and add 25 μl/well of 1× FluxOR solution to cells

5.

Incubate 90 minutes at room temperature (RT) in the dark

6.

Prepare 7.5× compound plates and control plates on Cybi-Well system; test compounds are prepared using assay buffer; controls are assay buffer (EC₀), and EC_max of SID17386958

7.

Remove FluxOR dye solution and add 20 μl/well of assay buffer to cells

8.

Add 4 μl of 7.5× compound stock into the cell plates via Cybi-Well system

9.

Incubate all cell plates for 20 minutes at RT in the dark

10.

Prepare 5× stimulus buffer containing 25 mM K₂SO₄ and 7 mM Tl₂SO₄

11.

Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader

12.

Measure fluorescence for 10 seconds at 1Hz to establish baseline

13.

Add 6 μl/well of stimulus buffer onto cells and continue measuring fluorescence for 180 seconds

14.

Calculate ratio readout as F_(max−min)/F₀

15.

Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z′ factors [15]

16.

Calculate B scores [16] for test compounds

17.

In SAR analysis, IC₅₀ and Hill Constant calculations from replicates were generated using Microcal Origin 6.0

Selectivity assay for the identification of compounds that inhibit the two-pore domain potassium channel TASK1 using TASK3-expressing cells

1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 μg/mL streptomycin, 15 μg/mL Blasticidin S and 400 μg/mL hygromycin
2. Cell plating: Add 50 μl/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% FBS and 1μg/μl Tetracycline
3. Incubate overnight at 37°C and 5% CO₂
4. Remove medium and add 25 μl/well of 1× FluxOR solution to cells
5. Incubate 90 minutes at room temperature (RT) in the dark
6. Prepare 7.5× compound plates and control plates on Cybi-Well system; test compounds are prepared using assay buffer; controls are assay buffer (EC₀), and EC_max of SID17386958
7. Remove FluxOR dye solution and add 20 μl/well of assay buffer to cells
8. Add 4 μl of 7.5× compound stock into the cell plates via Cybi-Well system
9. Incubate all cell plates for 20 minutes at RT in the dark
10. Prepare 5× stimulus buffer containing 25 mM K₂SO₄ and 7 mM Tl₂SO₄
11. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
12. Measure fluorescence for 10 seconds at 1Hz to establish baseline
13. Add 6 μl/well of stimulus buffer onto cells and continue measuring fluorescence for 110 seconds
14. Calculate ratio readout as F_(max−min)/F₀
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z′ factors
16. IC₅₀ and Hill Constant calculations from replicates were generated using Microcal Origin 6.

Selectivity assay for the identification of compounds that inhibit the two-pore domain potassium channel TASK1 using cells expressing non-K2P potassium channels

1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50μg/ml streptomycin, and 500μg/ml G418
2. Cell plating: Add 50 μl/well of 300,000 cells/ml re-suspended in DMEM/F12 medium with 10% FBS
3. Incubate overnight at 37°C and 5% CO₂
4. Remove medium and add 25 μl/well of 1× FluxOR solution to cells
5. Incubate 90 minutes at room temperature (RT) in the dark
6. Prepare 7.5× compound plates and control plates on Cybi-Well system; test compounds are prepared using assay buffer
7. Remove FluxOR dye solution and add 20 μl/well of assay buffer to cells
8. Add 4 μl of 7.5× compound stock into the cell plates via Cybi-Well system
9. Incubate all cell plates for 20 minutes at RT in the dark
10. Prepare 5× stimulus buffer containing 25 mM K₂SO₄ and 7 mM Tl₂SO₄
11. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
12. Measure fluorescence for 10 seconds at 1Hz to establish baseline
13. Add 6 μl/well of stimulus buffer onto cells and continue measuring fluorescence for 110 seconds
14. Calculate ratio readout as F_(max−min)/F₀
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z′ factors
16. Calculate percent inhibition by normalizing fluorescent ratios to EC₀ controls. IC₅₀ and Hill Constant calculations from replicates were generated using Microcal Origin 6.

TASK1 validation assay using automated electrophysiology

1. Cell culture: Cells are routinely cultured in F12 medium, supplemented with 10% Fetal Bovine Serum (HiFBS), 10 μg/mL Blasticidin S and 400 μg/mL hygromycin
2. The stable CHO cell line expressing inducible TASK1 expression was seeded and induced (1 μg/ml Tetracycline) in 15 cm dish two days before testing on QPatch 16X automated patch clamp system andTASK1 cells were incubated at 30 °C overnight before recording.
3. Cells were detached into single cell suspension and allowed to recover for at least 1 hr before running in single-hole mode on QPatch.
4. 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. Whole cell currents were filtered at 1 kHz (4-pole Bessel) and sampled at 5 kHz. The series resistance was compensated by 60% with a cut-off frequency of 0.8 kHz.
5. TASK1 cells were in 4 mM potassium external buffer (4K buffer) for 2 minutes after which time external solution was switched to 140 mM potassium external buffer (140K buffer). TASK1 currents were measured by brief voltage ramps applied every 5 seconds. During voltage ramps, cells were held at −80 mV for 50 ms, then at −100 mV for 50 ms followed by a 750 ms ramp to +50 mV, cells were then held at +50 mV for 5 ms, and then back to −80 mV for 40 ms.
6. In order to evaluate effects of test compounds as inhibitors of TASK1 channels, cells were stabilized in 4K buffer for 2 minutes. After application of 140K buffer, cells were recorded for 3 minutes. Then test compound in 140K buffer was added to cells in a dual addition mode in which the external solution was completely exchanged twice for each concentration. Compound effects were determined after a 5 minute incubation-recording period. Three test compound concentrations were recorded during each protocol. Compound was washed off by application of 140K buffer for 2 minutes after testing the highest concentration. A reference 140 mM potassium solution containing 2 mM barium at pH 5.8 was then added to cells and recorded for 3 minutes to define the level of 100% inhibition.
7. Percent inhibition of TASK1 currents was calculated at the end of each test period as 100 × (1−(Itest-Ibarium/I140k -Ibarium)) where I is the measured current at −30 mV.
8. Percent inhibition values calculated at each test compound concentration for a number of cells were combined and fitted with Hill equation to provide IC₅₀ values.

Selectivity assay for the identification of compounds that inhibit the two-pore domain potassium channel TASK1 using automated electrophysiology

1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Fetal Bovine Serum (HiFBS), 15 μg/mL Blasticidin S and 400 μg/mL hygromycin
2. The stable HEK 293 cell line expressing inducible TASK3 expression was seeded and induced (0.5μg/ml Tetracycline) in 15 cm dish two days before testing on QPatch 16X automated patch clamp system.
3. Cells were detached into single cell suspension and allowed to recover for at least 1 hr before running in single-hole mode on QPatch.
4. 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. Whole cell currents were filtered at 1 kHz (4-pole Bessel) and sampled at 5 kHz. The series resistance was compensated by 20% with a cut-off frequency of 0.2 kHz.
5. TASK3 cells were in 4 mM potassium external buffer (4K buffer) for 2 minutes after which time external solution was switched to 140 mM potassium external buffer (140K buffer). TASK3 currents were measured by brief voltage ramps applied every 5 seconds. During voltage ramps, cells were held at −80 mV for 50 ms, then at −100 mV for 50 ms followed by a 600 ms ramp to +20 mV, cells were then held at +20 mV for 5 ms, and then back to −80 mV for 40 ms.
6. In order to evaluate effects of test compounds as inhibitors of TASK3 channels, cells were stabilized in 4K buffer for 2 minutes. After application of 140K buffer, cells were recorded for 3 minutes. Then test compound in 140K buffer was added to cells in a dual addition mode in which the external solution was completely exchanged twice for each concentration. Compound effects were determined after a 5 minute incubation-recording period. Three test compound concentrations were recorded during each protocol. Compound was washed off by application of 140K buffer for 2 minutes after testing the highest concentration. A reference 140 mM potassium solution containing 2 mM barium at pH 5.8 was then added to cells and recorded for 3 minutes to define the level of 100% inhibition.
7. Percent inhibition of TASK3 currents was calculated at the end of each test period as 100 × (1−(Itest-Ibarium/I140k -Ibarium)) where I is the measured current at −30 mV.
8. Percent inhibition values calculated at each test compound concentration for a number of cells were combined and fitted with Hill equation to provide IC₅₀ values.