A small molecule activator of KCNQ2 and KCNQ4 channels

2.1. Assays

2.2. Probe Chemical Characterization

Probe compound ML213 (CID: 3111211, SID: 104223736) was prepared according to the above scheme and provided the following characterization data: LCMS (>98%) m/z = 258.4 [M + H]⁺ (0.766 min retention time, 254 nm). ¹H NMR (400 MHz, CDCl₃): δ 6.89 (s, 2H), 6.59 (bs, 1H), 4.26 (dddd, J = 11.6, 10.4, 6.0, 1.6 Hz, 1H), 2.59 (d, J = 5.6 Hz, 1H), 2.27 (s, 3H), 2.19 (s, 3H), 2.18 (s, 3H), 1.78-1.72 (m, 2H), 1.67-1.21 (m, 8H).

Solubility: Solubility in PBS was determined to be 38.6 μM, which is more than 100-fold higher that the EC₅₀ for KCNQ2 channel activation.

GSH Conjugates: No glutathione conjugates detected.

Stability: Stability was determined for ML213 at 23°C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) and is shown in Table 3 and Figure 2 presented below. Due to the nature of the stability curve, it appears the instability may in fact be due to solubility issues with the compound. This can be concluded by the drop in stability between 2 hour and 24 hour and the plateau between 24 and 48 hours. However, further studies would be needed to determine this absolutely.

Table 3

Stability data for ML213.

Figure 2

Stability data for ML213.

Compounds added to the SMR collection (MLS#s): 003370512 (ML213, CID 311121, 28.3 mg), 003370511 (CID 2852569, 7.3 mg), 003370510 (CID 4296446, 5.0 mg), 003370509 (CID 3843095, 8.3 mg), 003370508 (CID 4658599, 5.0 mg), 003370507 (CID 3473417, 5.2 mg).

2.3. Probe Preparation

N-mesitylbicyclo[2.2.1]heptane-2-carboxamide (ML213, CID 3111211). To a solution of 2-endo-norbornanecarbonylchloride (1.0 eq, 250 mg, 1.6 mmol) in DMF (1 mL) was added 2,4,6-trimethylaniline (213 mg, 1.6 mmol) and triethylamine (440 μL, 3.2 mmol). After stirring 24 h, the reaction mixture was filtered and the supernatant purified by mass directed chromatography to provide ML213 (124.3 mg, 0.5 mmol, 30%) as a white crystalline solid. The final product was evaluated via chiral SFC (supercritical fluid chromatography) and it was shown to be a mixture of 4 diastereomers (see attached spectral analysis, Appendix 3). The endo starting material is the only isomer commercially available.

3.1. Summary of Screening Results

Figure 3Flow chart for generation of KCNQ2 probe candidates

Screening Summary:

1. 305k compounds initially screened in HTS assay (AID 2239) → 1644 hits
2. 1189 available compounds were retested (AID 2287) → 807 compounds validated as KCNQ2 activators using assay from primary screen
3. 1189 compounds were tested in a counterscreen against parental cells (AID 2282) → 676 compounds showed no activity in the parental cell line, yielding 468 validated KCNQ2 activator hits
4. Based on confirmatory and counter-screens, an expanded set of 944 compounds were tested on KCNQ2/W236Lchannels in an automated patch clamp assay (AID 2558) in order to possibly select for compounds with mechanisms differing from retigabine, in which 91 compounds tested as active.
5. A set of 58 compounds were selected for titration experiments in automated electrophysiology on KCNQ2 channels. 38 compounds afforded clear dose-response activation of KCNQ2 channels (AID 2603 and AID 2654).
6. From this list, Vanderbilt Specialized Chemistry Center selected two classes of compounds for SAR optimization (AID 2654).
7. CID 3111211 was identified as a probe candidate from one structural class.

3.2. Dose Response Curves for Probe

ML213 activation of KCNQ2 channels was evaluated in both electrophysiological and fluorescent assays. For the fluorescence assay, the activity of KCNQ2 potassium channels was monitored by the influx of a surrogate ion, Tl⁺, for potassium. Tl⁺ influx was detected with a fluorescent dye (FluxOR). This assay can be implemented in high-throughput formats and was used in the primary screen. As shown in Figure 4, ML213 produced a concentration-dependent increase in Tl⁺ influx, with an EC₅₀ value of 0.36 μM and a maximal increase of 56% (Figure 4A and 4B).

Figure 4

ML213 activation of KCNQ2 channels in a Tl⁺ flux assay. (A) Typical fluorescence intensity traces in the Tl⁺ flux assay were recorded in vehicle control (black line) and in the probe compound ML213 at 10 μM (red line). (B) Dose response curve (more...)

Currents through KCNQ2 channels were recorded using an IonWorks automated electrophysiology instrument in Population Patch Clamp mode (Figure 5); the results show ML213 displays a concentration dependent enhancement of KCNQ2 currents. Membrane currents provide a linear measure of channel activity and provide a direct determination of compound effects on channel function. KCNQ2 currents were activated by depolarizing voltages and after the addition of 5 μM ML213 to the KCNQ2 cells, a greater than 4-fold increase in currents was produced at −10 mV (Figure 5A). To evaluate EC₅₀ values for ML213 at different test potentials, 8-point concentration response curves for ML213 were generated at a variety of voltage steps. The EC₅₀ values were similar at voltages greater than −10 mV (Figure 5B), although the relative increases in current were decreased at higher levels of depolarization due to higher levels of channel open probability in control. At −10 mV step potential, ML213 exhibited a concentration-dependent enhancement of KCNQ2 currents with an EC₅₀ value of 0.23 μM and a maximal increase of 445% (Figure 5C). The close agreement between EC₅₀ values in the electrophysiological and fluorescent assays supports use of both assay methods in selectivity studies. To evaluate the mechanism for KCNQ2 potentiation by ML213 and to determine an estimate of potency that was not dependent on test potential, voltage activation curves were determined in the absence and presence of ML213. In the presence of 5 μM ML213, the V_1/2 (voltage required for half-maximal activation) of KCNQ2 was left-shifted by 37.42 ± 3.0 mV (mean±s.e.m) compared with control values in the same cells (Figure 5D). ML213 caused a concentration dependent shift in the V_1/2 for KCNQ2 activation with an EC₅₀ 0.34 ± 0.07 μM and a maximal shift of 37 mV (Figure 5E).

Figure 5

ML213 effects on voltage-dependent activation of KCNQ2 channels using automated electrophysiology. (A) KCNQ2 currents traces were recorded in the absence (left panel) and presence (middle panel) of 5 μM ML213. From a holding potential of −100mV, (more...)

3.3. Scaffold/Moiety Chemical Liabilities

No chemical liabilities for the probe molecule have been identified at the present time.

3.4. SAR Tables

Based on the distribution of KCNQ2 in the CNS, and previous work showing efficacy of KCNQ2 activators in a number of CNS related in vivo animal models – ML213 represents an attractive lead molecule for a CNS indication. The calculated properties of ML213 are in line with most known molecules with CNS exposure (MW < 300, cLogP = 2 – 4, tPSA <75). In addition, ML213 represents a novel chemical scaffold, as this compound does not include a heteroaryl moiety (as in ICA-27243) and displays a unique activity profile (KCNQ2 and KCNQ4 activator). Using ML213 as a starting point, the SAR evaluation was centered on the aniline portion (blue) and amide portion (red).

Keeping the bicyclo[2.2.1]heptylamide constant, a variety of anilines were investigated. The 2,4-dimethyl derivative (SID 103911606) was equipotent with ML213 (220 nM; 194% maximal change). Moving the methyl groups around the ring revealed interesting SAR, the 2,5-dimethyl compound (SID 103073347) was also equipotent (400 nM; 251% maximal change), but the 2,6-dimethyl and 2,3-dimethyl derivatives were at least 3 to 10-fold less potent (3,240 nM; 198% maximal change and 1,000 nM; 426% maximal change, respectively). Moving the dimethyl groups to the meta positions (3,5-dimethyl) as in SID 103911620 led to a much less active compound (47.4% maximal change at 30 μM). Removal of two of the methyl groups, leaving the 4-substituted compound retained activity (SID 103911615, 750 nM; 167% maximal change). The simple phenyl derivative also showed a potentiation of the maximal change (136% at 30 μM; EC₅₀ not determined). Extending the substitution in the 4-position to either an ethyl group (SID 103911605, 920 nM; 305% maximal change), an isopropyl group (SID: 103911608, 720 nM; 191% maximal change) or a 2-naphthyl group (SID 103911607; 600 nM; 143% maximal change) were all tolerated. Interestingly, replacing the 4-methyl with a 4-trifluoromethyl group led to an inactive compound (SID 103911618). Finally, addition of halogens (2,4-dichloro, SID 103911621; 1,520 nM, 171% maximal change; 2,4-difluoro, SID 103911617, 10,050 nM, 147% maximal change; 4-fluoro, SID 103911616, 61% maximal change, EC₅₀ not determined) was not tolerated. Replacement of the phenyl with a pyridyl was also not tolerated (SID 103911604, inactive), although a substituted pyridyl was active (SID 103911624, 1,810 nM, 500% maximal change).

Table 4

SAR Evaluation of the right-hand portion of ML213.

The next round of SAR kept the mesityl (2,4,6-trimethyl) aniline constant and a variety of alky, cycloalkyl and aryl amides were evaluated. Groups replacing the bicyclo[2.2.1]heptane were not well tolerated. Replacement of the bicyclo[2.2.1]heptane group with a simple cyclohexyl group (i.e., removal of the bridgehead group) led to a 5-fold loss in activity (1,570 nM; 230% maximal change). Simple alkyl or branched alkyl groups were not tolerated (SID: 103911622, 103% maximal change, EC₅₀ not determined; SID 103911597, 79% maximal change, EC₅₀ not determined; SID 103911596, 38% maximal change, EC₅₀ not determined), nor was the bulkier adamantyl group (SID 103911602, inactive). The only group that gave comparable activity to ML213 was the cyclopentylethyl group (SID 103911613, 490 nM, 310% maximal change). Replacement with aryl groups or substituted aryl groups led to inactive compounds. It is recognized that ML213 is a mixture of diastereomers, although this probe should still be a valuable tool for in vitro experiments.

Table 5

SAR Evaluation of the left-hand amide portion of ML213.

3.5. Cellular Activity

All assays for KCNQ2 and other ion channels described in this report are cell-based assays. The activity of ML213 in these assays indicates that the probe compound exhibits adequate cell permeability to support further studies with native cells or tissues. No acute toxicity was observed in the cell-based assays.

3.6. Profiling Assays

ML213 (CID 3111211) has been tested in 247 assays performed within the MLPCN network and was active in only one assay (Cycloheximide Counter-screen for Small Molecule Inhibitors of Shiga Toxin [Primary Screening: AID 2314]) that was not dependent on KCNQ2.

ML213 was evaluated in fluorescent and electrophysiological assays against six related channels from the KCNQ family and against two distantly related potassium channels, hERG and Kir2.1. The results are listed in Table 6. For comparison, CID 3111211 displayed EC₅₀ values for KCNQ2 activation of 0.36 μM in a fluorescent Tl⁺ influx assay and 0.23 μM in an IonWorks electrophysiological assay.

Table 6

Selectivity of ML213 for activation of KCNQ2 channels compared with activation or block of other potassium channels. EC₅₀ values are calculated from dose-dependent activation curves for KCNQ channels and for inhibition of Kir2.1 and hERG channels.

To more fully characterize this novel KCNQ2 and KCNQ4 probe molecule, ML213 was tested on 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 bind with any of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM). Thus, from the Ricerca selectivity profile, inactivity in the PubChem assays and the selectivity versus closely related ion channels in which ML213 affords greater than 25-fold selectivity for activation or block of related KCNQ channels and other distantly related potassium channel except for KCNQ4 channels, ML213 (CID 3111211) is highly selective and can be used to dissect the role of KCNQ2 and KCNQ4 activators in vitro and, potentially, in vivo.

Table 7

Ricerca Profiling of SID 104223736/VU0448090-1.

ML213 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 assays, ML213 was clean against all of the CYP enzymes evaluated (>30 μM). Other analogs of ML213 also displayed a favorable profile against CYP inhibition with the exception of SID 103911607, which showed inhibition of CYP1A2 (830 nM) and CYP2C9 (7,680 nM) and SID 103911623, which inhibited CYP1A2 (7,750 nM). All other compounds tested were >10 μM against all CYPs.

Table 8

CYP₄₅₀ Inhibition for ML213 and analogs.

The metabolic stability of ML213 and analogs were evaluated in assays which can predict rat and human hepatic clearance from in vitro microsomal clearance values (Table 9). This allows for a rank ordering of compounds that would be predicted to have poor stability in in vivo testing protocols (after 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 or naphthyl group which is known to undergo oxidation by liver microsomes. However, ML213 did display an acceptable free fraction profile (6.5% and 1.2% free unbound for human and rat, respectively). Other analogs also showed free fraction profile >1–3% as well.

Table 9

Intrinsic clearance and protein binding data of ML213 and analogs.

This data suggests that ML213 would likely not be appropriate for oral dosing; however, several other dosing options are available for compounds that undergo significant first-pass metabolism (e.g., subcutaneous dosing, intravenous dosing). Thus, due to the therapeutic importance and distribution of KCNQ2 and KCNQ4 in the CNS, we further profiled ML213 in a snapshot in vivo PK study to assess the plasma and brain levels after a single time point (1 hr, 10 mg/kg) following subcutaneous administration (Table 10). ML213 displayed plasma levels after 1 hour time point greater than 1 μM (1.03 μM); however, ML213 displayed poor brain levels at the same time point (0.095 μM); resulting in a very low B/P (0.09). ML213 does exhibit lower solubility than desired (~40 μM) and this could be contributing to the low brain levels. Further optimization of ML213 is planned in order to address the issues of metabolic stability and solubility in order to refine the in vivo PK properties.

Table 10

Brain:plasma ratio after subcutaneous administration in rat.

Table 11

Calculated Property Comparison of ML213 with MDDR Compounds.

4.1. Comparison to Existing Art and How the New Probe is an Improvement

A number of structural classes of compounds have been described in the scientific and patent literature that act as KCNQ channel activators (vide supra) (2, 11, 31). Broad selectivity analysis has not been presented for many described KCNQ-activating compounds. BMS-204352 and retigabine have been evaluated for activation of KCNQ2, KCNQ2/3, KCNQ3/4 and KCNQ4 channels and both compounds exhibit similar levels of activation for all channel subtypes (10, 33–35). More recent examples from Bristol-Myers Squibb also have been reported as pan-KCNQ activators; although the potency against the other members of the family has not been reported. Icagen’s most recent compound (ICA-27243) shows selectivity for KCNQ2/Q3 versus KCNQ4 (25-fold) and KCNQ3/Q5 (>75-fold), although data against KCNQ1 was not reported. Compound 6 is selective for KCNQ2/Q3 versus KCNQ1 + KCNE1; however, no other data have been reported. Selectivity data is lacking for other compounds disclosed in the patent literature.

In contrast to other reported activators, ML213 exhibits nanomolar activation of KCNQ2 and KCNQ4 channels, and exhibits selectivity against other subtypes (>80 fold selective versus KCNQ1, KCNQ3, KCNQ5, KCNQ1 + KCNE1). In addition, ML213 was inactive against a profiling assay of 68 GPCRs, transporters and ion channels (Ricerca Lead Profiling), and was 27-fold selective against another potassium channel (Kir2.1). ML213 also shows an excellent profile against the major CYP enzymes (>80-fold selectivity). Unfortunately, ML213 shows poor stability in liver microsomes and suboptimal brain levels after subcutaneous dosing. However, ML213 represents the most potent and selective KCNQ2/Q4 activator to date.

4.2. Mechanism of Action Studies

The anticonvulsant, retigabine, activates KCNQ2 and KCNQ2/3 heteromultimeric channels. A single amino acid substitution, W236L, removes sensitivity of KCNQ2 channels to retigabine activation (34, 35). ML213 was evaluated for effects on KCNQ2/W236L channels to evaluate the possibility of a shared binding site or mechanism with retigabine. ML213 was inactive on KCNQ2/W236L channels, suggesting possible similar interaction sites with retigabine. ML213 largely shifts the conductance-voltage curve to hyperpolarizing potentials. The combination of increased conductance and hyperpolarizing shift of half maximal activation contributes to the overall augmentation of current amplitude.

4.3. Planned Future Studies

ML213 is suitable for in vitro studies of the functional properties and roles of KCNQ2 and KCNQ4 channels. These studies could include, but are not limited to, evaluation of the roles of these channels in controlling excitability in isolated neurons and in tissue slices. Since mutations in KCNQ2 channels are involved in some forms of human epilepsy, ML213 would be useful for examining mechanisms underlying epileptogenesis in tissue slices. ML213 is unique in displaying selectivity for activation of KCNQ4 over KCNQ3 and KCNQ5 channels. KCNQ4 channels are expressed in neurons in the auditory system and ML213 may provide a tool to investigate the role of these channels in auditory processing. ML213 may be a useful probe for mapping channel domains involved in gating by constructing chimeric channels composed of domains from related KCNQ channels with differing sensitivities to activation by ML213. With respect to improving the liver metabolic profile, more expanded SAR may be applied. However, our data have clearly shown that benzylmethyl or naphthyl group cannot be replaced.

Improvements in the metabolic properties of ML213, while retaining or improving potency and selectivity, would provide a useful probe for in vivo analysis of the roles of KCNQ2 and KCNQ4 channels in CNS function. In addition, the diastereomeric ratio will be further evaluated in the extended probe phase of the project. Chemistry will be developed to synthesize and characterize each of the isomers; or, the [2.2.1]-bicyclo will be eliminated altogether in order to improve on the calculated properties and minimize the lipophilicity of the molecule.

Appendix 1. Cell-based FDSS Tl⁺ flux assay

1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50 μg/ml streptomycin, and 500 μg/ml G418.
2. Cell plating: Add 50 μl/well of 120,000 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 darkness.
6. Prepare 7.5× compound plates and control plates on the Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (IC0), ICmax of ZTZ240(36) (all with DMSO concentrations matched to that of test compounds).
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 darkness.
10. Prepare 5× stimulus buffer containing 12.5 mM K₂SO₄ and 12.5 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. Depolarize cells with 6 μl/well of stimulus buffer 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 B scores for test compounds using ratios calculated in Step 14.
17. Outcome assignment: If the B score of the test compound is more than 3 times the standard deviation (SD) of the B scores of ratios of the library compounds (>=3*SD), AND the B score of initial fluorescence intensity is within 3 times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as an active (Value=2) potentiator of the KCNQ2 channels. Otherwise, it is designated as inactive (value=1).
18. Score assignment: An active test compound is assigned a score between 0 and 100 by calculation of INT(100*LOG(B Score Potentiator Ratio), they are normalized to the smallest and largest LOG(B Score Potentiator Ratio), B Score Potentiator Ratio.

Appendix 2. KCNQ potassium channel IonWorks electrophysiology assay

KCNQ2 activity was examined in an electrophysiological assay using the population patch clamp mode on the Ionworks Quattro (MDC, Sunnyvale, CA), an automated patch clamp instrument. The CHO cells stably expressing KCNQ2 channels were freshly dislodged from flasks and dispensed into a 384-well population patch clamp (PPC) plate. The cell plating density was 7,000 cells/well suspended in the extracellular solution, composed of (in mM): 137 NaCl, 4 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 HEPES, and 10 glucose, pH 7.4 adjusted with NaOH.

After dispensing, seal resistance of cells was measured for each well and cells were perforated by incubation with 50μg/ml amphotericin B (Sigma, St. Louis, MO), which was dissolved in the internal solution composed of (in mM): 40 KCl, 100 K-Gluconate, 1 MgCl₂, 5 HEPES, 2 CaCl₂, pH 7.2 adjusted with KOH. Activity of KCNQ2 was then measured with the recording protocol as followings. Leak currents were linear subtracted extrapolating the current elicited by a 100-ms step to −100 mV from a holding potential of −90 mV. During the voltage pulse protocol, cells were held at −90 mV, followed by by 2,000 ms depolarizing step from −90 mV to −10 mV, and then back to −90mV for 2000 ms. The currents were measured at the end of the depolarization pulse before and after the application of compounds for 3 min. Only cells with a current amplitude more than 100 pA at −10 mV and a seal resistance >30 MΩ were included in the data analysis.

Compound effects were assessed by the percentage changes in the KCNQ2 steady state currents, which were calculated by dividing the difference between pre- and post-compound KCNQ2 currents by the respective pre-compound currents in the same well. When constructing conductance-voltage curves, conductance values were calculated by dividing the steady state outward currents measured during the voltage steps by the driving force (step voltage minus the calculated potassium reversal potential).

The KCNQ2 protocol was also used for KCNQ2/KCNQ3 recording. But for KCNQ1 and KCNQ4, the cells were depolarized to +40mV from the holding potential −70 mV. Currents were measured at the step current at +40 mV. And for KCNQ1/KCNE1, cells were stimulated by 3,000 ms depolarizing step from −70 mV to +40 mV, followed by hyperpolarization to −20 mV for 500 ms. Currents were measured at the steady state of +40mV voltage step.

No corrections for liquid junction potentials (estimated as −20 mV by comparing the KCNQ2 reversal potential with the calculated Nernst potential for potassium) were applied. The current signal was sampled at 0.625 kHz.

Appendix 3

ML213 was in parallel resynthesized using two commercially available starting materials, an “endo-prefering” carboxylic was used to make SDT-I-35 and an “endo” carboxylic acid was used to make SDT-I-117. The products were analyzed by chiral supercritical fluid chromatography (SFC) analysis (see Figures A1 and A2). Both products exhibited multiple peaks indicating that both products were composed of similar mixtures of diasteromers.

Figure A1

SFC analysis of STD-I-117.

Figure A2

SFC analysis of SDT-I-35.