2.1. Assays

• Summary AID 2262: Summary of probe development for inhibitors of KCNQ2 potassium channel
• AID 2156: Primary cell-based high-throughput screening assay for identification of compounds that inhibit KCNQ2 potassium channels
• AID 493025: Confirmatory screen for compounds that inhibit KCNQ2 potassium channels
• AID 493029: Counter screen against parental CHO cells for compounds that inhibit KCNQ2 potassium channels
• AID 588531: Confirmatory screen for compounds that inhibit KCNQ2 potassium channels using automated patch clamp
• AID 588637: Dose response assay for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay II
• AID 493026: Specificity screen against KCNQ1 for compounds that inhibit KCNQ2 potassium channels
• AID 588426: SAR analysis for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay, CRC2
• AID 588425: Dose response assay of SAR compounds for the identification of selective inhibitors of KCNQ2 potassium channels in the KCNQ1 expressing cells on automated patch clamp
• AID 504837: SAR analysis for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay
• AID 504839: Dose response assay for compounds that inhibit KCNQ2 potassium channels on automated electrophysiological assay
• AID 588635: SAR-Confirmatory assay to confirm a potent KCNQ2 inhibitor using manual electrophysiology

2.2. Probe Chemical Characterization

Probe compound ML252 (CID: 50985951, SID: 116933760) was prepared according to the scheme in Figure 1 and provided the following characterization data: LCMS (>98%) m/z = 309.3 [M + H]⁺ (0.66 min retention time, 215 and 254 nm). ¹H NMR (400 MHz, DMSO-d₆) δ 10.10 (br s, 1H), 7.52–7.41 (m, 3H), 7.41 – 7.30 (m, 3H), 7.31–7.10 (m, 3H), 3.76 (t, J = 7.5 Hz, 1H), 3.72–3.42 (m, 1H), 3.84 (br s, 4H), 2.20–2.02 (m, 1H), 1.88 (br s, 4H), 1.81–1.66 (m, 1H), 0.89 (t, J = 7.3 Hz, 3H). Chiral HPLC: 94.2% (S) RT = 3.97 min: 5.75% (R) RT = 4.29 min; 88.5% ee.

Figure 1

Synthesis of ML252.

Solubility: Solubility in PBS was determined to be 14 μM, which is more than 200-fold higher than the EC₅₀ for KCNQ2 channel inhibition.

Stability: Stability was determined for ML252 at 23°C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%; see appendix 4). After 48 hours, 66% of the initial concentration of ML252 remained (Table 1; Figure 2).

Table 1

Stability assessment in PBS (Absorption Systems).

Figure 2

Stability assessment in PBS (Absorption Systems).

Compounds added to the SMR collection (MLS#s): MLS003871689 (ML252, CID 50985951, 20.0 mg); MLS003871690 (CID 53313402, 5.3 mg); MLS003871691 (CID 53313363, 5.0 mg); MLS003871692 (CID 53313387, 5.4 mg); MLS003871693 (CID 53313379, 5.0 mg); MLS003871694 (CID 53313381, 6.1 mg)

2.3. Probe Preparation

(S)-2-Phenyl-N-(2-(pyrrolidin-1-yl)phenyl)butanamide (ML252, CID: 50985951). To a stirred solution of (S)-(−)-2-phenylbutyric acid (50 mg, 0.30 mmol), HATU (116 mg, 0.304 mmol) in DMF (1.5 mL) was added ethyldiisopropylamine (79 μL, 0.46 mmol) followed by 2-(1-pyrrolidinyl)aniline (49 mg, 0.30 mmol) and the reaction mixture was stirred at room temperature overnight. The reaction was diluted with water (1.5 mL) and extracted with EtOAc (2 × 2 mL). The organic extracts were combined, concentrated and the residue was purified by RP prepHPLC eluting with 10 to 90% CH₃CN/H₂O (0.1% TFA) to give the product as the TFA salt (60 mg, 57%).

3.1. Dose Response Curves for Probe

ML252 displayed potent block of KCNQ2 channels in automated electrophysiology experiments using IonWorks instruments. The protocol used to evaluate block of KCNQ2 channels is shown in Fig. 3A. Currents from a representative well (Fig. 3A) were significantly inhibited by 0.3 μM ML252. In dose-response experiments (Fig. 3B), such as the one shown in Fig. 3B, ML252 displayed an average IC₅₀ value of 69 ± 6 nM (mean ± SD; n=6) and produced nearly complete block at concentrations above 1 μM.

Figure 3

A) KCNQ2 currents from a single well in the absence and presence of 0.3 μM ML252 are shown along with the voltage protocol used to elicit currents. B) ML252 inhibited KCNQ2 currents in a dose-dependent manner with IC₅₀ value of 59 nM in this experiment (more...)

3.2. Cellular Activity

The primary HTS assay and all secondary assays are cell-based assays, indicating that ML252 can gain access to its molecular target when applied to cells. The compound did not exhibit acute toxicity in cell-based assays at concentrations up to 30 μM.

3.3. Profiling Assays

ML252 (CID 50985951) has been tested in 301 assays performed within the MLPCN network and was active in only four assays that were not dependent on KCNQ2. ML252 showed >300-fold selectivity versus these assays (Table 2).

Table 2

Selectivity profile from PubChem.

To more fully characterize this novel KCNQ2 inhibitor, ML252 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 bind with only 1 of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM) (Table 3). Included in the Ricerca screening panel are a number of ion channels (Calcium Channel, L-Type and N-Type; Potassium channel [K_ATP]; Potassium channel [hERG]) showing the selectivity profile for ML252.

Table 3

Ricerca Profiling of ML252 (CID 50985951).

ML252 selectivity for blocking KCNQ channel family members was examined using automated electrophysiology as described in AID588426 (Table 5). ML252 displayed approximately 40-fold selectivity for block of KCNQ2 channels compared with KCNQ1 channels (IC₅₀ = 2.92 μM). The heteromultimeric combination of KCNQ1 with KCNE1 forms the I_ks current [3], which is a key repolarization component in cardiac myocytes. ML252 afforded greater than 100-fold selectivity for block of KCNQ2 compared with KCNQ1/E1 (IC₅₀ = 8.12 μM). ML252 displayed little selectivity for block of KCNQ2/Q3 heteromultimeric channels (IC₅₀ = 0.12 μM) and modest selectivity for block of KCNQ4 channels (IC₅₀ = 0.20 μM).

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

The prior art consists of three known compounds (XE991, Linopridine, and UCL2077). Each of these compounds show mid-nanomolar to micromolar activity against KCNQ2 based on published values (Table 4, right column) [4][5]. The newly reported probe (ML252) is >10-fold more potent at KCNQ2 than known compounds based on literature values, which were generated using different experimental approaches. In order to directly compare ML252 with previously described KCNQ2 blockers using a common assay system, we evaluated these compounds using IonWorks automated electrophysiology (AID588426) for block of KCNQ2 and other KCNQ family members (Table 4). ML252 affords equivalent or higher potency along with increased selectivity against blocking KCNQ1 channels. ML252 displayed approximately 40-fold selectivity for block of KCNQ2 channels compared with KCNQ1 channels. In contrast, XE991 and linopiridine were also potent blockers of KCNQ2 channels, but displayed reduced selectivity versus KCNQ1 channels. The heteromultimeric combination of KCNQ1 with KCNE1 forms the I_ks current [3], which is a key repolarization component in cardiac myocytes. ML252 afforded greater than 100-fold selectivity for block of KCNQ2 compared with KCNQ1/E1, while XE991 and linopiridine displayed 26-fold and 3-fold selectivity, respectively. Linopiridine and ML252 blocked KCNQ2 channels at submicromolar concentrations, with less than two-fold selectivity for blocking KCNQ2/Q3 heteromultimeric channels, while XE991 also blocked KCNQ2 channels, but displayed five-fold selectivity against KCNQ2/Q3 channels.

Table 4

Selectivity table showing IC₅₀ (μM) values for ML252 and previously identified KCNQ2 channel blockers for inhibition of a panel of KCNQ channels measured using IonWorks electrophysiology.

Unlike previously described KCNQ2 inhibitors, ML252 displays striking structure-activity properties, in which small structural changes in ML252 can lead to a functional switch between antagonist and agonist behavior. These properties may allow ML252 to serve as a useful tool for molecular studies of KCNQ2 channel gating.