KCNQ voltage-gated potassium channels are involved in regulating excitability, action potential duration and transport processes in a variety of cell types. A high throughput fluorescent screen based depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the Molecular Libraries Probe Production Centers Network (MLPCN) library to identify activators of KCNQ1 channels. Identified hits were characterized using IonWorks automated electrophysiology. An initial racemic hit (EC50 = 1.7 μM) served as a starting point for optimization studies of newly synthesized compounds. ML277 was identified as a potent (EC50 = 0.26 μM) activator of KCNQ1 channels with more than 100-fold selectivity for activation of KCNQ1 channels compared with closely related KCNQ2 and KCNQ4 channels and with distantly related hERG potassium channels. ML277 provides a novel and selective chemical probe to investigate the role of KCNQ1 channels.
Assigned Assay Grant #: 1 R03 MH090837-01
Screening Center Name & PI: Johns Hopkins Ion Channel Center, Min Li
Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley
Assay Submitter & Institution: Meng Wu, Johns Hopkins University School of Medicine
PubChem Summary Bioassay Identifier (AID 2699)
Probe Structure & Characteristics
1. Recommendations for Scientific Use of the Probe
Human genetic studies have identified a link between expression of certain KCNQ1 alleles and susceptibility to diabetes , pancreatic insulin secretion , and insulin signaling. ML277 would provide a novel pharmacological tool to investigate the role of KCNQ1 channels in insulin secretion from human pancreatic islets or islets from other species that express a similar set of channels in islets. ML277 offers a complementary tool to use of nonselective KCNQ1 inhibitors. Additional pharmacological characterization of the effects of ML277 on islet ion channels would be a prerequisite to these studies. KCNQ1 is also expressed in some epithelial tissues plays a role in salt, glucose, and vitamin transport . ML277 may provide a new pharmacological probe to investigate the role of these channels in fluid and salt transport. Co-assembly of KCNQ1 and KCNE1 forms the Iks current, which regulates action potential duration. Characterization of the effects of ML277 on KCNQ1/E1 and KCNQ1/KCNEx channels would provide a possible additional tool for investigation of the roles of KCNQ1 channels in a variety of tissues.
2. Materials and Methods
- CHO-KCNQ1, Parental CHO, CHO-KCNQ2 and CHO-KCNQ1/KCNE1 cell lines
- FluxOR dye kit (Invitrogen, F10017)
- BD Biocoat, Poly-D-Lysine coated, black/clear bottom 384-well assay plates
- Hamamatsu FDSS 6000 fluorescent plate reader
- Population Patch Clamp patch plates from Molecular Devices
- Summary AID 2699: Summary of assays for compounds that potentiate/activate KCNQ1 potassium channels
- AID 2648: Primary cell-based high-throughput screening assay for identification of compounds that potentiate/activate KCNQ1 potassium channels
- AID 493006: Counter screen assay of the parental CHO cells for identification of compounds that potentiate KCNQ1 potassium channels
- AID 493007: Validation assay for identification of compounds that potentiate KCNQ1 potassium channels
- AID 493009: Specificity screen assay against KCNQ2 for identification of compounds that potentiate KCNQ1 potassium channels
- AID 493184: Secondary automated electrophysiology assay of compounds that potentiate KCNQ1 potassium channels
- AID 493186: Specificity screen assay against KCNQ1/E1 for identification of compounds that potentiate KCNQ1 potassium channels
- AID 588673: SAR analysis for compounds that activate KCNQ1 potassium channels in the KCNQ1 expressing cells on automated patch clamp
- AID 602121: SAR analysis for compounds that activate KCNQ1 potassium channels in the KCNQ1 expressing cells on automated patch clamp 2
- AID 602120: Manual patch clamp assay to confirm a potent KCNQ1 activator
2.2. Probe Chemical Characterization
Probe compound ML277 (CID 53347901, SID 125081894) was prepared according to the scheme in Figure 1 and provided the following characterization data: 1H NMR: (400 MHz, CDCl3) δ (ppm): 9.98 ( br s, 1H), 7.81 (m, 4H), 7.39 (d, J = 8.1 Hz, 2H), 7.05 (s, 1H), 6.98 (dt, J = 2.6, 8.9 Hz, 2H), 4.75 (d, J = 4.8 Hz, 1H), 4.08 (dd, J = 2.2, 14.7 Hz, 1H), 3.88 (s, 3H), 3.14 (td, J = 2.4, 14 Hz, 1H), 2.48 (s, 3H), 2.33 (d, J = 14 Hz, 1H), 1.63-1.12 (m, 6H); 13C NMR: (400 MHz, CDCl3) δ (ppm): 168.06, 159.55, 156.86, 149.71, 144.18, 136.77, 130.11, 127.33, 127.19, 126.93, 114.05, 105.90, 56.21, 55.28, 43.94, 23.54, 23.07, 21.55, 19.75. LCMS: RT = 0.845 min., m/z = 472.1 [M + H]+ (>99% @ 215 and 254 nm).
Solubility: Powder samples were diluted to 10 mM solution at Analiza. The solubility as determined in PBS buffer is shown in Table 1. Kinetic solubility measurements are performed after 18 hours. The stability data shown in Table 1 suggests that ML277 is soluble for up to 90 minutes. These results suggest that ML277 display adequate solubility for in vitro experiments in which ML277 is tested less than one hour after preparation.
Stability: Stability was determined for ML277 at 23° C in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) and is shown in Table 2. ML277 is stable up to 90 min.; however, since there is a significant time gap between the 90 min. and 24 hour measurement, it is difficult to state the half-life of the molecule. ML277 shows significant instability at the 24 and 48 hour time points, which may result from chemical instability or low solubility.
Compounds added to the SMR collection (MLS#s): MLS003875054 (ML277, CID 53347902, 20.1 mg); MLS003875049 (CID 53488270, 3.6 mg); MLS003875050 (CID 25541644, 5.1 mg); MLS003875051 (CID 2313552, 4.8 mg); MLS003875052 (CID 26915367, 4.8 mg); MLS003875053 (CID 25993147, 5.1 mg)
2.3. Probe Preparation
(R)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide (ML277, CID 53347902). To a solution of 4-(4-methoxyphenyl)thiazol-2-amine, 2, in DMF (0.65 mmol, 0.4M) was added in order O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU; 0.72 mmol), N,N-Diisopropylethylamine (DIEA; 2 mmol), and (R)-1-(tert-butoxycarbonyl)piperidine-2-carboxylic acid, 1, (0.65 mmol). The mixture was stirred overnight at room temperature. The reaction mixture was dissolved in 5× volume H2O and extracted with dichloromethane (DCM), then concentrated in vacuo. The product was purified by column chromatography (ethyl acetate and hexanes, ramp to 50% ethyl acetate).
The Boc-protected intermediate was subsequently dissolved in minimal DCM. To this was added equivolume TFA and the deprotection proceeded at room temperature for 2 hours, at which time the reaction mixture was concentrated in vacuo.
The final compound was afforded by dissolving the deprotected intermediate (0.57 mmol) in DCM (0.1 M). To this solution was added triethylamine (1.14 mmol) and p-Toluenesulfonyl chloride (TsCl; 0.63 mmol). After stirring overnight at room temperature, the reaction mixture was washed with saturated sodium bicarbonate and brine, and passed through a phase separator. The mixture was concentrated in vacuo and purified by HPLC.
3.1. Dose Response Curves for Probe
ML277 displayed potent activation of KCNQ1 channels in automated electrophysiology experiments using IonWorks instruments. The protocol used to evaluate activation of KCNQ1 channels is shown in Figure 2A. Currents from a representative well (Figure 2A) were significantly increased by 0.3 μM ML277. In dose-response experiments (Figure 2B) ML277 displayed an EC50 value of 260 nM and produced a 266% enhancement of KCNQ1 current amplitudes at +40 mV.
Scaffold/Moiety Chemical Liabilities
No chemical liabilities for the probe molecule have been identified at the present time.
3.2. Cellular Activity
The primary HTS assay and all secondary assays are cell-based assays, indicating that ML277 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
ML277 (CID 53347901) is a newly synthesized compound and has not been tested in assays performed within the MLPCN network.
The selectivity profile of ML277 was examined in 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 6 of the 68 assays conducted (no inhibition of radio ligand binding > 50% at 10 μM) (Table 3). ML277 did have activity against several targets (see Table 1); 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.” This is highlighted by the fact that ML277 was shown to have 80% inhibition @ 10 μM against the hERG channel. However, further profiling studies at Johns Hopkins University showed that in a functional assay ML277 had no activity against hERG up to 30 μM. In addition, ML277 was shown to weakly inhibit the Calcium L-Type channel (~55% at 10 μM); this will be further evaluated in the extended probe phase. These discrepancies are not uncommon and as such, these binding activities only point to receptors to be evaluated.
ML277 selectivity for blocking KCNQ channel family members was examined using automated electrophysiology as described in AID 493009. ML277 caused no significant changes (<25% up to 30 μM) in the amplitudes of KCNQ2 or KCNQ4 currents indicating greater than 100-fold selectivity for activation of KCNQ1 channels compared with KCNQ2 and KCNQ4 channels. In addition, ML277 produced no significant block of hERG potassium channels up to 30 μM indicating more than 100-fold selectivity for KCNQ1 compared with hERG. The recombinant cells expressing other KCNQ and hERG channels were made from the same parental CHO-K1 cells used to express KCNQ1, which indicates that the compound ML277 doesn’t affect endogenous channels or membrane properties of the host CHO-K1 cells.
4.1. Comparison to Existing Art and How the New Probe is an Improvement
Few potent and selective KCNQ1 channels have been described in the scientific or patent literature (Table 5). R-L3 (L-363,373) enhances KCNQ1 currents expressed in Xenopus oocytes [5, 6] at micromolar concentrations, but selectivity data for this compound against other KCNQ isoforms are not available. In IonWorks automated electrophysiology experiments, R-L3 produced modest activation of KCNQ1 channels, displaying a 0.96 μM EC50 value for channel activation and produced a 68% maximal increase in current magnitude. Zinc pyrithione (ZnPy) activates KCNQ1 channels (EC50=3.5 μM), but additionally activates KCNQ2, KCNQ4 and KCNQ5 channels. Phenyl boronic acid (PBA) modulated KCNQ1 channels with a complex phenotype with kinetically distinct inhibitory and stimulatory phases. The EC50 for enhancement of KCNQ1 currents (0.1 mM) occurred at lower concentrations than for other KCNQ channels, but the low potency, complex time-course for modulation, and effects on other voltage-gated potassium channels will limit the use of this molecule in pharmacological experiments. Amato and colleagues  describe a series of N-pyridyl benzamide analogues that activate KCNQ2/3 channels. A few of these compounds also activate KCNQ1 channels at micromolar concentrations, but show increased activity for KCNQ2/3 channels. As can be seen in Table 5, these compounds are either weak KCNQ1 activators, display no selectivity for the KCNQ1 channel, or are chemical structures that cannot be used in a meaningful manner to determine channel function due to other reactive functional groups.
Searches of the patent literature at USPTO.GOV and using Google Patents revealed few applications or issued US patents claiming compounds that activate KCNQ1 channels. US2011/0257146 claims ZnPy as a KCNQ1 activator, but this compound is also described in the scientific literature.
- Yasuda K, et al. Variants in KCNQ1 are associated with susceptibility to type 2 diabetes mellitus. Nat Genet. 2008;40(9):1092–7. [PubMed: 18711367]
- Salata JJ, et al. A novel benzodiazepine that activates cardiac slow delayed rectifier K+ currents. Mol Pharmacol. 1998;54(1):220–30. [PubMed: 9658209]
- Seebohm G, et al. Pharmacological activation of normal and arrhythmia-associated mutant KCNQ1 potassium channels. Circ Res. 2003;93(10):941–7. [PubMed: 14576198]
- Solubility (PBS at pH = 7.4), Stability and Reactivity experiments were conducted at Absorption Systems. For additional information see: https://www
- For information on the Ricerca Lead Profiling Screen see: https://www
Haibo Yu,1 Zhihong Lin,1 Kaiping Xu,1 Xiaofang Huang,1 Shunyou Long,1 Meng Wu,1 Owen B. McManus,1 Julie Le Engers,2 Margrith E. Mattmann,2 Darren W. Engers,2 Uyen M. Le,2 Craig W. Lindsley,2 Corey R. Hopkins,2 and Min Li1.
Received: December 16, 2011; Last Update: March 7, 2013.
National Center for Biotechnology Information (US), Bethesda (MD)
Yu H, Lin Z, Xu K, et al. Identification of a novel, small molecule activator of KCNQ1 channels. 2011 Dec 16 [Updated 2013 Mar 7]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.