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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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A potent and selective small molecule Kir2.1 inhibitor

, , , , , , , , , and .

Received: ; Last Update: October 20, 2010.

Inwardly rectifying potassium channels (Kir) have key regulatory roles controlling electrical activity of diverse cell types. For example, Kir2.1 channels are important in cardiac muscle, where genetic and modeling studies suggest that they control resting potential and contribute to the terminal phase of action potential repolarization. Mutations in the Kir2.1 gene lead to cardiac, developmental and other pathologies. One factor that has hindered development of more complete knowledge of the Kir function has been the lack of specific small molecule tools to dissect Kir2 channel function in intact tissues. Thus, the overall goal of the project is to discover an inhibitor of Kir2.1 channels with an IC50 < 1 µM and selectivity versus other related ion channels to provide the first Kir2.1 small molecule, in vitro probe. The current small molecule probe, ML133 (CID-781301) can be used for in vitro and electrophysiological studies of Kir2.x functions with sub-micromolar potency at pH 8.5, and without significant inhibition of the closely related inwardly rectifying potassium channel ROMK. ML133 also displays modest selectivity versus hERG and possesses fair ancillary pharmacology against a larger panel of GPCRs, ion channels and transporters. In vivo PK has not yet been performed.

Assigned Assay Grant #: 1 R03 DA026212-01

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: Elena Makhina, University of Pittsburgh

PubChem Summary Bioassay Identifier (AID): AID-1843, AID-1672, AID-2032, AID-2105, AID-2236, AID-2329, AID-2345, AID-2404, AID-2581, AID-2591, AID-2594, AID-463252

Probe Structure & Characteristics

Image ml133fu1
CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 44247466/ML133Kir2.1Manual patch clamp, 290 [SID-85281105, AID-2591]ROMKManual patch clamp, >300 [SID-85281105, AID-2404]>100Manual patch clamp Kir2.1 (pH 7.4), 1800, [SID-85281105, AID 2404] Automated Patch Clamp Kir2.1, 13600 [SID-85281105, AID-2581]

Recommendations for scientific use of the probe

Currently, few confirmed, specific, small molecule blockers of classical Kir channels have been reported in scientific literature or in issued patents or patent applications available at the US Patent Office. Tamoxifen[1] and chloroquine[2, 3] block Kir2.1 channels at low micromolar concentration, but display prominent effects on other targets. Celastrol[4], 22G and 48F10[5] block Kir2.1 channels in the 10–100 μM range and limited selectivity data is available for these compounds. Gambogic[6] acid and pentamidine[7] potently block Kir2.1 channels following chronic exposure, but are less efficacious acutely. Thus, there are clear unmet needs for more specific and potent inhibitor and/or activator probes for Kir2.x channels.

The current small molecule probe (ML133, CID 44247466, Parental CID 781301) can be used for in vitro and electrophysiology studies of Kir2.x functions with sub-micromolar potency at pH 8.5 and without significant inhibition of the closely related inwardly rectifying potassium channel ROMK. ML133 also displays modest selectivity versus hERG and possesses fair ancillary pharmacology against a larger panel of GPCRs, ion channels and transporters. In vivo PK has not yet been performed. As the HCl salt, ML133 is quite soluble (>10 mg/mL in saline) and >100 μM in DMSO.

The probe will be useful to researchers investigating the role of Kir2 family channels, particularly in tissues in which it is expressed along with other inward rectifier channels from the Kir1 and Kir4 families.

1. Introduction

The overall goal of the project is to discover an inhibitor of Kir2.1 channels with an IC50 < 1 μM and selectivity versus other related ion channels to provide the first Kir2.1 small molecule, in vitro probe.

Potassium (K+) channels are well recognized as targets for treatment of cardiovascular, neurological, renal and metabolic disorders[8, 9]. Recent advances in ion channel screening technologies have enabled efficient, target-based screening efforts to identify additional, specific ion channel modulators. Inwardly rectifying potassium channels (Kir) have key regulatory roles controlling electrical activity of diverse cell types. Kir2.1 channels are important in cardiac muscle where genetic and modeling studies suggest that they control resting potential and contribute to the terminal phase of action potential repolarization[10]. Mutations in the Kir2.1 gene lead to cardiac, developmental and other pathologies[11]. A factor that has hindered development of more complete knowledge of the Kir function has been a lack of specific small molecule tools to dissect Kir2 channel function in intact tissues.

To enable high throughput screening for Kir2.1 channels, an assay was developed based on K+ uptake in yeast via ectopically expressed mammalian Kir2.1 channels. However, the yeast assay could not be scaled to 384 well format, which is critical for implementing large screens with >300,000 compounds. With the consent of the Assay Provider and NIH Scientific Officer, JHICC developed and optimized a Tl+ flux assay to screen for human Kir2.1inhibitors, with confirmation assays performed using patch clamp of whole-cell currents of Kir2.1.

2. Materials and Methods

2.1. Assays

The purpose of this assay is to identify test compounds that inhibit/block the inward rectifying potassium ion channel, Kir2.1. This assay employs a HEK293 cell line that stably expresses Kir2.1 channels. The cells are treated with test compounds, followed by measurement of intracellular thallium uptake, as monitored by a thallium-sensitive, fluorescent dye, FluxOR. The thallium assay was based on the FluxOR detection kit sold by Molecular Devices Corporation. HEK293 cells stably expressing Kir2.1 channels were plated into 384-well plates. On the following day, cells were loaded with a thallium-sensitive dye, FluxOR, and then incubated with assay buffer. Compounds were added to the assay buffer. Cells were incubated with 10 μM compound for 20 minutes, and Tl+ influx was triggered by the addition of stimulus solution (5 mM K2SO4 and 1.4 mM Tl2SO4). The fluorescence of FluxOR was measured on a Hamamatsu FDSS 6000 kinetic imaging plate reader. Compound effect was evaluated by the calculated FluxOR fluorescence ratio, normalized with negative controls. If the compound caused less than three times the standard deviation of the B-scores of the library compounds, the compound was considered to be active as an inhibitor/blocker of the Kir2.1 channels.

Protocol for the Kir2.1 primary screening assay

  1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/mL penicillin, 50ug/ml streptomycin, and 500ug/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 1x FluxOR solution to cells
  5. Incubate 90 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 (IC0), and IC100 of chlorpromaizne (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 7.5x 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 5x 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 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 prime factors[12].
  16. Calculate B scores for test compounds using ratios calculated in Step 14[13].
  17. Outcome assignment: If the B score of the test compound is less than minus three 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 two times the standard deviation of the B scores of the library compounds, the compound is designated in the Outcome as active as an inhibitor/blocker of the Kir2.1 channels. Otherwise, it is designated as inactive.
  18. Score assignment: An active test compound is assigned a score between 0 and 100 by calculation of 100*(0- Integer ([B Score Inhibitor Ratio]))/20, B Score Inhibitor Ratio, as in the result definition. Among the active compounds in the assay, the activity score range is 95-20. All inactive test compounds are assigned to the score 0.

List of reagents

  1. Kir2.1 HEK293 cell lines (provided by JHICC)
  2. PBS: pH7.4 (Gibco, Cat #10010)
  3. Medium: DMEM/F12 50/50 (Mediatech, Ca t#15-090-CV)
  4. Fetal Bovine Serum (Gemini, Cat #100-106)
  5. 200 mM L-Glutamine (Gibco, Cat #25030)
  6. 100x Penicillin-Streptomycin (Mediatech, Cat #30-001-CI)
  7. 0.05% Trypsin-EDTA (Gibco, Cat #25300)
  8. G418 (Geneticin): (Gibco, Cat #11811-031)
  9. HEPES (Sigma, Cat #H4034)
  10. Chlorpromazine hydrochloride (Sigma, C8138)
  11. FluxOR detection kit (Invitrogen, Cat #F10017): FluxOR, assay buffer and stimulus buffer.
  12. Triple-layer flask (VWR, Cat #62407-082)
  13. BD Biocoat 384-well plates (BD, Cat# (35)6663 and Lot #8163495)
  14. 10x HBSS (Gibco, Cat #14065)

Possible artifacts of this assay can include, but are not limited to: non-intended chemicals, or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.

Protocol for secondary electrophysiological assay

In manual electrophysiology experiments, whole-cell currents of Kir2.1 were recorded from HEK293 cells stable expressing Kir2.1 channels using an Axopatch 200B amplifier. The electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). Pipette resistance was maintained around 3–4 mΩ. The bath solution contained 140 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM Hepes (pH 8.5, 7.4 or 6.5 with KOH). The bath solution contained 140 mM KCl, 2 mM EDTA, 10 mM Hepes (pH 7.4). To record Kir2.1 currents, voltage steps were applied from 0 mV holding potential to −100mV (500 ms) with an interval of 30 seconds. Capacitance and access resistance were monitored and 75% series resistance compensation was applied. Currents were filtered at 1 kHz, and data were acquired at 5 kHz with a Digidata 1322A computer interface and pClamp 9.2 software (Axon Instruments). Data were analyzed using pClamp 8 and Origin 6. To apply the compound, 7–10 mL of compound solution was injected by a 10 mL syringe into recording chamber containing 0.5 mL bath solution. Excessive solution was be removed by suction. To check the quality of seal during recording, each voltage step was followed by a ramp protocol (500ms, from −100mV to 100 mV). Cells losing seal during recording were identified by a sudden increase of outward current in ramp protocol, and were not used. ROMK currents were recorded using identical protocols using HEK293 cells transiently transfected with an expression vector for the ROMK gene.

In automated electrophysiology experiments used to confirm Kir2.1 block, whole cell currents were recorded from HEK293 cells stably expressing Kir2.1 channels using an IonWorks instrument in population patch clamp (PPC) mode as described in AID-2581. A counterscreen against hERG potassium channels was performed using HEK293 cells stably expressing hERG channels, as described in AID-2236. A counterscreen against KCNK9 potassium channels was performed using a Tl+ influx assay, in which the fluorescent signal was measured using a Hamamatsu FDSS 6000 plate reader (AID-2329).

2.2. Probe Chemical Characterization

Kir2.1 Absorption Systems

Table 1Stability at room temperature (23ºC) in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%)

Percent Remaining (%)
Compound (CID/SID/ML#)0 Min15 Min30 Min1 Hour2 Hour24 Hour48 Hour
CID 44247466/ SID-85281105/ ML13310090981009573105

Table 2PBS solubility at pH 7.4

Compound (CID/SID/ML#)Solubility (μM)
CID 44247466/ SID-85281105/ ML133964

ML133: CID 44247466, Parental CID 781301; SID-85281105 (assay data) and SID-87457855 (deposited Probe)

MLS#s: 002699048 (Probe, 500 mg); analogues: 002699049, 002699050, 002699051, 002699052, 002699053

2.3. Probe Preparation

Image ml133fu2

ML133. N-(4-methoxybenzyl)-1-(naphthalen-1-yl)methanamine: To a solution of (4-methoxyphenyl)methanamine (1.15 mL, 8.83 mmol) in DCE (25 mL) was added 1-naphthaldehyde (0.69 mL, 5.08 mmol). After 15 min, NaBH(OAc)3 (2.80 g, 13.23 mmol) was added portion wise over 5 min. After 12 h, the water (25 mL) was added to the rxn and after another additional 1h, transferred to DCM:water (1:1; 250 mL). The organic layer was separated, washed with water (2 x 50 mL), and passed through a Phase separator. After concentration, the desired product was purified by preparative HPLC to afford an off-white solid (0.70 g, 50%): Analytical LCMS: single peak (214 nm), 1.123 min; 1H NMR (400 MHz, d6-DMSO): δ 9.25 (br s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.68-7.48 (m, 6H), 7.01 (d, J = 8.4 Hz, 2H), 4.59 (s, 2H), 4.26 (s, 2H), 3.77 (s, 3H); HRMS, calculated for C19H20NO (M+ H+ ), 278.1545; found 278.1544.

HCl Salt Formation: To a solution of the amine in DCM (0.2M) at 0°C was added 4M HCl in 1,4-dioxane (5 eq.) dropwise. After 15 min, the ice bath was removed. The solvent was removed after additional 30 min at RT to provide a pure HCl salt of the appropriate amine.

3. Results

Summary of Screen: This screen was performed using a Tl+ flux assay (Z’ = 0.74) with 305,616 compounds in the MLSMR library, and 2,592 actives were identified. Confirmation screening was performed on 2,265 compounds in duplicate affording 927 confirmed actives. Counter-screening against the parental HEK293 cells eliminated 426 compounds from consideration. Further counter-screening against hERG and KCNQ9 led to selection of CID 17367817 for re-synthesis and SAR evaluation at Vanderbilt Specialized Chemistry Center. CID 44247466 (ML133) met probe criteria, blocking Kir2.1 with an IC50 = 290 nM at pH 8.5 (AID-2404) and good selectivity against a related inward rectifier channel, ROMK (Kir1.1; IC50 >30 μM, AID-2404) at similar pH values.

3.1. Summary of Screening Results

Figure 1 illustrates a flowchart of the process used to identify ML133 as a Kir2.1 inhibitor. The summary AID-1843 lists the assays used in this project. The MLSMR library of 305,616 compounds was screened at a single test compound concentration of 10 μM using a Tl+ influx assay to identify inhibitors of Kir2.1 channels. Tl+ influx was used as a surrogate marker for potassium flux through Kir2.1 channels using a Tl+-sensitive fluorescent dye (FluxOR) measured on a Hamamatsu FDSS 6000 kinetic imaging plate reader (AID-1672). The primary screen afforded an adequate screening window (Z’ = 0.74) and 2,592 actives were identified based on plate-normalized B-score ratios. Confirmation screening was performed on 2,265 compounds in duplicate yielding 927 actives (AID-2032). Counter-screening against the parental HEK293 cells using a similar Tl+-based assay (AID-2105) eliminated 426 false actives in the Tl+ assay. A set of 320 compounds was evaluated using IonWorks automated electrophysiology at a single concentration (AID-463252) and further counter-screened against hERG channels (AID-2236) and KCNK9 channels (AID-2329). A set of 25 compounds was then further evaluated for block of Kir2.1 in dose-response experiments (AID-2594) using the using the Tl+ influx assay that was used in the primary HTS campaign. One lead class exemplified by CID 17367817 (parental CID 781301; SID-49645137) was selected for re-synthesis and SAR investigation at VU Specialized Chemistry Center.

Figure 1. Flowchart of generation of Kir2.x probe candidates.

Figure 1

Flowchart of generation of Kir2.x probe candidates.

3.2. Dose Response Curves for Probe

Block of Kir2.1 channels by ML133 was evaluated in experiments using manual patch clamp recording methods. Whole cell recordings with high membrane resistance values were used to allow separation of inward rectifier currents from background leak currents. Figure 2B shows a recording from a cell stably expressing Kir2.1 channels. The voltage protocol includes a ramp increase in membrane potential from −100 mV to +100 mV. A large inward current was observed at negative potentials, as expected for an inward rectifier channel and little outward current was observed at positive potentials, indicating a lack of background leak currents. ML133 blocked Kir2.1 channels in manual patch clamp experiments in a pH-dependent manner. At neutral pH (7.4), the IC50 for Kir2.1 block was 1.8 μM, which increased to 9.1 μM at pH 6.5. Submicromolar block (IC50=0.29 μM) was observed at pH 8.5. The Hill slopes for block at each pH level were greater than one suggesting a possible mechanism for channel block involving more than one inhibitor molecule.

Figure 2. Inhibitory effects of ML133 on Kir2.1 channels at different pH levels.

Figure 2

Inhibitory effects of ML133 on Kir2.1 channels at different pH levels. A) Representative currents from a HEK293 cell expressing mKir2.1 channels are shown before and after exposure to 3 μM ML133. The voltage clamp protocol is shown below the current (more...)

3.3. Scaffold/Moiety Chemical Liabilities

None

3.4. SAR Tables

Probe Chemical Lead Optimization Strategy: Our probe lead optimization strategy focused initially on a re-synthesis of screening hit CID 781301. As we have done in the past, we re-synthesized the original hit in a library format with 10 new analogs (Scheme 1) wherein the p-OMe benzyl moiety was held constant and the naphthyl group was varied. For the library, p-OMe benzyl amine 1 was treated with 10 different benzaldehydes 2 to produce analogs 3. Clear SAR was established (Table 3) using automated electrophysiology and manual patch clamp measurements of Kir2.1 currents at pH 8.5, and the re-synthesized HTS hit (3a), now assigned CID 44247466 (SID-85281105 / SID-87457855) confirmed with a Kir2.1 IC50 of 290 nM in manual patch clamp experiments – the most potent Kir2.1 inhibitor reported to date. The IC50 for Kir2.1 inhibition increased to 1.8 μM at pH 7.4. From the analogs in Table 3, the naphthyl moiety appears critical for Kir2.1 potency, and a library holding that moiety has been submitted for screening. However, five of the ten new analogs 3bf possessed Kir2.1 IC50s < 5 μM. A p-Cl benzyl congener (3b, CID 44483168) displayed submicromolar potency (Kir2.1 IC50 = 770 nM), while the m-Cl benzyl congener (3c, CID 44483172) was weaker (Kir2.1 IC50 = 1.06 μM). A p-tBu benzyl analog (3e, CID 44483167) was comparable to the m-Cl congener (Kir2.1 IC50 = 1.39 μM). Substitution in the 2-position, in the form of either a 2-Me (3f, CID 44483176) or a 2,4-diCl (3d, CID 44483171) analog led to diminished potency, 5.15 μM and 3.89 μM, respectively. Other analogs in Table 1 led to either inactive compounds, or much reduced potency compared to 3a (CID 44247466).

Scheme 1. Synthesis of analogs 3.

Scheme 1

Synthesis of analogs 3.

Table 3. Structure and activity of analogs of 3.

Table 3

Structure and activity of analogs of 3.

To assess the right-hand SAR, naphthalen-1-ylmethylamine, 4, was held constant and a variety of aldehydes were reacted under reductive amination conditions to afford the desired compounds, 5. Unfortunately, this SAR did not prove fruitful since none of the 17 compounds were as potent as 3a.

Scheme 2. Synthesis of analogs 5.

Scheme 2Synthesis of analogs 5

Table 4Structure and activity of analogs of 5

Image ml133fu24.jpg
CmpdCIDSIDVU#*RKir2.1 IC50 (nM) – Manual Patch ClampKir2.1 IC50 (nM) – Ion Works
3a4424746685281105VU0404943-1S
Image ml133fu25.jpg
29013630
5a4454349087225317VU0409040-1S
Image ml133fu26.jpg
23620
5b4454349287225318VU0409041-1S
Image ml133fu27.jpg
18080
5c82977987225311VU0409034-1S
Image ml133fu28.jpg
22970
5d87211387225314VU0409037-1S
Image ml133fu29.jpg
38490
5e87132687225310VU0409033-1S
Image ml133fu30.jpg
37880
5f77545287225309VU0409032-1S
Image ml133fu31.jpg
28160
5g78130387225312VU0409035-1S
Image ml133fu32.jpg
37280
5h4454350187225325VU0409048-1S
Image ml133fu33.jpg
no fit
5i4454348887225316VU0409039-1S
Image ml133fu34.jpg
46270
5j4454349587225321VU0409044-1S
Image ml133fu35.jpg
no fit
5k4454349887225323VU0409046-1S
Image ml133fu36.jpg
no fit
5l4454350087225324VU0409047-1S
Image ml133fu37.jpg
101940
5m4454349687225322VU0409045-1S
Image ml133fu38.jpg
199300
5n4454350587225327VU0409050-1S
Image ml133fu39.jpg
no fit
5o4454350387225326VU0409049-1S
Image ml133fu40.jpg
161890
5p4454349387225319VU0409042-1S
Image ml133fu41.jpg
40330
5q4454349487225320VU0409043-1S
Image ml133fu42.jpg
37160
5r4454348787225315VU0409038-1S
Image ml133fu43.jpg
no fit

Lastly, the middle-core of the compound was evaluated by replacement of the NH with an oxygen 6, reducing the basicity of the nitrogen (7 and 8), introduction of steric bulk around the nitrogen (9 and 10), and alkylation of the nitrogen (1116). All of these modifications led to inactive compounds. In total, 43 compounds were synthesized/purchased for SAR determination and compound 3a (CID 44247466/ML133) proved to be the most potent compound.

Table 5Structure and activity of analogs of 3

Image ml133fu44.jpg
CmpdCIDSIDVU#*Kir2.1 – Manual Patch ClampKir2.1 IC50 (nM) – Ion Works
64548954793617951VU0417906-1S
Image ml133fu45.jpg
5.8% @ 30 μMno fit
72806198193617952VU0417907-1P
Image ml133fu46.jpg
8.5% @ 30 μMno fit
885476693617955VU0033754-1S
Image ml133fu47.jpg
0.26% @ 30 μMno fit
94548954893617953VU0417908-1S
Image ml133fu48.jpg
16.1% @ 30 μMno fit
104548954993617954VU0417909-1S
Image ml133fu49.jpg
4.2% @ 30 μMno fit
1182898287225313VU0409036-1S
Image ml133fu50.jpg
23530
12284524287225305VU0409028-1S
Image ml133fu51.jpg
38230
13284522987225304VU0018224-2S
Image ml133fu52.jpg
62770
14137570987225307VU0409030-1S
Image ml133fu53.jpg
inactive
15284526687225306VU0409029-1S
Image ml133fu54.jpg
inactive
16137562987225303VU0409027-1S
Image ml133fu55.jpg
inactive

3.5. Cellular Activity

In silico properties of ML133 were also calculated using TRIPOS software and compared to the MDDR database of compounds entering both Phase I and launched drugs (Table 6), and found to have an overall favorable profile.

Table 6. Calculated Property Comparison with MDDR Compounds.

Table 6

Calculated Property Comparison with MDDR Compounds.

The screening data described in this report utilize cell-based assays suggesting that ML133 is cell permeable and exhibits not acute toxicity over the experimental times (up to 30 minutes) and doses used (up to 30 μM).

3.6. Profiling Assays

The effects of ML133 on other ion channels were evaluated to establish an ion channel selectivity profile for this compound. At JHICC, the team evaluated ML133 against a panel of inward rectifier potassium channels at pH 7.4 using manual whole cell patch clamp methods (Table 7). ML133 showed little or no selectivity among Kir2.x family channels with the IC50 values falling within a two-fold range. In contrast, ML133 afforded only weak block of Kir1.1, Kir4.1 and Kir7.1 channels providing 17-100-fold selectivity for block of these channels compared with block of Kir2.1. Modest selectivity (4-fold) was observed for block of Kir6.2 channels.

Table 7. Selectivity profile of CID 44247466 for inward rectifier potassium channels.

Table 7

Selectivity profile of CID 44247466 for inward rectifier potassium channels.

The effects of ML133 on hERG potassium channels were determined in whole cell voltage clamp experiments using an IonWorks automated electrophysiology instrument. ML133 blocked hERG currents with an IC50 value of 4.3 μM, indicating modest selectivity against hERG channels compared with block of Kir2.1 channels at neutral pH.

ML133 was evaluated in the Lead Profile Screen at MDS Pharma at a concentration of 10 μM against 68 GPCRs, ion channels and transporters in a radioligand binding assay panel. Despite the low molecular weight of ML133, it had rather clean ancillary pharmacology (only 6 targets with >50% inhibition at 10 μM). Interestingly, ML133 had no effect on hERG binding (34% at 10 μM), although it blocked hERG channels in electrophysiological experiments. ML133 also had no effect on L- and N-type calcium channels, sodium channels and the KATP potassium channel in this ligand binding profile. ML133 had significant effects on the adrenergic family of receptors (α1A, α1B, α1D, α2A and β1 – all >80%@10 μM). The only other significant ancillary pharmacology was against the H2 receptor (81%@10 μM). It is not known whether these activities in binding assays are translated into functional modification of the activities of these receptors.

4. Discussion

ML133 was identified following an HTS screen of the MLSMR library at the Johns Hopkins Ion Channel Center and SAR chemistry efforts at the Vanderbilt Specialized Chemistry Center. ML133 blocks Kir2.1 inward rectifier potassium channels in cell-based electrophysiology experiments with an IC50 value of 1.8 μM at pH 7.4 and more potently, with an IC50 value of 0.29 μM at pH 8.5. This compound exhibits greater than 100-fold selectivity for block of Kir2.1 channels compared with block of Kir1.1 inward rectifier potassium channels, and 17–40-fold selectivity against Kir7.1 and Kir4.1 channels. Lower levels of selectivity were observed for block of other Kir2 family channels, Kir6.2 and hERG channels. Mapping studies suggest that the compound interacts with the M1 and M2 transmembrane domains and the compound may serve as a probe for structure-function studies of these regions. The compound exhibits adequate water solubility and cell penetration for use in examining the roles of Kir2 channels in cell and tissue preparations.

4.1. Comparison to existing art and how the new probe is an improvement

Few potent or specific probes of Kir2 family channels exist in the public domain. Tamoxifen[1] and chloroquine[2, 3] block Kir2.1 channels at micromolar concentrations but additionally affect other targets at similar or lower concentrations, and thus provide limited opportunities for exploring the roles of Kir2 channel function at the cellular, tissue or in vivo levels. Other compounds either block at higher concentrations (celastrol[4] and 22G and 48F10[5]) or block selectively after chronic exposure [6, 7]. Mutations in the gene encoding Kir2.1 lead to Andersen syndrome, which is characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features[11]. The function of Kir2 inward rectifier channels has been examined in cardiac muscle, where Kir2.1 apparently encodes the IK1 current and thereby regulates resting membrane potential and action potential properties[10]. Kir2.x channels are widely expressed in the cerebellum, hippocampus, forebrain, microvilli of Swan cells and other areas in the nervous systems as evidenced by in situ hybridization, histochemistry and immunohistochemistry, but the precise roles of these channels in the nervous system are not determined due to lack of specific probes. Development of more potent and specific probes of Kir2 family channels, and specifically Kir2.1 channels, would lead to an improved understanding of the roles of these channels in cardiac and neuronal function.

4.2. Mechanism of Action Studies

ML133 exhibited very weak block of ROMK (Kir1.1) inward rectifier potassium channels (Figure 3). No apparent block of ROMK currents by ML133 was observed at up to 300 μM at pH 7.4 and only very weak block (IC50>30 μM) was seen at pH 8.5 (Figure 3A AND 3B). A set of chimeric channels were constructed from ROMK and Kir2.1 in order to identify channel domains that may control sensitivity to ML133. Whole cell manual voltage clamp recordings were made at pH 7.4 from cells transiently transfected with the various chimeric channel constructs and the compared with data obtained from the parental channels. Chimeras used to study the critical region for high affinity inhibition in the Kir2.1 family are shown in Figure 3C. Both Kir1.1 (blue) and Kir2.1 (orange) consist of four identical subunits surrounding a central pore. Based on the crystal structure of Kir2.2, we divided Kir1.1 and Kir2.1 subunits into seven homologous regions: the N-terminus, Kir2.1 Met 1-Leu85; the outer transmembrane domain (M1), Kir2.1 Val86-Leu109; the MH region which lies between M1 and H5, Kir2.1 His110-Thr130; the pore region (H5), Kir2.1 Ala131-Arg148; the HM region which lies between H5 and M2, Kir2.1 Cys149-Ala157; the inner transmembrane segment (M2), Kir2.1 Val158-Ala181; and finally the cytoplasmic C-terminus, Kir2.1 Lys158-Ile428. From Chm 1 to Chm4, Kir2.1 region(s) were gradually displaced by corresponding Kir1.1 region as indicated. The effects of 30 μM ML133 at pH7.4 on the corresponding constructs are shown in the right panel of Figure 3C. Chm1 and Chm2 remain sensitive to 30 μM ML133 with inhibition above 80%, while Chm 3 and Chm 4 are insensitive to 30 μM ML133 with inhibition below 20%. These results identify the M1 and M2 regions of Kir2.1 as the critical region(s) for high affinity inhibition by ML133.

Figure 3. Mapping studies of critical regions for ML133 block of Kir2.1 channels.

Figure 3

Mapping studies of critical regions for ML133 block of Kir2.1 channels. See text above for description

4.3. Planned Future Studies

ML133 can serve as a starting point for future studies in three different areas: 1) SAR optimization studies are planned to improve the potency and selectivity of ML133 and also reducing the pH-dependence of the compound for channel inhibition; 2) Mapping studies employing mutagenesis, expression and electrophysiological analysis are aimed at identifying the specific residue responsible for differential block of Kir2.1 channels compared with Kir1.1 and other inward rectifier channels. These studies may help improve understanding of the architecture of the pore region of these channels in a physiological context; 3) ML133 will be used in pharmacological experiments to examine the roles of Kir2 channels in cardiac and neuronal function. We have developed methods for maintaining and recording from embryonic stem cells and iPS cells differentiated into cardiomyocytes. ML133 will be used to modulate Kir2 channels in these cells and determine effects of cell electrical properties, although effects on hERG channels at slightly higher concentrations will limit this analysis to instances in which hERG function is not predominant. ML133 will also be used to investigate the roles of Kir2 channels in the electrical properties of cultured hippocampal neurons. Electrophysiological and fluorescent methods will be used in conjunction with peptide toxins and shRNA to examine the proposed role of Kir2 channels in regulating neuronal excitability.

5. References

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Sun H, et al. Chronic inhibition of cardiac Kir2.1 and HERG potassium channels by celastrol with dual effects on both ion conductivity and protein trafficking. J Biol Chem. 2006;281(9):5877–84. [PubMed: 16407206]
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Zaks-Makhina E, et al. Novel neuroprotective K+ channel inhibitor identified by high-throughput screening in yeast. Mol Pharmacol. 2004;65(1):214–9. [PubMed: 14722253]
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Zaks-Makhina E, et al. Specific and slow inhibition of the kir2.1 K+ channel by gambogic acid. J Biol Chem. 2009;284(23):15432–8. [PMC free article: PMC2708840] [PubMed: 19366693]
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de Boer TP, et al. The anti-protozoal drug pentamidine blocks KIR2.x-mediated inward rectifier current by entering the cytoplasmic pore region of the channel. Br J Pharmacol. 159(7):1532–41. [PMC free article: PMC2850409] [PubMed: 20180941]
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Wulff H, Castle NA, Pardo LA. Voltage-gated potassium channels as therapeutic targets. Nat Rev Drug Discov. 2009;8(12):982–1001. [PMC free article: PMC2790170] [PubMed: 19949402]
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Kaczorowski GJ, et al. Ion channels as drug targets: the next GPCRs. J Gen Physiol. 2008;131(5):399–405. [PMC free article: PMC2346569] [PubMed: 18411331]
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Plaster NM, et al. Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome. Cell. 2001;105(4):511–9. [PubMed: 11371347]
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Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen. 1999;4(2):67–73. [PubMed: 10838414]
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Malo N, et al. Statistical practice in high-throughput screening data analysis. Nat Biotechnol. 2006;24(2):167–75. [PubMed: 16465162]

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