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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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Discovery of a small molecule inhibitor of ROMK and Kir7.1

, , , , and 1.

Received: ; Last Update: October 20, 2010.

The specific aim of this project is to identify small molecule inhibitors of Renal Outer Medullary Potassium Channel (ROMK, Kir1.1, KCNJ1), a potassium channel located in the renal tubule, where it critically regulates sodium and potassium balance. However, due to the lack of selective small molecule inhibitors of ROMK, understanding of the molecular pharmacology of ROMK, and indeed that of the entire inward rectifier K+ channel family (kir), is undeveloped, precluding assessment of ROMK's potential as a diuretic target. Furthermore, at least 6 other members (Kir2.3, Kir4.1, Kir4.2, Kir5.1, Kir7.1) of the Kir channel family are expressed in the nephron, but their physiological functions are not well understood. This project aimed to develop ROMK inhibitors with submicromolar potency, cell penetrance and greater than 10-fold selectivity versus other members of the Kir channel family (Kir2.3, Kir4.1, Kir4.2, Kir5.1 and Kir7.1). The currently identified probe, ML111 (CID-4536383) can be used for in vitro and electrophysiology studies to probe the role of ROMK inhibition without inhibition of closely related channels Kir2.1 and Kir4.1. Moreover, ML111 is the first small molecule known to inhibit Kir7.1; thus, the probe also can be used to explore the role of Kir7.1. A second probe from this screen, ML112 (CID-44123657) was a potent ROMK inhibitor with complete selectivity versus Kir2.1, Kir4.1, Kir7.1, Kir2.3 or hERG.

Assigned Assay Grant #: NS057041-01

Screening Center Name & PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver

Chemistry Center Name & PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley

Assay Submitter & Institution: Jerod S. Denton, Vanderbilt University

PubChem Summary Bioassay Identifier (AID): AID-2436

Probe Structure & Characteristics

7,13-bis(4-nitorbenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane

MW = 488.5, ClogP = 3.06

Image ml111fu1
CID/ML#Target NameIC50/E C50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 4536383 ML111ROMK294 [SID-84975334, AID-1917, AID-1918]Kir2.1, Kir4.1,>10 μM [SID-84975334, AID-1916, AID-1924]>30Electrophysiology [SID-84975334, AID-1922]
CID 4536383 ML111ROMKKir7.170% @ 10 μM [SID-84975334, AID-1916, AID-1924

Recommendations for the scientific use of this probe

This probe (CID 4536383) can be used for in vitro and electrophysiology studies to probe the role of ROMK inhibition without inhibition of closely related channels Kir2.1 and Kir4.1. Moreover CID 4536383 is the first small molecule known to inhibit Kir7.1; thus, CID 4536383 can also be used to explore the role of Kir7.1. A second probe from this screen, CID 44123657 was a potent ROMK inhibitor with complete selectivity versus Kir2.1, Kir4.1, Kir7.1, Kir2.3 or hERG.

Specific AIM

To identify small molecule inhibitors of Renal Outer Medullary Potassium Channel (ROMK, Kir1.1, KCNJ1), a potassium channel located in the renal tubule where it critically regulates sodium and potassium balance. There are no selective small molecule inhibitors of ROMK. We aimed to develop ROMK inhibitors with submicromolar potency, cell penetrance and greater than 10-fold selectivity versus other members of the Kir channel family (Kir2.3, Kir4.1, Kir4.2, Kir5.1 and Kir7.1).

Significance

The Renal Outer Medullary potassium (K) channel (ROMK, Kir1.1, KCNJ1) is expressed in the kidney tubule where it plays key roles in regulating fluid and electrolyte homeostasis. (1,2) In the thick ascending limb of Henle, luminal K recycling by ROMK supports NaCl reabsorption by the Na-K-2Cl co-transporter and loop diuretic (e.g. furosemide) target NKCC2, which in turn promotes osmotic water reabsorption in the distal nephron. (3–5) In the connecting tubule, distal convoluted tubule and collecting duct, ROMK mediates the final step in K secretion and functions to match dietary K intake with urinary K excretion. (6,7) A growing body of genetic evidence (8–10) suggests that pharmacological antagonists of ROMK could have potent diuretic effects while minimizing potentially dangerous urinary K loss caused by furosemide. (11,12) However, the molecular pharmacology of ROMK, and indeed that of the entire inward rectifier K+ channel family, is virtually undeveloped, precluding assessment of ROMK’s potential as a diuretic target.

At least 6 other members (Kir2.3, Kir4.1, Kir4.2, Kir5.1, Kir7.1) of the Kir channel family are expressed in the nephron, but their physiological functions are not well understood. (2,13–16) The newest family member, Kir7.1 (KCNJ13), is expressed in several nephron segments. In principal cells of the collecting duct, Kir7.1 is believed to contribute to basolateral K recycling necessary for Na-K-ATPase-dependent K secretion. (15) However, there is no direct evidence that Kir7.1 forms functional ion channels in the nephron. Kir7.1 has an unusually low unitary conductance (~50 fS), (17) making it difficult to identify in single-channel recordings, and there have been no pharmacological tools available with which to discriminate Kir7.1 from other channels in whole-cell recordings. The identification of pharmacological probes would be helpful in defining the functions of Kir7.1 in the nephron and other tissues. (17,25) To date, there are no potent and or selective ROMK inhibitors; however, known KATP blockers have been shown to exhibit low potency toward ROMK together with undesirable cardiovascular and metabolic side effects mediated by KATP channels. (37)

Rationale

In the present study, we developed and implemented a fluorescence-based assay for high-throughput screening (HTS) of chemical libraries for novel modulators of ROMK function, and developed the appropriate counter screens within the Kir family and electrophysiology assays to rapidly confirm and optimize putative ROMK inhibitor hits into selective ROMK inhibitors. (18–25)

Assay Implementation and Screening

PubChem Bioassay Name

Identification of Novel Modulators of ROMK K+ Channel Activity

List of PubChem bioassay identifiers generated for this screening project (AIDs)

AID-1916, AID-1917, AID-1918, AID-1922, AID-1924, AID-2436, AID-2753

PubChem Primary Assay Description

We used C1 cells to develop a fluorescence-based assay of ROMK function for high-throughput screening (HTS). The assay reports flux of the K+ congener thallium (Tl+) through ROMK channels using the fluorescent dye Fluozin-2. (22–24) Figure 1A shows representative fluorescence traces recorded from individual wells of a 384-well plate containing uninduced (−Tet) or Tet-induced cells bathed in control (+Tet) or TPNQ-containing assay buffer (Tet + TPNQ). Tl+ evoked a rapid fluorescence increase in induced cells, but not in uninduced cells or induced cells pre-treated with the ROMK blocker TPNQ. The slope of the Fluozin-2 fluorescence increase between 7 and 12 seconds after Tl+ addition was used as a measure of Tl+ flux. TPNQ dose-dependently decreased Tl+ flux in induced cells with an IC50 of 5.2 nM (Fig. 1C), indicating the assay is sensitive and capable of reporting graded changes in ROMK activity. To assess the suitability of the assay for HTS, every other well of a 384-well plate was pre-treated with control assay buffer or TPNQ-containing buffer before Tl+ addition. The rate of Tl+ flux varied little within treatments, but was dramatically different between control- and TPNQ-treated wells (Fig. 1B). For this plate the calculated Z′ value, a statistical indicator of the assay’s ability to correctly identify hits in a screen, was 0.68. Assays with Z′ values between 0.5 and 1.0 are considered suitable for HTS. (26)

Figure 1. High-throughput assay of ROMK function.

Figure 1

High-throughput assay of ROMK function. (A) Fluozin-2 fluorescence traces recorded fram C1 cells before and after addition of extracellular Tl+ (shaded area); (B) Plot of the slope of Tl+-induced fluorescence increase recorded from individual wells of (more...)

Summary of Screen

We developed a robust HTS assay for small-molecule ROMK modulators, which enabled the identification of a novel blocker of ROMK and Kir7.1 channels. The assay extends previous work using Tl+ flux to detect potassium channel and transporter activities (22–25) and overcomes ROMK-specific technical hurdles associated with reportedly poor channel expression in mammalian cells (27–33) by using an inducible expression system and point mutation (S44D) that promotes cell surface expression. (21–35) The identification by HTS of numerous ROMK antagonists, some of which preferentially block other Kir channels, is a significant step toward developing the molecular pharmacology of the inward rectifier channel family. Using the Tl+ flux assay we screened 126,009 compounds in 384-well plates at a single dose of 10 μM. DMSO at a final concentration 0.1% was used as the compound vehicle. During assay development, we established that Tl+ flux through ROMK was insensitive to DMSO concentrations up to 10% (data not shown). Four rows in each plate were filled with control or TPNQ-containing buffer for determination of Z′ (e.g. Fig. 3C). Plates with Z′ values less than 0.45 were not included in the data analysis. The mean ± SD Z′ of plates passing quality control was 0.72 ± 0.08. The processed primary data resulted in 1,758 compounds designated as inhibitors. After re-orders from ChemDiv and confirmation screening in electrophysiology experiments, we were left with three hits (Figure 2): SID-17510129 (IC50 = 3 μM), SID-24802482 (80% block @ 10 μM) and SID-17409040 (30% block @ 10 μM).

Figure 3. Lead Optimization of SID-17510129.

Figure 3

Lead Optimization of SID-17510129.

Figure 2. Confirmed ROMK HTS hits.

Figure 2

Confirmed ROMK HTS hits.

Chemical Probe Lead Optimization

Based on the initial activity at inhibiting ROMK and the fact that PubChem data indicated that it had been tested in 181 bioassays, and only active in the ROMK screen, we focused on optimization of SID-17510129, a bis-benzylated kryptofix analog. Under our standard paradigm, (36) we resynthesized SID-17510129 in a library formpat with 21 other analogs wherein we varied the aryl moiety (Figure 3A) utilizing solution phase parallel synthesis (Figure 3B). In the event, kryptofix 1 underwent a bis-reductive amination with 22 different aldehydes 2 employing MP-B(OAc)3H to deliver a total of 21 analogs 3 and analytically pure, fresh stock of SID-17510129 (CID 4536383). Upon testing fresh powder of analytically pure CID 4536383, we were pleased to find that the IC50 was 294 nM, a potency meeting MLPCN probe criteria. Moving the NO2 group from the 4-position to the 3-position (CID 44123652) led to a significant decrease in potency (IC50 = 14.2 μM) and further migration to the 2-position led a complete loss of activity (IC50 = >100 μM). SAR proved to be quite shallow (Figure 3B), with any other substituents on the aryl ring leading to ROMK inhibitors in the micromolar range (IC50s from 6.6 M to >100 μM). We then synthesized three analogs (Figure 3C) in which we maintained the 4-nitrophenyl moiety, but attenuated the basicity of the nitrogen atoms by capping as an amide 4, a urea 5 or a sulfonamide 6; however, all were inactive (IC50 = >100 μM), suggesting the basic nitrogen moieties are required for ROMK activity. Since CID 4536383 possessed the potency requirements for an MLPCN probe (IC50 = 294 nM) in both Tl+ flux assay (Figure 4B) and the in confirmatory electrophysiology assay (Figure 4C), we next evaluated Kir selectivity. We were pleased to find that CID 4536383 was selective versus the closely related Kir2.1 and Kir4.1 potassium channels. Interestingly, low micromolar concentrations of CID 4536383 inhibit Kir7.1 (70% @ 10 μM). Other than non-selective cationic pore blockers, CID 4536383 is the first small molecule inhibitor of Kir7.1, the newest Kir family member. Kir7.1 (KCNJ13), is expressed in several nephron segments. In principal cells of the collecting duct, Kir7.1 is believed to contribute to basolateral K+ recycling necessary for Na-K-ATPase-dependent K secretion. However, there is no direct evidence that Kir7.1 forms functional ion channels in the nephron. Kir7.1 has an unusually low unitary conductance (~50 fS), making it difficult to identify in single-channel recordings, and there have been no pharmacological tools available with which to discriminate Kir7.1 from other channels in whole-cell recordings. The identification of CID 4536383 as the first pharmacological probe of Kir7.1 will be helpful in defining the functions of Kir7.1 in the nephron and other tissues.

Figure 4. (A) CID 4536838; (B) CRC in Tl+ Flux assay; (C) Electrophysiology.

Figure 4

(A) CID 4536838; (B) CRC in Tl+ Flux assay; (C) Electrophysiology.

Figure 5. CID 4536838 selectivity versus Kir members.

Figure 5CID 4536838 selectivity versus Kir members

This intriguing Kir selectivity profile led to additional experiments to determine a mode of action for ROMK inhibition. The rate of ROMK inhibition by 10 M CID 4536383 was slow (time constant = 40 ± 0.5 sec; n = 8), and washout was incomplete, suggesting the compound might first cross the plasma membrane to reach an intracellular binding site. Furthermore, we noted a large hyperpolarizing shift in the rectification potential during the onset of inhibition. Because rectification in Kir channels is caused by block of outward current by intracellular solutes, this observation raised the possibility that CID 4536383 binds in the cytoplasmic pore. (28–31) We therefore tested if CID 4536383 could be displaced from the cytoplasmic pore with inwardly directed K+ ions through a mechanism known as “knock-off”. (32,33) Experiments were performed by pre-blocking ROMK with 10 μM CID 4536383 and voltage clamping the cell to potentials more negative than the K+ equilibrium potential (EK) to drive K+ ions intracellularly through the ROMK channel pore. In the presence of 5 mM extracellular K+ and 135 mM intracellular K+ (EK = −83 mV), block by CID 4536383 was virtually insensitive to repetitive pulses to −120 mV. However, increasingly stronger pulses led to a knock-off of CID 4536383 that was both time- and voltage-dependent (Fig. 7B′). To confirm that CID 4536383 knock-off is caused by permeant ions and not some voltage-dependent conformational change in the channel, we raised extracellular K+ concentration to 50 mM (EK = −25 mV) and repeated the -120 mV step protocol. CID 4536383 was readily displaced from the pore under these conditions. The most parsimonious interpretation of these data is that CID 4536383 is an intracellular pore blocker of ROMK. (25)

Solubility

~ 100 M in DMSO. Homogeneous/microsuspension at 10 mg/mL in : 20% β-cyclodextrin, 10% cremaphor, 40% PEG-400/H2O. As bis-HCl salt, soluble in saline at 10 mg/mL.

Synthetic procedure and Spectral data for CID 4536383

Image ml111fu2

7,13-bis(4-nitrobenzyl)-1,4,10-trioxa-7,13-diazacyclopentadecane(CID 4536383) [ML111]

To an 8 mL vial was added 4-nitrobenzaldehyde (70 mg, 0.46 mmol), Kryptofix (50 mg, 0.23 mmol) and MP-BH(OAc)3 (500 mg) in DCM (3 mL). The mixture was rotated for 12 h, filtered and concentrated. Purification of the crude oil by ISCO flash chromatography [10% MeOH/DCM containing 1% NH4OH (12CV)] provided the title compound CID 4536383 as an orange oil (39 mg, 35%). 1H-NMR (400 MHz, CDCl3) δ 8.18 (d, J=8.8 Hz, 4H), 7.60 (d, J=8.8 Hz, 4H), 3.79 (s, 4H), 3.65–3.62 (m, 12H), 2.86–2.79 (m, 8H); HRMS (calculated for C24H32N4O7+H) 489.2349; Found 489.2349.

MLS#s

002474506 (CID 4536383, 500 mg), 002474503, 002474504, 002474505, 002765789, 002765790

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APPENDIX I. Solubility, Stability and Reactivity data as determined by Absorption Systems

Solubility

Solubility in PBS (at pH = 7.4) for ML111 was 58.2 μM.

Stability

Stability (at room temperature = 23 °C) for ML111 in PBS (no antioxidants or other protectorants and DMSO concentration below 0.1%) is shown in the table below. After 48 hours, the percent of parent compound remaining was 91%, but the assay variability over the course of the experiment ranged from a low of 91% (at 48 hours) to a high of 107% (at 30 minutes).

Percent Remaining (%)
Compound0 Min15 Min30 Min1 Hour2 Hour24 Hour48 Hour
ML111---10010799979591

Reactivity

As assessed through a glutathione (GSH) trapping experiment in phosphate buffered saline (with a substrate concentration of typically 5–50 μM and a GSH concentration of 5 mM, at t = 60 minutes) ML111 was found to not form any detectable GSH adducts.*

Footnotes

*

Solubility (PBS at pH = 7.4), Stability and Reactivity experiments were conducted at Absorption Systems. For additional information see: https://www​.absorption.com/site

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