High-throughput Discovery of Novel Modulators of ROMK K+ Channel Activity
The Renal Outer Medullary Potassium channel (ROMK, Kir1.1) is expressed in the renal tubule where it critically regulates fluid and electrolyte homeostasis (Hebert, 2005). An emerging body of evidence suggests that ROMK could be a target for a novel class loop diuretic that lowers blood pressure while preserving plasma potassium levels (Ji, 2008). Furthermore, homozygous loss-of-function more ..
Assay Provider: Jerod Denton
Assay Provider Affiliation: Vanderbilt University
Grant Title: High throughput discovery of novel modulators of ROMK K+ channel activity
Grant Number: R21 NS057041-01
The Renal Outer Medullary Potassium channel (ROMK, Kir1.1) is expressed in the renal tubule where it critically regulates fluid and electrolyte homeostasis (Hebert, 2005). An emerging body of evidence suggests that ROMK could be a target for a novel class loop diuretic that lowers blood pressure while preserving plasma potassium levels (Ji, 2008). Furthermore, homozygous loss-of-function mutations in the gene encoding ROMK (KCNJ1) cause antenatal Bartter syndrome, a severe salt and water wasting disease in infants (Simon,1996). ROMK is thus an important pharmacological target for the management of disease. Its actual therapeutic value and drugability, however, are unknown due to the lack of small-molecule probes targeting the channel. The discovery of ROMK modulators will provide important new tools for studying the structure, function and therapeutic potential of ROMK and other inward rectifying potassium channels.
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 (Fallen, 2009; Weaver, 2004; Delpire, 2009; Lewis, 2009) and overcomes ROMK-specific technical hurdles associated with reportedly poor channel expression in mammalian cells (Schulte, 1999; Lopatin, 1994; Lu, 1995; Wible, 1994) by using an inducible expression system and point mutation (S44D) that promotes cell surface expression (Fallen, 2009). 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 uM (AID 1918). DMSO at a final concentration 0.1 percent was used as the compound vehicle. During assay development, we established that Tl+ flux through ROMK was insensitive to DMSO concentrations up to 10 percent. Four columns in each plate were filled with control or TPNQ-containing buffer for determination of Z'. 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 (AID 1917). After re-orders from ChemDiv and confirmation screening in electrophysiology experiments, we were left with three hits: SID 17510129 (IC50 = 3 uM), SID 24802482 (80percent block @ 10 uM) and SID 17409040 (30percent block @ 10 uM).
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 (Kennedy, 2008) we resynthesized SID 17510129 in a library format with 21 other analogs wherein we varied the aryl moiety utilizing solution phase parallel synthesis. In the event, kryptofix underwent a bisreductive amination with 22 different aldehydes employing MP-B(OAc)3H to deliver a total of 21 analogs and analytically pure, fresh stock of SID 17510129. Upon testing fresh powder of analytically pure SID 17510129, we were pleased to find that the IC50 was 294 nM, a potency meeting probe criteria. Moving the NO2 group from the 4- position to the 3-position (SID 17510129) led to a significant decrease in potency (IC50 = 14.2 uM) and further migration to the 2-position led a complete loss of activity (IC50 = >100 uM). SAR proved to be quite shallow, with any other substituents on the aryl ring leading to ROMK inhibitors in the micromolar range (IC50s from 6.6 uM to >100 uM). We then synthesized three analogs in which we maintained the 4-nitrophenyl moiety, but attenuated the basicity of the nitrogen atoms by capping as an amide, a urea or a sulfonamide; however, all were inactive (IC50 = >100 uM), suggesting the basic nitrogen moieties are required for ROMK activity. Since SID 17510129 possessed the potency requirements for an MLPCN probe (IC50 = 294 nM) in both Tl+ flux assay and the in confirmatory electrophysiology assay, we next evaluated Kir selectivity. We were pleased to find that SID 17510129 was selective versus the closely related Kir2.1 and Kir4.1 potassium channels (AID 1922). Interestingly, low micromolar concentrations of SID 17510129 inhibit Kir7.1 (70 percent @ 10 uM). Other than nonselective cationic pore blockers, SID 17510129 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 SID 17510129 as the first pharmacological probe of Kir7.1 will be helpful in defining the functions of Kir7.1 in the nephron and other tissues.
The first probe from this effort was based on SID 17510129. Resynthesis of the parent in the context of a small parallel library (Kennedy, 2008) resulted in 'flat' SAR, but fresh powder of analytically pure SID 17510129/CID 4536383 generated a ROMK inhibitor with an IC50 of 294 nM, with selectivity versus Kir2.1 and Kir4.1. However, SID 17510129 was also the first reported small molecule inhibitor of Kir7.1 (70 percent @ 10 uM) (Lewis, 2009). In order to truly probe the role of ROMK and determine therapeutic potential of ROMK inhibition, a more selective ROMK inhibitor was required.
Efforts focused on SID 24802482, a bisbenzimidazole tethered by a propyl chain that displayed 80% inhibition of ROMK at 10 uM. The parent molecule was resynthesized by heating a suspension of glutaric acid and 4-nitrobenzene-1,2-diamine in 5 N HCl in a sealed tube at 120C to provide SID 24802482 in 48% isolated yield, which was assigned a new SID for this new lot, SID 84975339. When evaluated in the Tl+ flux assay, fresh powder of analytically pure SID 84975339 possessed an IC50 of 60 uM for inhibition of ROMK. This was concerning as the HTS stock afforded 80% inhibition at 10 uM. Upon LCMS inspection of the original HTS DMSO stock solution, we found that the sample contained several impurities. However, we noticed a structural similarity between SID 84975339 and the first generation ROMK probe SID 17510129. Our initial thought on SID 24802482, based on a kryptofix scaffold, was that it chelated potassium ions (K+). We postulated that if we replaced the central methylene moiety of SID 24802482 with an oxygen atom, we would enhance its ability to chelate K+ through a three-point chelate, and therefore increase inhibition of ROMK. In order to test this hypothesis, we synthesized SID 24802482 by heating a suspension of diglycolic acid and 4-nitrobenzene-1,2-diamine in 5 N HCl in a sealed tube at 120C to provide SID 84975340 in 51% isolated yield. When evaluated in the Tl+ assay, fresh powder of analytically pure SID 84975340 possessed an IC50 of 220 nM for inhibition of ROMK, an increase in ROMK potency of over >250-fold.
At this point, the Lead Profiling Screen (68 GPCRs, ion channels and transporters) from MDS Pharma was performed on SID 84975340 to determine a broader ancillary pharmacology profile for this probe. In addition to selectivity versus the Kir family, SID 84975340 possessed clean ancillary pharmacology, displaying inhibition of only 4 targets: Dopamine D4 (75% @ 10 uM), GABAA (77% @ 10 uM), DAT (59% @ 10 uM) and NET (56% @ 10 uM). Upon obtaining full CRCs for these four off-target activities, only GABAA proved to afford an IC50<10 uM (GABAA IC50 = 6.2 uM), 28-fold selective for ROMK. Notably, SID 84975340 was also inactive on hERG and both L- and N-type calcium channels in this panel. Thus, SID 84975340, a ROMK inhibitor with unprecedented selectivity was declared a probe for studying the selective inhibition of ROMK in vitro.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 2
MLP Probe ML# for probe 1: ML111
PubChem Substance ID (SID) for probe 1: 84975334
PubChem Compound ID (CID) for probe 1: 4536383
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 294
Target for probe 1: ROMK (gi: 223460826)
Disease relevance for probe 1: Research Tool for Diuresis
Anti-target for probe 1: Kir2.1, Kir4.1
Fold selectivity for probe 1: >30
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK50685/ (ID: 2376246)
Grant number for probe 1: NS057041-01
MLP Probe ML# for probe 2: ML112
PubChem Substance ID (SID) for probe 2: 84975340
PubChem Compound ID (CID) for probe 2: 44123657
Probe type for probe 2: Inhibitor
IC50/EC50 (nM) for probe 2: 220
Target for probe 2: ROMK (gi: 223460826)
Disease relevance for probe 2: Research Tool for Diuresis
Anti-target for probe 2: Kir2.1, Kir4.1, Kir7.1, Kir2.3
Fold selectivity for probe 2: >50
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK50699/ (ID: 2377530)
Grant number for probe 2: NS057041-01
PubMed Publication ID (PMID) for probe 1: 19706730
NCBI Book chapter title for probe 1: Discovery of a small molecule inhibitor of ROMK and Kir7.1
NCBI Book chapter title for probe 2: Discovery of a small molecule inhibitor of ROMK with unprecedented selectivity