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ScienceDirect Targeting the inward-rectifier potassium channel ROMK in cardiovascular disease Maria L Garcia and Gregory J Kaczorowski The kidney plays a critical role in blood pressure homeostasis as a result of the integrated activity of different mechanisms that ensure proper salt and water reabsorption. Diuretics, developed more than four decades ago, are used to treat hypertension and/or congestive heart failure, although there are therapeutic issues that limit their use. Human and rodent genetic studies provide a large body of evidence which suggests that inhibitors of the kidney potassium channel, ROMK, will represent novel diuretics for the treatment of hypertension. The search for potent and selective ROMK inhibitors has recently yielded compounds that display efficacy in animal models, providing the first pharmacological validation of ROMK as a novel diuretic target. Addresses Kanalis Consulting, L.L.C., 5 Ashbrook Drive, Edison, NJ 08820, USA Corresponding authors: Garcia, Maria L ([email protected]) Current Opinion in Pharmacology 2014, 15:1–6 This review comes from a themed issue on Cardiovascular and renal Edited by Gregory J Kaczorowski and Olaf Pongs For a complete overview see the Issue and the Editorial Available online 26th November 2013 1471-4892/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coph.2013.11.005
Introduction Kidneys play a critical role in control and regulation of blood pressure as a result of the integrated operation of ion channels, transporters and pumps that are located along the nephron to ensure proper salt and water reabsorption. Mutations in some of these mechanisms that lead to increased salt reabsorption, such as in Liddle syndrome, raise blood pressure, whereas those mutations that decrease salt reabsorption, such as in Bartter’s or Gitelman’s syndromes, lower blood pressure [1]. Pharmacological intervention(s) at some of these critical salt reabsorption steps is used clinically to treat hypertension (thiazide-class diuretics) and/or congestive heart failure (CHF; loop diuretics). These medications, developed more than four decades ago, have some issues associated with efficacy (loop diuretics lose efficacy with time) [2], and adverse effects (mainly effects on serum potassium levels) [3] that can limit their use in Man. Interestingly, no new diuretics have entered clinical development recently. To be of clinical interest, an agent should display equivalent or superior efficacy to current drugs, be potassium neutral, www.sciencedirect.com
and be amenable to combination therapy. In this context, the renal outer medullary potassium channel, ROMK, product of the KCNJ1 gene, represents a novel target for development of such new diuretics.
Biological validation of ROMK as a drug target ROMK (Kir1.1) is a member of the inwardly-rectifying family of potassium (Kir) channels [4], and is almost exclusively localized at the apical surface of epithelial cells in two parts of the nephron (Figure 1): thick ascending limb of Henle (TALH) and cortical collecting duct (CCD) [5,6]. At the TALH, ROMK participates in potassium recycling across the apical membrane, a process critical for proper function of furosemide-sensitive, Na+/ K+/2Cl co-transporter, the rate-determining step for salt reabsorption in this part of the nephron. At the CCD, ROMK represents one of the major potassium secretory pathways, together with the high-conductance, calciumactivated potassium (BK) channel [7]. ROMK activity is tightly coupled to the amiloride-sensitive, epithelial sodium channel (ENaC), the final sodium reabsorption pathway in nephrons that is highly regulated by the reninangiotensin system [1]. Inhibition of ROMK in TALH is predicted to cause diuresis/natriuresis, similar to furosemide. However, because of presence of ROMK in CCD, hypokalemia that can develop with use of loop and thiazide-like diuretics, as a consequence of increased sodium delivery to CCD and activation of ENaC, should be ameliorated when ROMK is inhibited. Furthermore, although ROMK represents a major potassium secretory pathway in CCD, activity of BK channels should contribute to preventing hyperkalemia that is associated with ENaC inhibition [7]. Thus, selective ROMK inhibitors would be predicted to display diuretic/natriuretic efficacy, without altering serum potassium levels. Human and rodent genetic studies have provided strong evidence which support ROMK as a target for novel diuretics to treat hypertension and/or heart failure. Bartter’s syndrome type II, caused by loss of function mutations in the KCNJ1 gene, is an autosomal recessive life-threatening disorder associated with renal salt wasting, hypotension, and mild hypokalemia [8]. Importantly, heterozygous carriers of ROMK mutations associated with Bartter’s syndrome type II, identified in the Framingham Heart Study, have reduced blood pressure and decreased risk of developing hypertension by age 60 [9]. In addition, ROMK/ mice [10,11] and rats [12] display Bartter’s syndrome type II phenotypes, and Dahl saltsensitive, ROMK+/ heterozygous rats exhibit reduced Current Opinion in Pharmacology 2014, 15:1–6
2 Cardiovascular and renal
Figure 1
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Current Opinion in Pharmacology
Physiological role of ROMK in kidney. ROMK is present at the luminal surface of epithelial cells in two regions of the nephron: the thick ascending limb of Henle (TAHL) and the cortical collecting duct (CCD) where it contributes to salt reuptake and potassium homeostasis.
blood pressure compared to wild-type when challenged with a 4% salt diet, as well as, increased protection from salt-induced blood pressure elevation and from renal injury when challenged with an 8% salt diet, illustrating the important role of ROMK in regulating blood pressure in this model.
electrophysiology and constructing a polarized cell system by growing Madin–Darby canine kidney cells expressing Kir1.1 in permeable Transwell supports are useful for studying Kir1.1 inhibitor mechanisms [18]. Together, these assays can provide valuable information during lead identification and/or optimization of Kir1.1 drug candidates.
Discovery of small molecule ROMK inhibitors Despite substantial genetic evidence, pharmacological validation of the ROMK target is just underway to determine short and long term consequences of Kir1.1 inhibition on renal function and blood pressure. Unlike other ion channel families, molecular pharmacology of Kir1.1, and in general the entire Kir family, is presently quite limited [13]. The only previously disclosed Kir1.1 inhibitor is a bee venom peptide, tertiapin (TPN). TPN is a selective and potent inhibitor of the rat channel [14], but it inhibits human isoform with at least 100-fold reduced potency [15]. In addition, TPN inhibits Kir1.1 by binding to residues at the extracellular surface, which is equivalent to glomerular filtrate, making it a challenging tool for studying ROMK in vivo. For this reason, high-throughput screening (HTS) assays employing heterologous expressed Kir1.1 channels were developed to interrogate large chemical libraries and identify lead small molecules that potentially could be optimized as drug candidates by Medicinal Chemistry. Such assays that function in high density formats, 384-well or 1536-well plates, and support screening >1.5 million samples are based on either membrane potential [16] or thallium flux fluorescence technologies [17,18]. Other assays of intermediate capacity, up to 384-well plates, evaluate channel activity by either automated electrophysiology, or by 86Rb flux, and can be used to support Medicinal Chemistry [18]. The 86Rb flux protocol provides advantage of determining compound potency in absence or presence of up to 100% serum, and is therefore a useful surrogate for more time consuming protein binding measurements. Additionally, manual Current Opinion in Pharmacology 2014, 15:1–6
Using a fluorescence-based thallium flux 384-well assay, a small molecule was identified at Vanderbilt University in a screening campaign of 126 000 molecules. This compound, VU590 (7,13-bis(4-nitrobenzyl)-1,4,10-trioxa7,13-diazacyclopentadecane; 1) [17], and structurally related VU591 (2,20 -oxybis(methylene)bis(5-nitro-1Hbenzo-[d]imidazole); 2), identified later by synthetic exploration of VU590 [19], inhibit rat Kir1.1 with IC50 values of 290 and 240 nM (Figure 2), respectively, and appear to block at the channel’s intracellular pore. Although VU590 has no activity against Kir2.1 and Kir4.1, it inhibits Kir7.1, a channel that is also expressed in kidney. However, VU591 appears to be selective for Kir1.1 over other Kir channels, as well as, other ion channels and receptors. VU591 displays an unfavorable degree of protein binding (>98%) in human and rat serum, but good microsomal stability. Although neither compound has been tested in vivo, VU591 was evaluated in isolated-perfused rat collecting distal tubules. Luminal perfusion with VU591 inhibited net potassium transport with no effect on net sodium transport, suggesting that VU591 inhibits native ROMK in distal nephron that contribute to basal potassium secretion. Independent of the Vanderbilt group, scientists at Merck performed a HTS of Kir1.1 with an internal sample collection (1.5 M compounds) using a membranepotential based fluorescence assay in 1536-well format [16]. A very small number of hits were identified, but one sample, (4-nitrophenethyl)piperazine, was prioritized www.sciencedirect.com
ROMK drug development Garcia and Kaczorowski 3
Figure 2
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Discovery of ROMK inhibitors at Vanderbilt University. Compound 1 was identified by HTS of a chemical library, while compound 2 was derived from 1 by medicinal chemistry investigation.
based on its selectivity against two other Kir channels; cardiac Kir2.1 (whose block would cause serious negative cardiovascular consequences [20]) and kidney Kir2.3. When the screening sample was subjected to re-purification, Kir1.1 inhibitory activity was lost. Subsequently, it was discovered that original sample activity was due to a minor impurity (1%; Figure 3) representing the disubstituted piperazine (3), 1,4-bis(4-nitrophenethyl)piperazine [21]. This finding was confirmed by synthesis. When monitoring activity by either thallium flux or electrophysiology, 3 inhibits human Kir1.1 with IC50 values of 20 and 33 nM, respectively. In 86Rb+ efflux assay, 3 inhibits Kir1.1 with an IC50 of 47 nM (n = 50), and, importantly, its potency is not significantly affected in presence of 100% human serum, suggesting a low degree of serum protein binding. 3 had no significant effect on Kir2.1 or Kir2.3 at concentrations of up to 100 mM, and, other than causing potent inhibition of Kv11.1 (hERG), IC50 of 5 nM, displayed remarkable selectivity against other ion channels, as well as, a broad panel of receptors and enzymes.
Progress in developing ROMK inhibitors as drugs Historically, it has been challenging to identify potent and selective small molecule Kir inhibitors. The general finding is that HTS directed against targets in this family has yielded few interesting or chemically tractable hits to date, impeding drug development. Therefore, it is remarkable that a simple di-substituted piperazine, 3, present as a contaminant, potently blocks Kir1.1 with marked selectivity over other Kir. There may be some www.sciencedirect.com
similarity between 3 and the Kir1.1 VU blockers (Figure 2), discovered independently, since both series contain symmetrical bis-nitrophenyl substituents; whether this feature is related to mechanism of channel inhibition is unclear, but will only be resolved with certainty by co-crystallization of representative inhibitors with Kir1.1. However, the Merck screening lead is not without issues that must be addressed by Medicinal Chemistry: first, aromatic nitro groups are subject to metabolism in vivo which generate both chemically reactive species and agents with carcinogenic potential; second, the lead is 10-fold more potent as a Kv11.1 than Kir1.1 blocker, and this potential hERG activity is a major cardiovascular safety liability [22]; third, symmetrical nature of the lead structure, together with the observation that the mono-substituted piperazine is completely inactive, might limit the ability to introduce chemical diversity into this chemical series. Nonetheless, despite these and other potential issues typically associated with drug development, initial success of Merck Chemistry efforts focused on this lead (Figure 3) have been highlighted recently in three publications [21,23,24] and five patent applications [25–29]. Initial Merck structure–activity relationship (SAR) studies focused on three areas [21]; identifying bioisosteric replacements for nitro groups; modifying linkers between the core piperazine and terminal aromatic substituents; replacement of the core. Since both nitro groups are necessary for maintaining Kir1.1 potency, bioisosteres were introduced into both ends of the molecule, one at a time (Figure 3). Some appropriate replacements were benzonitriles (A), a 5-benzo(2,1,3-oxadiazole) (B) and, interestingly, a 4-phthalide group (C), but several other potential alternatives caused substantial loss of Kir1.1 inhibitory activity. The 4-phthalide substitution was noted to weaken hERG interactions, suggesting that SAR between Kir1.1 and Kv11.1 block might be divergent within this series, and substitution of both nitrophenyl moieties with 4-phthalide groups, 4, maintained Kir1.1 block, while decreasing Kv11.1 inhibition by 20fold, a very encouraging result. Other studies demonstrated that distance between bis-nitro groups is critical for activity, as shortening distance by one carbon abolished channel block completely, while lengthening distance by one carbon also significantly decreased Kir1.1 inhibition. As well, small modifications of the core structure resulted in some loss of Kir1.1 activity. These results, taken together with studies of asymmetrical molecules possessing both 4-phthalide and benzonitrile groups, indicate the following trends: first, it is possible to replace both nitrophenyl groups in the initial lead with either symmetrical or asymmetrical pharmacophore substitutions that are much more suitable for drug development; second, Kir1.1 blocking potency and selectivity over other Kir can be maintained, while at the same time, Kv11.1 inhibition can be reduced; third, overall shape and Current Opinion in Pharmacology 2014, 15:1–6
4 Cardiovascular and renal
Figure 3
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Discovery and development of ROMK inhibitors at Merck. Compound 3 was discoverd by HTS of a very large corporate sample collection, while compounds 4 and 5 were synthesized by Merck medicinal chemistry during SAR investigation of lead 3. Data from both ROMK (Kir1.1) and hERG (Kv11.1), the major off-target liability of this lead series are shown. Compounds A–D are bioisosteric replacements for a nitro group that were used in the Merck SAR studies.
size of the molecule must be maintained to keep Kir1.1 inhibitory potency. The latter result might predict a tight SAR in this series and, interestingly, potency was not significantly increased in initial studies over that of the original lead. However, some analogs prepared had markedly improved pharmacokinetic (PK) properties than those observed with 1,4-bis(4-nitrophenethyl)piperazine [21], another encouraging finding. That Kir1.1 inhibitors can be developed from the initial Merck lead with maintained Kir1.1 potency, but much improved Kv11.1 selectivity, was demonstrated within a novel sub-class of di-substituted piperazines displaying an asymmetric substitution pattern (Figure 3), characterized by a 4-phthalidyl ethyl group as one N-substituent, and a 4-(1H-tetrazol-1-yl)phenyl methyl amide as the other N-substituent [23]. This discovery resulted from attempts to replace the 4-nitrophenyl group with small heterocyclic bioisosteres and noting that a compound Current Opinion in Pharmacology 2014, 15:1–6
containing 4-phenyl tetrazole (D) retained moderate Kir1.1 blocking activity. However, this substitution also resulted in increasing Kv11.1 potency. To generally diminish Kv11.1 blocking activity characteristic of most molecules in this series, an amide substituent was incorporated into one scaffold linker to reduce basicity of the piperazine nitrogens, and while this reduced Kir1.1 potency of molecules containing nitrophenyl, benzonitrile or 4-phthalide pharmacophores, surprisingly it markedly improved the Kir1.1 potency of the phenyl tetrazole containing piperazine, while concurrently diminishing its Kv11.1 blocking activity. This finding was further capitalized on by substituting the other nitrophenyl group with a 4-phthalate substituent, which resulted in even greater selectivity for Kir1.1 over Kv11.1 block. The phenyl-tetrazole moiety appears critical because exploration of many other five-membered heterocycles as tetrazole replacements resulted in significant loss of Kir1.1 potency. Thus, new Kir1.1 blockers with markedly www.sciencedirect.com
ROMK drug development Garcia and Kaczorowski 5
improved properties have been identified [23], as typified by 5-(2-(4-(2-(4-(1H-tetrazol-1yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one, 5; this molecule maintains Kir1.1 inhibitory activity, is selective for Kir1.1 over Kir2.1, Kir2.3, Kir4.1 and Kir7,1, has no off target activity against Nav1.5 and Cav1.2, and has much reduced inhibitory activity, 100-fold, with respect to Kv11.1. It also displays good oral bio-availability with moderate oral exposures, and a short half-life. Oral dosing of 5 in volume loaded Sprague-Dawley (SD) normotensive rats and dogs caused concentration-dependent increase in urinary flow and urinary sodium excretion compared to vehicle treated animals [24]. The magnitude of these effects was comparable to that caused by oral dosing of 25 mg/kg hydrochlorothiazide, a diuretic recommended as first line therapy in treatment of hypertension. Unlike hydrochlorothiazide, however, 5 did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These results strengthen the notion that ROMK inhibitors will be novel diuretic and natriuretic agents with similar efficacy to that of clinically used diuretics, but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics, and are encouraging in terms of supporting future efforts to optimize potency, selectivity, PK and bioactivity of the bis N-substituted piperazine Kir1.1 inhibitor class. Five Merck patent applications [25–29] have published covering approximately 400 specific examples of molecules from the lead series represented by the template: pharmacophore-linker-core-linker-pharmacophore, with pharmacophores primarily containing previously reported nitro bioisosteres in either symmetrical or asymmetrical arrangements. Specific Kir1.1 blocking potencies are only reported for select agents, but some values noted are in low nM range, and IC50’s for all Kir1.1 inhibitors listed are 1 mM. Examples are presented of modification and replacement of the central piperazine core, flexible, rigid and conformationally restricted linker moieties, introduction of stereochemical sites into linker scaffolds, amide and heteroatom containing linkers, and structural modifications to control overall shape of resulting molecules. However, no data are given on important ancillary pharmacology properties, such as Kv11.1 block, so without these comparative results, it is difficult to judge progress that has been made with this lead series based solely on present documentation. There is, however, indication of oral bio-activity of some of these examples. Certain molecules [illustrated in [25]] are reported to display diuretic activity, with twofold to ninefold increase over vehicle treated animals, 4 hours after oral dose to SD rats, at either 1 or 3 mg/kg test compound. In addition, compounds from the same application were reported to lower blood pressure when dosed orally in spontaneous hypertensive rats with telemetry implanted devices that allow continuous monitoring of blood pressure and heart www.sciencedirect.com
rate in freely moving animals. Compounds were dosed once a day for a period of 3–14 days at either 3 or 10 mg/ kg. When compared to vehicle treated animals, specific compounds displayed typical reductions in daily mean systolic blood pressure ranging from 8 to 32 mmHg by last day of study.
Conclusions It is an exciting period as pharmacology of the ROMK target is finally being characterized. Questions regarding physiologic responses elicited by chronic treatment with Kir1.1 inhibitors and their effects on blood pressure, either alone, or in combination with other anti-hypertensive drugs [30], may soon be answered. Moreover, evaluation of therapeutic potential of Kir1.1 blockers in humans with hypertension or CHF will gauge the predictive value of the human genetic phenotypes. Hopefully, small molecule Kir1.1 inhibitors currently under development will be instrumental in addressing all these issues.
Acknowledgement We thank Dr. Jed Fisher for help with preparing the chemical structure’s art work.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Lifton RP, Gharavi AG, Geller DS: Molecular mechanisms of human hypertension. Cell 2001, 104:545-556.
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De Bruyne LKM: Mechanisms and management of diuretic resistance in congestive heart failure. Postgrad Med J 2003, 79:268-271.
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Palmer BF, Naderi AS: Metabolic complications associated with use of thiazide diuretics. J Am Soc Hypertens 2007, 1:381-392.
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Nichols CG, Lopatin AN: Inward rectifier potassium channels. Annu Rev Physiol 1997, 59:171-191.
5.
Wang W: Renal potassium channels: recent developments. Curr Opin Nephrol Hypertens 2004, 13:549-555.
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Herbert SC, Desir G, Giebisch G, Wang W: Molecular diversity and regulation of renal potassium channels. Physiol Rev 2005, 85:319-371.
7.
Rieg T, Vallon V, Sausbier M, Sausbier U, Kaissling B, Ruth P, Osswald H: The role of the BK channel in potassium homeostasis and flow-induced renal potassium excretion. Kidney Int 2007, 72:566-573.
8.
Simon DB, Karet FE, Rodrguez-Soriano J, Hamdan JH, DiPietro A, Trachtman H, Sanjad SA, Lifton RP: Genetic heterogeneity of Bartter’s syndrome revealed by mutations in the K+ channel, ROMK. Nat Genet 1996, 14:152-156.
9.
Ji W, Foo JN, O’Roak BJ, Zhao H, Larson MG, Simon DB, NewtonCheh C, State MW, Levy D, Lifton RP: Rare independent mutations in renal salt handling genes contribute to blood pressure variation. Nat Genet 2008, 40:592-599.
10. Lorenz JN, Baird NR, Judd LM, Noonan WT, Andringa A, Doetschman T, Manning PA, Liu LH, Miller ML, Shull GE: Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter’s syndrome. J Biol Chem 2002, 277:37871-37880. Current Opinion in Pharmacology 2014, 15:1–6
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11. Lu M, Wang T, Yan Q, Yang X, Dong K, Knepper MA, Wang W, Giebisch G, Shull GE, Hebert SC: Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter’s) knockout mice. J Biol Chem 2002, 277:37881-37887. 12. Zhou X, Zhang Z, Shin MK, Horwitz SB, Levorse JM, Zhu L, Sharif Rodriguez W, Streltsov DY, Dajee M, Hernandez M et al.: Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure. Hypertension 2013, 62:288-294. Using a ROMK knockout rat model with Dahl salt-sensitive background, authors demonstrate that ROMK plays a critical role in blood pressure regulation. This is the first illustration that ROMK/+ rats recapitulate the blood pressure phenotype of heterozygous humans carrying ROMK mutations associated with Bartter’s syndrome type II, identified in the Framingham Heart Study. 13. Wulff H, Zhorov BS: K+ channel modulators for the treatment of neurological disorders and autoimmune diseases. Chem Rev 2008, 108:1744-1773. 14. Jin W, Lu Z: A novel high-affinity inhibitor for inward-rectifier K+ channels. Biochemistry 1998, 37:13291-13299. 15. Felix JP, Liu J, Schmalhofer WA, Bailey T, Bednarek MA, Kinkel S, Weinglass AB, Kohler M, Kaczorowski GJ, Priest BT, Garcia ML: Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin. Biochemistry 2006, 45:10129-10139. 16. Solly K, Cassaday J, Felix JP, Garcia ML, Ferrer M, Strulovici B, Kiss L: Miniaturization and HTS of a FRET-based membrane potential assay for Kir channel inhibitors. Assay Drug Dev Technol 2008, 6:225-234. 17. Lewis LM, Bhave G, Chauder BA, Banerjee S, Lornsen KA, Redha R, Fallen K, Lindsley CW, Weaver CD, Denton JS: Highthroughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1. Mol Pharmacol 2009, 76:1094-1103. 18. Felix JP, Priest BT, Solly K, Bailey T, Brochu RM, Liu CJ, Kohler MG, Kiss L, Alonso-Galicia M, Tang H et al.: The inwardly rectifying potassium channel Kir1.1: development of functional assays to identify and characterize channel inhibitors. Assay Drug Dev Technol 2012, 10:417-431. A wide variety of Kir1.1 functional assays that can support evaluation of large chemical libraries (>1.5 M) to identify leads and assist Medicinal Chemsitry studies during lead optimization, as well as, aid mechanism of action studies are described and validated. 19. Bhave G, Chauder BA, Liu W, Dawson ES, Kadakia R, Nguyen TT, Lewis LM, Meiler J, Weaver CD, Satlin LM et al.: Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel. Mol Pharmacol 2011, 79:42-50. The first selective Kir1.1 inhibitor, VU591, is revealed. Using isolatedperfused rat collecting distal tubules, VU591 inhibits net potassium transport with no effect on net sodium transport, suggesting that VU591 inhibits native ROMK channels in the distal nephron that contribute to basal potassium secretion.
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20. Dhamoon AS, Jalife J: The inward rectifier current (IK1) controls cardiac excitability and is involved in arrhythmogenesis. Heart Rhythm 2005, 2:316-324. 21. Tang H, Walsh SP, Yan Y, de Jesus RK, Shahripour A, Teumelsan N, Zhu Y, Ha S, Owens KA, Thomas-Fowlkes B et al.: Discovery of selective small molecule ROMK inhibitors as potential new mechanism diuretics. ACS Med Chem Lett 2012, 3:367-372. First SAR study of the Merck potent, di-substituted piperazine ROMK inhibitor screening lead which reveals discovery of analogs with potent ROMK blocking activity and selectivity over other Kir channels, as well as, improved selectivity against Kv11.1 and improved PK properties over that of the original parent compound. 22. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT: A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 1995, 80:795-803. 23. Tang H, de Jesus RK, Walsh SP, Zhu Y, Yan Y, Priest BT, Swensen AM, Alonso-Galicia M, Felix JP, Brochu RM et al.: Discovery of a novel sub-class of ROMK channel inhibitors typified by 5-(2-(4-(2-(4-(1H-tetrazol-1yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)one. Bioorg Med Chem Lett 2013, 23:5829-5832. A sub-class of ROMK inhibitor developed from the original Merck screening lead with good ROMK functional activity and improved hERG selectivity is revealed; two members of this class are used to demonstrate for the first time in vivo proof of concept that ROMK blockers represent a new mechanism diuretic and natriuretic agent. 24. Garcia ML, Priest BT, Alonso-Galicia M, Zhou X, Felix JP, Brochu RM, Bailey T, Thomas-Fowlkes B, Liu J, Swensen A et al.: Pharmacological inhibition of the renal outer medullary potassium channel causes diuresis and natriuresis in the absence of kaliuresis. J Pharmacol Exp Therap 2013 http:// dx.doi.org/10.1124/jpet.113.208603. First demonstration that in vivo dosing of a selective ROMK inhibitor to normotensive rats and dogs cause concentration-dependent increases in urinary flow and urinary sodium excretion which are comparable to hydrochlorothiazide, but however unlike hydrochlorothiazide, these changes are not associated with potassium losses or changes in plasma electrolyte levels. 25. Merck & Co. Inhibitors of the renal outer medullary potassium channel, US Patent Application: US2010/0286123A1; 2010 26. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2011/057346; 2011 27. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2012/054354; 2012 28. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2012/061274; 2012 29. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2012/061896; 2012 30. Sood N, Reinhart KM, Baker WL: Combination therapy for the management of hypertension: a review of the evidence. Am J Health Syst Pharm 2010, 67:885-894.
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ScienceDirect Targeting the inward-rectifier potassium channel ROMK in cardiovascular disease Maria L Garcia and Gregory J Kaczorowski The kidney plays a critical role in blood pressure homeostasis as a result of the integrated activity of different mechanisms that ensure proper salt and water reabsorption. Diuretics, developed more than four decades ago, are used to treat hypertension and/or congestive heart failure, although there are therapeutic issues that limit their use. Human and rodent genetic studies provide a large body of evidence which suggests that inhibitors of the kidney potassium channel, ROMK, will represent novel diuretics for the treatment of hypertension. The search for potent and selective ROMK inhibitors has recently yielded compounds that display efficacy in animal models, providing the first pharmacological validation of ROMK as a novel diuretic target. Addresses Kanalis Consulting, L.L.C., 5 Ashbrook Drive, Edison, NJ 08820, USA Corresponding authors: Garcia, Maria L ([email protected]) Current Opinion in Pharmacology 2014, 15:1–6 This review comes from a themed issue on Cardiovascular and renal Edited by Gregory J Kaczorowski and Olaf Pongs For a complete overview see the Issue and the Editorial Available online 26th November 2013 1471-4892/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coph.2013.11.005
Introduction Kidneys play a critical role in control and regulation of blood pressure as a result of the integrated operation of ion channels, transporters and pumps that are located along the nephron to ensure proper salt and water reabsorption. Mutations in some of these mechanisms that lead to increased salt reabsorption, such as in Liddle syndrome, raise blood pressure, whereas those mutations that decrease salt reabsorption, such as in Bartter’s or Gitelman’s syndromes, lower blood pressure [1]. Pharmacological intervention(s) at some of these critical salt reabsorption steps is used clinically to treat hypertension (thiazide-class diuretics) and/or congestive heart failure (CHF; loop diuretics). These medications, developed more than four decades ago, have some issues associated with efficacy (loop diuretics lose efficacy with time) [2], and adverse effects (mainly effects on serum potassium levels) [3] that can limit their use in Man. Interestingly, no new diuretics have entered clinical development recently. To be of clinical interest, an agent should display equivalent or superior efficacy to current drugs, be potassium neutral, www.sciencedirect.com
and be amenable to combination therapy. In this context, the renal outer medullary potassium channel, ROMK, product of the KCNJ1 gene, represents a novel target for development of such new diuretics.
Biological validation of ROMK as a drug target ROMK (Kir1.1) is a member of the inwardly-rectifying family of potassium (Kir) channels [4], and is almost exclusively localized at the apical surface of epithelial cells in two parts of the nephron (Figure 1): thick ascending limb of Henle (TALH) and cortical collecting duct (CCD) [5,6]. At the TALH, ROMK participates in potassium recycling across the apical membrane, a process critical for proper function of furosemide-sensitive, Na+/ K+/2Cl co-transporter, the rate-determining step for salt reabsorption in this part of the nephron. At the CCD, ROMK represents one of the major potassium secretory pathways, together with the high-conductance, calciumactivated potassium (BK) channel [7]. ROMK activity is tightly coupled to the amiloride-sensitive, epithelial sodium channel (ENaC), the final sodium reabsorption pathway in nephrons that is highly regulated by the reninangiotensin system [1]. Inhibition of ROMK in TALH is predicted to cause diuresis/natriuresis, similar to furosemide. However, because of presence of ROMK in CCD, hypokalemia that can develop with use of loop and thiazide-like diuretics, as a consequence of increased sodium delivery to CCD and activation of ENaC, should be ameliorated when ROMK is inhibited. Furthermore, although ROMK represents a major potassium secretory pathway in CCD, activity of BK channels should contribute to preventing hyperkalemia that is associated with ENaC inhibition [7]. Thus, selective ROMK inhibitors would be predicted to display diuretic/natriuretic efficacy, without altering serum potassium levels. Human and rodent genetic studies have provided strong evidence which support ROMK as a target for novel diuretics to treat hypertension and/or heart failure. Bartter’s syndrome type II, caused by loss of function mutations in the KCNJ1 gene, is an autosomal recessive life-threatening disorder associated with renal salt wasting, hypotension, and mild hypokalemia [8]. Importantly, heterozygous carriers of ROMK mutations associated with Bartter’s syndrome type II, identified in the Framingham Heart Study, have reduced blood pressure and decreased risk of developing hypertension by age 60 [9]. In addition, ROMK/ mice [10,11] and rats [12] display Bartter’s syndrome type II phenotypes, and Dahl saltsensitive, ROMK+/ heterozygous rats exhibit reduced Current Opinion in Pharmacology 2014, 15:1–6
2 Cardiovascular and renal
Figure 1
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Current Opinion in Pharmacology
Physiological role of ROMK in kidney. ROMK is present at the luminal surface of epithelial cells in two regions of the nephron: the thick ascending limb of Henle (TAHL) and the cortical collecting duct (CCD) where it contributes to salt reuptake and potassium homeostasis.
blood pressure compared to wild-type when challenged with a 4% salt diet, as well as, increased protection from salt-induced blood pressure elevation and from renal injury when challenged with an 8% salt diet, illustrating the important role of ROMK in regulating blood pressure in this model.
electrophysiology and constructing a polarized cell system by growing Madin–Darby canine kidney cells expressing Kir1.1 in permeable Transwell supports are useful for studying Kir1.1 inhibitor mechanisms [18]. Together, these assays can provide valuable information during lead identification and/or optimization of Kir1.1 drug candidates.
Discovery of small molecule ROMK inhibitors Despite substantial genetic evidence, pharmacological validation of the ROMK target is just underway to determine short and long term consequences of Kir1.1 inhibition on renal function and blood pressure. Unlike other ion channel families, molecular pharmacology of Kir1.1, and in general the entire Kir family, is presently quite limited [13]. The only previously disclosed Kir1.1 inhibitor is a bee venom peptide, tertiapin (TPN). TPN is a selective and potent inhibitor of the rat channel [14], but it inhibits human isoform with at least 100-fold reduced potency [15]. In addition, TPN inhibits Kir1.1 by binding to residues at the extracellular surface, which is equivalent to glomerular filtrate, making it a challenging tool for studying ROMK in vivo. For this reason, high-throughput screening (HTS) assays employing heterologous expressed Kir1.1 channels were developed to interrogate large chemical libraries and identify lead small molecules that potentially could be optimized as drug candidates by Medicinal Chemistry. Such assays that function in high density formats, 384-well or 1536-well plates, and support screening >1.5 million samples are based on either membrane potential [16] or thallium flux fluorescence technologies [17,18]. Other assays of intermediate capacity, up to 384-well plates, evaluate channel activity by either automated electrophysiology, or by 86Rb flux, and can be used to support Medicinal Chemistry [18]. The 86Rb flux protocol provides advantage of determining compound potency in absence or presence of up to 100% serum, and is therefore a useful surrogate for more time consuming protein binding measurements. Additionally, manual Current Opinion in Pharmacology 2014, 15:1–6
Using a fluorescence-based thallium flux 384-well assay, a small molecule was identified at Vanderbilt University in a screening campaign of 126 000 molecules. This compound, VU590 (7,13-bis(4-nitrobenzyl)-1,4,10-trioxa7,13-diazacyclopentadecane; 1) [17], and structurally related VU591 (2,20 -oxybis(methylene)bis(5-nitro-1Hbenzo-[d]imidazole); 2), identified later by synthetic exploration of VU590 [19], inhibit rat Kir1.1 with IC50 values of 290 and 240 nM (Figure 2), respectively, and appear to block at the channel’s intracellular pore. Although VU590 has no activity against Kir2.1 and Kir4.1, it inhibits Kir7.1, a channel that is also expressed in kidney. However, VU591 appears to be selective for Kir1.1 over other Kir channels, as well as, other ion channels and receptors. VU591 displays an unfavorable degree of protein binding (>98%) in human and rat serum, but good microsomal stability. Although neither compound has been tested in vivo, VU591 was evaluated in isolated-perfused rat collecting distal tubules. Luminal perfusion with VU591 inhibited net potassium transport with no effect on net sodium transport, suggesting that VU591 inhibits native ROMK in distal nephron that contribute to basal potassium secretion. Independent of the Vanderbilt group, scientists at Merck performed a HTS of Kir1.1 with an internal sample collection (1.5 M compounds) using a membranepotential based fluorescence assay in 1536-well format [16]. A very small number of hits were identified, but one sample, (4-nitrophenethyl)piperazine, was prioritized www.sciencedirect.com
ROMK drug development Garcia and Kaczorowski 3
Figure 2
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Discovery of ROMK inhibitors at Vanderbilt University. Compound 1 was identified by HTS of a chemical library, while compound 2 was derived from 1 by medicinal chemistry investigation.
based on its selectivity against two other Kir channels; cardiac Kir2.1 (whose block would cause serious negative cardiovascular consequences [20]) and kidney Kir2.3. When the screening sample was subjected to re-purification, Kir1.1 inhibitory activity was lost. Subsequently, it was discovered that original sample activity was due to a minor impurity (1%; Figure 3) representing the disubstituted piperazine (3), 1,4-bis(4-nitrophenethyl)piperazine [21]. This finding was confirmed by synthesis. When monitoring activity by either thallium flux or electrophysiology, 3 inhibits human Kir1.1 with IC50 values of 20 and 33 nM, respectively. In 86Rb+ efflux assay, 3 inhibits Kir1.1 with an IC50 of 47 nM (n = 50), and, importantly, its potency is not significantly affected in presence of 100% human serum, suggesting a low degree of serum protein binding. 3 had no significant effect on Kir2.1 or Kir2.3 at concentrations of up to 100 mM, and, other than causing potent inhibition of Kv11.1 (hERG), IC50 of 5 nM, displayed remarkable selectivity against other ion channels, as well as, a broad panel of receptors and enzymes.
Progress in developing ROMK inhibitors as drugs Historically, it has been challenging to identify potent and selective small molecule Kir inhibitors. The general finding is that HTS directed against targets in this family has yielded few interesting or chemically tractable hits to date, impeding drug development. Therefore, it is remarkable that a simple di-substituted piperazine, 3, present as a contaminant, potently blocks Kir1.1 with marked selectivity over other Kir. There may be some www.sciencedirect.com
similarity between 3 and the Kir1.1 VU blockers (Figure 2), discovered independently, since both series contain symmetrical bis-nitrophenyl substituents; whether this feature is related to mechanism of channel inhibition is unclear, but will only be resolved with certainty by co-crystallization of representative inhibitors with Kir1.1. However, the Merck screening lead is not without issues that must be addressed by Medicinal Chemistry: first, aromatic nitro groups are subject to metabolism in vivo which generate both chemically reactive species and agents with carcinogenic potential; second, the lead is 10-fold more potent as a Kv11.1 than Kir1.1 blocker, and this potential hERG activity is a major cardiovascular safety liability [22]; third, symmetrical nature of the lead structure, together with the observation that the mono-substituted piperazine is completely inactive, might limit the ability to introduce chemical diversity into this chemical series. Nonetheless, despite these and other potential issues typically associated with drug development, initial success of Merck Chemistry efforts focused on this lead (Figure 3) have been highlighted recently in three publications [21,23,24] and five patent applications [25–29]. Initial Merck structure–activity relationship (SAR) studies focused on three areas [21]; identifying bioisosteric replacements for nitro groups; modifying linkers between the core piperazine and terminal aromatic substituents; replacement of the core. Since both nitro groups are necessary for maintaining Kir1.1 potency, bioisosteres were introduced into both ends of the molecule, one at a time (Figure 3). Some appropriate replacements were benzonitriles (A), a 5-benzo(2,1,3-oxadiazole) (B) and, interestingly, a 4-phthalide group (C), but several other potential alternatives caused substantial loss of Kir1.1 inhibitory activity. The 4-phthalide substitution was noted to weaken hERG interactions, suggesting that SAR between Kir1.1 and Kv11.1 block might be divergent within this series, and substitution of both nitrophenyl moieties with 4-phthalide groups, 4, maintained Kir1.1 block, while decreasing Kv11.1 inhibition by 20fold, a very encouraging result. Other studies demonstrated that distance between bis-nitro groups is critical for activity, as shortening distance by one carbon abolished channel block completely, while lengthening distance by one carbon also significantly decreased Kir1.1 inhibition. As well, small modifications of the core structure resulted in some loss of Kir1.1 activity. These results, taken together with studies of asymmetrical molecules possessing both 4-phthalide and benzonitrile groups, indicate the following trends: first, it is possible to replace both nitrophenyl groups in the initial lead with either symmetrical or asymmetrical pharmacophore substitutions that are much more suitable for drug development; second, Kir1.1 blocking potency and selectivity over other Kir can be maintained, while at the same time, Kv11.1 inhibition can be reduced; third, overall shape and Current Opinion in Pharmacology 2014, 15:1–6
4 Cardiovascular and renal
Figure 3
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Discovery and development of ROMK inhibitors at Merck. Compound 3 was discoverd by HTS of a very large corporate sample collection, while compounds 4 and 5 were synthesized by Merck medicinal chemistry during SAR investigation of lead 3. Data from both ROMK (Kir1.1) and hERG (Kv11.1), the major off-target liability of this lead series are shown. Compounds A–D are bioisosteric replacements for a nitro group that were used in the Merck SAR studies.
size of the molecule must be maintained to keep Kir1.1 inhibitory potency. The latter result might predict a tight SAR in this series and, interestingly, potency was not significantly increased in initial studies over that of the original lead. However, some analogs prepared had markedly improved pharmacokinetic (PK) properties than those observed with 1,4-bis(4-nitrophenethyl)piperazine [21], another encouraging finding. That Kir1.1 inhibitors can be developed from the initial Merck lead with maintained Kir1.1 potency, but much improved Kv11.1 selectivity, was demonstrated within a novel sub-class of di-substituted piperazines displaying an asymmetric substitution pattern (Figure 3), characterized by a 4-phthalidyl ethyl group as one N-substituent, and a 4-(1H-tetrazol-1-yl)phenyl methyl amide as the other N-substituent [23]. This discovery resulted from attempts to replace the 4-nitrophenyl group with small heterocyclic bioisosteres and noting that a compound Current Opinion in Pharmacology 2014, 15:1–6
containing 4-phenyl tetrazole (D) retained moderate Kir1.1 blocking activity. However, this substitution also resulted in increasing Kv11.1 potency. To generally diminish Kv11.1 blocking activity characteristic of most molecules in this series, an amide substituent was incorporated into one scaffold linker to reduce basicity of the piperazine nitrogens, and while this reduced Kir1.1 potency of molecules containing nitrophenyl, benzonitrile or 4-phthalide pharmacophores, surprisingly it markedly improved the Kir1.1 potency of the phenyl tetrazole containing piperazine, while concurrently diminishing its Kv11.1 blocking activity. This finding was further capitalized on by substituting the other nitrophenyl group with a 4-phthalate substituent, which resulted in even greater selectivity for Kir1.1 over Kv11.1 block. The phenyl-tetrazole moiety appears critical because exploration of many other five-membered heterocycles as tetrazole replacements resulted in significant loss of Kir1.1 potency. Thus, new Kir1.1 blockers with markedly www.sciencedirect.com
ROMK drug development Garcia and Kaczorowski 5
improved properties have been identified [23], as typified by 5-(2-(4-(2-(4-(1H-tetrazol-1yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one, 5; this molecule maintains Kir1.1 inhibitory activity, is selective for Kir1.1 over Kir2.1, Kir2.3, Kir4.1 and Kir7,1, has no off target activity against Nav1.5 and Cav1.2, and has much reduced inhibitory activity, 100-fold, with respect to Kv11.1. It also displays good oral bio-availability with moderate oral exposures, and a short half-life. Oral dosing of 5 in volume loaded Sprague-Dawley (SD) normotensive rats and dogs caused concentration-dependent increase in urinary flow and urinary sodium excretion compared to vehicle treated animals [24]. The magnitude of these effects was comparable to that caused by oral dosing of 25 mg/kg hydrochlorothiazide, a diuretic recommended as first line therapy in treatment of hypertension. Unlike hydrochlorothiazide, however, 5 did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These results strengthen the notion that ROMK inhibitors will be novel diuretic and natriuretic agents with similar efficacy to that of clinically used diuretics, but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics, and are encouraging in terms of supporting future efforts to optimize potency, selectivity, PK and bioactivity of the bis N-substituted piperazine Kir1.1 inhibitor class. Five Merck patent applications [25–29] have published covering approximately 400 specific examples of molecules from the lead series represented by the template: pharmacophore-linker-core-linker-pharmacophore, with pharmacophores primarily containing previously reported nitro bioisosteres in either symmetrical or asymmetrical arrangements. Specific Kir1.1 blocking potencies are only reported for select agents, but some values noted are in low nM range, and IC50’s for all Kir1.1 inhibitors listed are 1 mM. Examples are presented of modification and replacement of the central piperazine core, flexible, rigid and conformationally restricted linker moieties, introduction of stereochemical sites into linker scaffolds, amide and heteroatom containing linkers, and structural modifications to control overall shape of resulting molecules. However, no data are given on important ancillary pharmacology properties, such as Kv11.1 block, so without these comparative results, it is difficult to judge progress that has been made with this lead series based solely on present documentation. There is, however, indication of oral bio-activity of some of these examples. Certain molecules [illustrated in [25]] are reported to display diuretic activity, with twofold to ninefold increase over vehicle treated animals, 4 hours after oral dose to SD rats, at either 1 or 3 mg/kg test compound. In addition, compounds from the same application were reported to lower blood pressure when dosed orally in spontaneous hypertensive rats with telemetry implanted devices that allow continuous monitoring of blood pressure and heart www.sciencedirect.com
rate in freely moving animals. Compounds were dosed once a day for a period of 3–14 days at either 3 or 10 mg/ kg. When compared to vehicle treated animals, specific compounds displayed typical reductions in daily mean systolic blood pressure ranging from 8 to 32 mmHg by last day of study.
Conclusions It is an exciting period as pharmacology of the ROMK target is finally being characterized. Questions regarding physiologic responses elicited by chronic treatment with Kir1.1 inhibitors and their effects on blood pressure, either alone, or in combination with other anti-hypertensive drugs [30], may soon be answered. Moreover, evaluation of therapeutic potential of Kir1.1 blockers in humans with hypertension or CHF will gauge the predictive value of the human genetic phenotypes. Hopefully, small molecule Kir1.1 inhibitors currently under development will be instrumental in addressing all these issues.
Acknowledgement We thank Dr. Jed Fisher for help with preparing the chemical structure’s art work.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Simon DB, Karet FE, Rodrguez-Soriano J, Hamdan JH, DiPietro A, Trachtman H, Sanjad SA, Lifton RP: Genetic heterogeneity of Bartter’s syndrome revealed by mutations in the K+ channel, ROMK. Nat Genet 1996, 14:152-156.
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10. Lorenz JN, Baird NR, Judd LM, Noonan WT, Andringa A, Doetschman T, Manning PA, Liu LH, Miller ML, Shull GE: Impaired renal NaCl absorption in mice lacking the ROMK potassium channel, a model for type II Bartter’s syndrome. J Biol Chem 2002, 277:37871-37880. Current Opinion in Pharmacology 2014, 15:1–6
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11. Lu M, Wang T, Yan Q, Yang X, Dong K, Knepper MA, Wang W, Giebisch G, Shull GE, Hebert SC: Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter’s) knockout mice. J Biol Chem 2002, 277:37881-37887. 12. Zhou X, Zhang Z, Shin MK, Horwitz SB, Levorse JM, Zhu L, Sharif Rodriguez W, Streltsov DY, Dajee M, Hernandez M et al.: Heterozygous disruption of renal outer medullary potassium channel in rats is associated with reduced blood pressure. Hypertension 2013, 62:288-294. Using a ROMK knockout rat model with Dahl salt-sensitive background, authors demonstrate that ROMK plays a critical role in blood pressure regulation. This is the first illustration that ROMK/+ rats recapitulate the blood pressure phenotype of heterozygous humans carrying ROMK mutations associated with Bartter’s syndrome type II, identified in the Framingham Heart Study. 13. Wulff H, Zhorov BS: K+ channel modulators for the treatment of neurological disorders and autoimmune diseases. Chem Rev 2008, 108:1744-1773. 14. Jin W, Lu Z: A novel high-affinity inhibitor for inward-rectifier K+ channels. Biochemistry 1998, 37:13291-13299. 15. Felix JP, Liu J, Schmalhofer WA, Bailey T, Bednarek MA, Kinkel S, Weinglass AB, Kohler M, Kaczorowski GJ, Priest BT, Garcia ML: Characterization of Kir1.1 channels with the use of a radiolabeled derivative of tertiapin. Biochemistry 2006, 45:10129-10139. 16. Solly K, Cassaday J, Felix JP, Garcia ML, Ferrer M, Strulovici B, Kiss L: Miniaturization and HTS of a FRET-based membrane potential assay for Kir channel inhibitors. Assay Drug Dev Technol 2008, 6:225-234. 17. Lewis LM, Bhave G, Chauder BA, Banerjee S, Lornsen KA, Redha R, Fallen K, Lindsley CW, Weaver CD, Denton JS: Highthroughput screening reveals a small-molecule inhibitor of the renal outer medullary potassium channel and Kir7.1. Mol Pharmacol 2009, 76:1094-1103. 18. Felix JP, Priest BT, Solly K, Bailey T, Brochu RM, Liu CJ, Kohler MG, Kiss L, Alonso-Galicia M, Tang H et al.: The inwardly rectifying potassium channel Kir1.1: development of functional assays to identify and characterize channel inhibitors. Assay Drug Dev Technol 2012, 10:417-431. A wide variety of Kir1.1 functional assays that can support evaluation of large chemical libraries (>1.5 M) to identify leads and assist Medicinal Chemsitry studies during lead optimization, as well as, aid mechanism of action studies are described and validated. 19. Bhave G, Chauder BA, Liu W, Dawson ES, Kadakia R, Nguyen TT, Lewis LM, Meiler J, Weaver CD, Satlin LM et al.: Development of a selective small-molecule inhibitor of Kir1.1, the renal outer medullary potassium channel. Mol Pharmacol 2011, 79:42-50. The first selective Kir1.1 inhibitor, VU591, is revealed. Using isolatedperfused rat collecting distal tubules, VU591 inhibits net potassium transport with no effect on net sodium transport, suggesting that VU591 inhibits native ROMK channels in the distal nephron that contribute to basal potassium secretion.
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20. Dhamoon AS, Jalife J: The inward rectifier current (IK1) controls cardiac excitability and is involved in arrhythmogenesis. Heart Rhythm 2005, 2:316-324. 21. Tang H, Walsh SP, Yan Y, de Jesus RK, Shahripour A, Teumelsan N, Zhu Y, Ha S, Owens KA, Thomas-Fowlkes B et al.: Discovery of selective small molecule ROMK inhibitors as potential new mechanism diuretics. ACS Med Chem Lett 2012, 3:367-372. First SAR study of the Merck potent, di-substituted piperazine ROMK inhibitor screening lead which reveals discovery of analogs with potent ROMK blocking activity and selectivity over other Kir channels, as well as, improved selectivity against Kv11.1 and improved PK properties over that of the original parent compound. 22. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, Keating MT: A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 1995, 80:795-803. 23. Tang H, de Jesus RK, Walsh SP, Zhu Y, Yan Y, Priest BT, Swensen AM, Alonso-Galicia M, Felix JP, Brochu RM et al.: Discovery of a novel sub-class of ROMK channel inhibitors typified by 5-(2-(4-(2-(4-(1H-tetrazol-1yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)one. Bioorg Med Chem Lett 2013, 23:5829-5832. A sub-class of ROMK inhibitor developed from the original Merck screening lead with good ROMK functional activity and improved hERG selectivity is revealed; two members of this class are used to demonstrate for the first time in vivo proof of concept that ROMK blockers represent a new mechanism diuretic and natriuretic agent. 24. Garcia ML, Priest BT, Alonso-Galicia M, Zhou X, Felix JP, Brochu RM, Bailey T, Thomas-Fowlkes B, Liu J, Swensen A et al.: Pharmacological inhibition of the renal outer medullary potassium channel causes diuresis and natriuresis in the absence of kaliuresis. J Pharmacol Exp Therap 2013 http:// dx.doi.org/10.1124/jpet.113.208603. First demonstration that in vivo dosing of a selective ROMK inhibitor to normotensive rats and dogs cause concentration-dependent increases in urinary flow and urinary sodium excretion which are comparable to hydrochlorothiazide, but however unlike hydrochlorothiazide, these changes are not associated with potassium losses or changes in plasma electrolyte levels. 25. Merck & Co. Inhibitors of the renal outer medullary potassium channel, US Patent Application: US2010/0286123A1; 2010 26. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2011/057346; 2011 27. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2012/054354; 2012 28. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2012/061274; 2012 29. Merck & Co. Inhibitors of the renal outer medullary potassium channel, WIPO Patent Application: PCT/US2012/061896; 2012 30. Sood N, Reinhart KM, Baker WL: Combination therapy for the management of hypertension: a review of the evidence. Am J Health Syst Pharm 2010, 67:885-894.
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