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The renal outer medullary potassium (ROMK) channel, located at the apical surface of epithelial cells in the thick ascending loop of Henle and cortical collecting duct, contributes to salt reabsorption and potassium secretion, and represents a target for the development of new mechanism of action diuretics. This idea is supported by the phenotype of antenatal Bartter's syndrome type II associated with loss-of-function mutations in the human ROMK channel, as well as, by cardiovascular studies of heterozygous carriers of channel mutations associated with type II Bartter's syndrome. Although the pharmacology of ROMK channels is still being developed, channel inhibitors have been identified and shown to cause natriuresis and diuresis, in the absence of any significant kaliuresis, on acute oral dosing to rats or dogs. Improvements in potency and selectivity have led to the discovery of MK-7145 [5,5'-((1R,1'R)-piperazine-1,4-diylbis(1-hydroxyethane-2,1-diyl))bis(4-methylisobenzofuran-1(3H)-one)], a potential clinical development candidate. In spontaneously hypertensive rats, oral dosing of MK-7145 causes dose-dependent lowering of blood pressure that is maintained during the entire treatment period, and that displays additive/synergistic effects when administered in combination with hydrochlorothiazide or candesartan, respectively. Acute or chronic oral administration of MK-7145 to normotensive dogs led to dose-dependent diuresis and natriuresis, without any significant urinary potassium losses or changes in plasma electrolyte levels. Elevations in bicarbonate and aldosterone were found after 6 days of dosing. These data indicate that pharmacological inhibition of ROMK has potential as a new mechanism for the treatment of hypertension and/or congestive heart failure. In addition, Bartter's syndrome type II features are manifested on exposure to ROMK inhibitors., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Considerable attention has focused on regulation of central tryptophan hydroxylase (TPH) activity and protein expression. At the time of these earlier studies, it was thought that there was a single central TPH isoform. However, with the recent identification of TPH2, it becomes important to distinguish between regulatory effects on the protein expression and activity of the two isoforms. We have generated a TPH2-specific polyclonal antiserum (TPH2-6361) to study regulation of TPH2 at the protein level and to examine the distribution of TPH2 expression in rodent and human brain. TPH2 immunoreactivity (IR) was detected throughout the raphe nuclei, in lateral hypothalamic nuclei and in the pineal body of rodent and human brain. In addition, a prominent TPH2-IR fiber network was found in the human median eminence. We recently reported that glucocorticoid treatment of C57/Bl6 mice for 4 days markedly decreased TPH2 messenger RNA levels in the raphe nuclei, whereas TPH1 mRNA was unaffected. The glucocorticoid-elicited inhibition of TPH2 gene expression was blocked by co-administration of the glucocorticoid receptor antagonist mifepristone (RU-486). Using TPH2-6361, we have extended these findings to show a dose-dependent decrease in raphe TPH2 protein levels in response to 4 days of treatment with dexamethasone; this effect was blocked by co-administration of mifepristone. Moreover, the glucocorticoid-elicited inhibition of TPH2 was functionally significant: serotonin synthesis was significantly reduced in the frontal cortex of glucocorticoid-treated mice, an effect that was blocked by mifepristone co-administration. This study provides further evidence for the glucocorticoid regulation of serotonin biosynthesis via inhibition of TPH2 expression, and suggest that elevated glucocorticoid levels may be relevant to the etiology of psychiatric diseases, such as depression, where hypothalamic-pituitary-adrenal axis dysregulation has been documented.