Preclinical validation of Kir4.1/5.1 inhibitors for overcoming diuretic resistance

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SUMMARY Edema is a common, life-threatening consequence of congestive heart failure (HF), chronic kidney disease (CKD), and chronic liver disease. Loop diuretics are often used as a first-line therapy to quickly reduce the fluid volume burden in HF, CKD, and liver failure patients. This class of diuretic works by inhibiting NaCl reabsorption in the thick ascending limb (TAL) of Henle’s loop and increasing the delivery of NaCl to the distal convoluted tubule (DCT) and cortical collecting duct (CCD) comprising the distal nephron. In response to the increased NaCl load, the DCT and CCD increase their NaCl reabsorption capacity through distal tubule remodeling involving cell/tissue hypertrophy and by upregulating the expression of ion transporters and channels involved in NaCl reabsorption. This compensatory mechanism diminishes the effectiveness of loop diuretics and gives rise to loop diuretic resistance, which is a common clinical problem in the treatment of HF, CKD, and liver failure. A growing consensus is that distally acting diuretics that inhibit sodium (Na+) reabsorption in the DCT (i.e., thiazide diuretics) or CCD (i.e., amiloride) downstream of the TAL should be administered to overcome loop diuretic resistance. However, both diuretic classes have serious liabilities that highlight the need for more effective, safer, and novel- mechanism distal nephron-targeted diuretics. Heteromeric Kir4.1/5.1 inward rectifier potassium (Kir) channels have emerged over the last decade as potential distal nephron diuretic targets for two main reasons. First, these basolateral membrane channels are expressed in the DCT and CCD and are essential for Na+ reabsorption in both nephron segments. Secondly, and importantly, the loss of Kir4.1/5.1 function in patients with SeSAME/EAST syndrome leads to renal salt wasting and low blood pressure, providing strong genetic validation for Kir4.1/5.1 as a diuretic target in humans. We recently performed an NIH-funded (R01DK120821; Denton PI) high-throughput screen (HTS) of 80,475 compounds from the Vanderbilt Institute of Chemical Biology Discovery Collection and identified hundreds of structurally diverse small-molecule inhibitors of Kir4.1/5.1. We employed iterative medicinal chemistry and functional analysis to drive the development of the first-in-class, moderately potent (IC50 = 0.24 µM), highly selective (>30 fold over 9 related Kir channels), in vitro inhibitor of Kir4.1/5.1, named VU6036720 (PMID 35246480). Unfortunately, however, VU6036720 failed to induce a diuretic response in mice due to high plasma protein binding and rapid metabolic clearance. In Aim 1 of this follow-up application, we propose to employ medicinal chemistry and established functional assays to further optimize the potency, selectivity, and drug metabolism and pharmacokinetic (DMPK) properties of VU6036720 and other backup scaffolds identified in our HTS campaign. In Aim 2, we will evaluate the in vivo activity of optimized inhibitors in mouse models of diuretic resistance and CKD. Completion of this program will provide pre-clinical validation of Kir4.1/5.1 as a viable diuretic target, generally, and for circumventing complications associated with diuretic resistance, specifically.