Kasper Lykke1, Kathrin Töllner2,3, Kerstin Römermann2,3, Peter W Feit2, Thomas Erker4, Nanna MacAulay1, Wolfgang Löscher2,3. 1. Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark. 2. Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany. 3. Center for Systems Neuroscience, Hannover, Germany. 4. Department of Medicinal Chemistry, University of Vienna, Vienna, Austria.
Abstract
BACKGROUND AND PURPOSE: The N-K-Cl cotransporters (NKCCs) mediate the coupled, electroneutral movement of Na+ , K+ and Cl- ions across cell membranes. There are two isoforms of this cation co-transporter, NKCC1 and NKCC2. NKCC2 is expressed primarily in the kidney and is the target of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure-activity studies on effects of bumetanide derivatives on NKCC2 are not available. EXPERIMENTAL APPROACH: In this study, the effect of a series of diuretically active bumetanide derivatives was investigated on human NKCC2 variant A (hNKCC2A) expressed in Xenopus laevis oocytes. KEY RESULTS: Bumetanide blocked hNKCC2A transport with an IC50 of 4 μM. There was good correlation between the diuretic potency of bumetanide and its derivatives in dogs and their inhibition of hNKCC2A (r2 = 0.817; P < 0.01). Replacement of the carboxylic group of bumetanide by a non-ionic residue, for example, an anilinomethyl group, decreased inhibition of hNKCC2A, indicating that an acidic group was required for transporter inhibition. Exchange of the phenoxy group of bumetanide for a 4-chloroanilino group or the sulfamoyl group by a methylsulfonyl group resulted in compounds with higher potency to inhibit hNKCC2A than bumetanide. CONCLUSIONS AND IMPLICATIONS: The X. laevis oocyte expression system used in these experiments allowed analysis of the structural requirements that determine relative potency of loop diuretics on human NKCC2 splice variants, and may lead to the discovery of novel high-ceiling diuretics.
BACKGROUND AND PURPOSE: The N-K-Cl cotransporters (NKCCs) mediate the coupled, electroneutral movement of Na+ , K+ and Cl- ions across cell membranes. There are two isoforms of this cation co-transporter, NKCC1 and NKCC2. NKCC2 is expressed primarily in the kidney and is the target of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure-activity studies on effects of bumetanide derivatives on NKCC2 are not available. EXPERIMENTAL APPROACH: In this study, the effect of a series of diuretically active bumetanide derivatives was investigated on humanNKCC2 variant A (hNKCC2A) expressed in Xenopus laevis oocytes. KEY RESULTS:Bumetanide blocked hNKCC2A transport with an IC50 of 4 μM. There was good correlation between the diuretic potency of bumetanide and its derivatives in dogs and their inhibition of hNKCC2A (r2 = 0.817; P < 0.01). Replacement of the carboxylic group of bumetanide by a non-ionic residue, for example, an anilinomethyl group, decreased inhibition of hNKCC2A, indicating that an acidic group was required for transporter inhibition. Exchange of the phenoxy group of bumetanide for a 4-chloroanilino group or the sulfamoyl group by a methylsulfonyl group resulted in compounds with higher potency to inhibit hNKCC2A than bumetanide. CONCLUSIONS AND IMPLICATIONS: The X. laevis oocyte expression system used in these experiments allowed analysis of the structural requirements that determine relative potency of loop diuretics on humanNKCC2 splice variants, and may lead to the discovery of novel high-ceiling diuretics.