Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl(-) cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.