Chemical library screening for WNK signaling inhibitors by using fluorescent correlation spectroscopy

With-no-lysine kinases (WNK) are the causative genes of a hereditary hypertensive disease, pseudohypoaldosteronism type II (PHA II), and form a signal cascade with oxidative stress-responsive 1 (OSR1)/STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and Slc12a transporters. We have shown that this signal cascade regulates blood pressure by controlling vascular tone as well as renal NaCl excretion. Therefore, agents that inhibit this signal cascade could be a new class of antihypertensive drugs. Since the binding of WNK to OSR1/SPAK kinases was postulated to be important for signal transduction, we sought to discover inhibitors of WNK-SPAK binding by screening chemical compounds that disrupt the binding. For this purpose, we developed a high-throughput screening method using fluorescent correlation spectroscopy. As a result of screening 17,000 compounds, we discovered two novel compounds that reproducibly disrupted the binding of WNK to SPAK. Both compounds mediated dose-dependent inhibition of hypotonicity-induced activation of WNK, namely the phosphorylation of SPAK and its downstream transporters NKCC1 and NCC in cultured cell lines. The two compounds could be the promising seeds of new types of antihypertensive drugs, and the method that we developed could be applied as a general screening method to identify compounds that disrupt the binding of two molecules.
Source: BJ Signal - Category: Biochemistry Authors: Tags: BJ ChemBio Source Type: research