title: Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy.
abstract: WNKs (with-no-lysine kinases) are the causative genes of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), and form a signal cascade with OSR1 (oxidative stress-responsive 1)/SPAK (STE20/SPS1-related proline/alanine-rich protein kinase) and Slc12a (solute carrier family 12) 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 17000 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 (Na/K/Cl cotransporter 1) and NCC (NaCl cotransporter) 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.
authors: *Department of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan.
authors: Takayasu
authors: T
authors: Mori
authors: Eriko
authors: E
authors: Kikuchi
authors: Yuko
authors: Y
authors: Watanabe
authors: Shinya
authors: S
authors: Fujii
authors: Mari
authors: M
authors: Ishigami-Yuasa
authors: Hiroyuki
authors: H
authors: Kagechika
authors: Eisei
authors: E
authors: Sohara
authors: Tatemitsu
authors: T
authors: Rai
authors: Sei
authors: S
authors: Sasaki
authors: Shinichi
authors: S
authors: Uchida
mesh_terms: D000818:Animals; D000959:Antihypertensive Agents / Q000737:chemistry / Q000494:pharmacology; D001794:Blood Pressure / Q000502:physiology; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D064506:Solute Carrier Family 12, Member 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D000959:Antihypertensive Agents; D064506:Solute Carrier Family 12, Member 2; C116037:PAS domain kinases; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human