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: Takayasu Mori, Eriko Kikuchi, Yuko Watanabe, Shinya Fujii, Mari Ishigami-Yuasa, Hiroyuki Kagechika, Eisei Sohara, Tatemitsu Rai, Sei Sasaki, Shinichi Uchida
• Journal: The Biochemical journal (Biochem J) Vol. 455 Issue 3 Pg. 339-45 (Nov 01 2013) ISSN: 1470-8728 [Electronic] England
• PMID: 23981180 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antihypertensive Agents
• Solute Carrier Family 12, Member 2
• PAS domain kinases
• Protein Serine-Threonine Kinases
• STK39 protein, human

Topics

• Animals
• Antihypertensive Agents (chemistry, pharmacology)
• Blood Pressure (physiology)
• Fluorescence
• High-Throughput Screening Assays
• Humans
• Phosphorylation
• Protein Serine-Threonine Kinases (genetics, metabolism)
• Rats
• Signal Transduction
• Solute Carrier Family 12, Member 2 (genetics, metabolism)