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.
Department of Nephrology, Graduate School of Medical and Dental Sciences.
Eriko
E
Kikuchi
Department of Nephrology, Graduate School of Medical and Dental Sciences.
Takayasu
T
Mori
Department of Nephrology, Graduate School of Medical and Dental Sciences.
Moko
M
Zeniya
Department of Nephrology, Graduate School of Medical and Dental Sciences.
Kiyoshi
K
Isobe
Chemical Biology Screening Center, and.
Mari
M
Ishigami-Yuasa
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan; and.
Shinya
S
Fujii
Chemical Biology Screening Center, and Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan; and.
Hiroyuki
H
Kagechika
Department of Analytical Chemistry, Faculty of Pharmacy, Keio University, Tokyo, Japan.
Tomoaki
T
Ishihara
Department of Analytical Chemistry, Faculty of Pharmacy, Keio University, Tokyo, Japan.
Tohru
T
Mizushima
Department of Nephrology, Graduate School of Medical and Dental Sciences.
Sei
S
Sasaki
Department of Nephrology, Graduate School of Medical and Dental Sciences.
Eisei
E
Sohara
Department of Nephrology, Graduate School of Medical and Dental Sciences.
Tatemitsu
T
Rai
Department of Nephrology, Graduate School of Medical and Dental Sciences, suchida.kid@tmd.ac.jp.
Shinichi
S
Uchida
D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006973:Hypertension / Q000188:drug therapy*; D017136:Ion Transport / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D015778:Minor Histocompatibility Antigens; D010766:Phosphorylation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011897:Random Allocation; D012458:Salicylanilides / Q000494:pharmacology; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000235:genetics; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D000076204:WNK Lysine-Deficient Protein Kinase 1
D000959:Antihypertensive Agents; D015778:Minor Histocompatibility Antigens; D012458:Salicylanilides; D028021:Sodium-Potassium-Chloride Symporters; C116037:PAS domain kinases; D017346:Protein Serine-Threonine Kinases; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498744:Wnk1 protein, mouse; C023342:closantel
cell physiology; cell signaling; hypertension; transport physiology