Development of SPAK kinase inhibitors

• Project/Area Number: 25670406
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Kidney internal medicine
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: UCHIDA SHINICHI 東京医科歯科大学, 医歯(薬)学総合研究科, 教授 (50262184)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2013: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 高血圧 / 創薬 / キナーゼ / ケミカルライブラリー / キナーゼ阻害薬

Outline of Final Research Achievements

WNK- STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)-SLC12a transporters cascade regulates blood pressure through NaCl reabsorption in kidney and vasoconstriction. Therefore, drugs that inhibit this signal cascade could become new antihypertensive drugs that have dual effects as a diuretic and vasodilator. We sought to discover SPAK kinase inhibitors by screening chemical compounds and existing drugs. We developed a new screening system using enzyme-linked immunosorbent assay (ELISA) and screened over 20,000 small-molecule compounds. As a result of this screening effort, we discovered one small-molecule compound and Closantel, an antiparasitic agent, which inhibited SPAK-regulated NCC and NKCC1 phosphorylation and activation not only in vitro but also in cultured cell lines and in mice.

Research Products

• [Journal Article] Discovery of novel SPAK inhibitors that block WNK kinase signaling to cation chloride transporters (2015)
  • Author(s): Kikuchi E, Mori T, Zeniya M, Isobe K, Ishigami-Yuasa M, Fujii S, Kagechika H, Ishihara T, Mizushima T, Sasaki S, Sohara E, Rai T, Uchida S
  • Journal Title: J. Am. Soc. Nephrol. Volume: 26 Pages: 1525-1536
  • DOI: 10.1681/asn.2014060560
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Kelch-Like Protein 2 Mediates Angiotensin II-With No Lysine 3 Signaling in the Regulation of Vascular Tonus. (2015)
  • Author(s): Zeniya M, Morimoto N, Takahashi D, Mori Y, Mori T, Ando F, Araki Y, Yoshizaki Y, Inoue Y, Isobe K, Nomura N, Oi K, Nishida H, Sasaki S, Sohara E, Rai T, Uchida S
  • Journal Title: J Am Soc Nephrol. Volume: 26 Pages: 2129-2138
  • DOI: 10.1681/asn.2014070639
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Aberrant glycosylation and localization of polycystin-1 cause polycystic kidney in an AQP11 knockout model (2014)
  • Author(s): Inoue Y, Sohara E, Kobayashi K, Chiga M, Rai T, Ishibashi K, Horie S, Su X, Zhou J, Sasaki S, Uchida S.
  • Journal Title: J Am Soc Nephrol Volume: 25 Pages: 2789-2799
  • DOI: 10.1681/asn.2013060614
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice. (2014)
  • Author(s): Susa K, Sohara E, Rai T, Zeniya M, Mori Y, Mori T, Chiga M, Nomura N, Nishida H, Takahashi D, Isobe K, Inoue Y, Takeishi K, Takeda N, Sasaki S, Uchida S
  • Journal Title: Hum Mol Genet. Volume: 23 (19) Pages: 5052-5060
  • DOI: 10.1093/hmg/ddu217
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] WNK4 is the major WNK positively regulating NCC in the mouse kidney. (2014)
  • Author(s): Takahashi D, Mori T, Nomura N, Khan MZ, Araki Y, Zeniya M, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Biosci Rep. Volume: 34 (3) Pages: 195-205
  • DOI: 10.1042/bsr20140047
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Regulation of with-no-lysine kinase signaling by Kelch-like proteins. (2014)
  • Author(s): Uchida S, Sohara E, Rai T, Sasaki S
  • Journal Title: Biol Cell. Volume: 106(2) Pages: 45-56
  • DOI: 10.1111/boc.201300069
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Regulation of blood pressure and renal electrolyte balance by Cullin-RING ligases. (2014)
  • Author(s): Uchida S.
  • Journal Title: Curr Opin Nephrol Hypertens. Volume: 23 Pages: 487-493
  • DOI: 10.1097/mnh.0000000000000049
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] SPAK deficiency corrects pseudohypoaldosteronism II caused by WNK4 mutation. (2013)
  • Author(s): Chu PY, Cheng CJ, Wu YC, Fang YW, Chau T, Uchida S, Sasaki S, Yang SS, Lin SH.
  • Journal Title: PLoS One Volume: 8
  • DOI: 10.1371/journal.pone.0072969
  • Peer Reviewed
• [Journal Article] Development of enzyme-linked immunosorbent assays for urinary thiazide-sensitive Na-Cl cotransporter (NCC) measurement. (2013)
  • Author(s): Isobe K, Mori T, Asano T, Kawaguchi H, Nonoyama S, Kumagai N, Kamada F, Morimoto T, Hayashi M, Sohara E, Rai T, Sasaki S, Uchida S.
  • Journal Title: Am J Physiol Renal Physiol. Volume: 305
  • Peer Reviewed
• [Journal Article] Dietary salt intake regulates WNK3-SPAK-NKCC1 phosphorylation cascade in mouse aorta through angiotensin II. (2013)
  • Author(s): Zeniya M, Sohara E, Kita S, Iwamoto T, Susa K, Mori T, Oi K, Chiga M, Takahashi D, Yang SS, Lin SH, Rai T, Sasaki S, Uchida S
  • Journal Title: Hypertension Volume: 62(5) Pages: 872-8
  • DOI: 10.1161/hypertensionaha.113.01543
  • Peer Reviewed
• [Journal Article] Chemical library screening for WNK signalling inhibitors using fluorescence correlation spectroscopy (2013)
  • Author(s): Mori, T.; Kikuchi, E.; Watanabe, Y.; Fujii, S.; Ishigami-Yuasa, M.; Kagechika, H.; Sohara, E.; Rai, T.; Sasaki, S.; Uchida, S.
  • Journal Title: Biochem J Volume: 455 Pages: 339-345
  • DOI: 10.1042/bj20130597
  • Peer Reviewed
• [Journal Article] Decrease of WNK4 ubiquitination by disease-causing mutations of KLHL3 through different molecular mechanisms. (2013)
  • Author(s): Mori Y, Wakabayashi M, Mori T, Araki Y, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Biochem Biophys Res Commun. Volume: 439(1) Pages: 30-4
  • DOI: 10.1016/j.bbrc.2013.08.035
  • Peer Reviewed
• [Journal Article] KLHL2 interacts with and ubiquitinates WNK kinases. (2013)
  • Author(s): Takahashi D, Mori T, Wakabayashi M, Mori Y, Susa K, Zeniya M, Sohara E, Rai T, Sasaki S, Uchida S
  • Journal Title: Biochem Biophys Res Commun. Volume: 437(3) Pages: 457-62
  • DOI: 10.1016/j.bbrc.2013.06.104
  • Peer Reviewed
• [Journal Article] Phosphorylation regulates NCC stability and transporter activity in vivo. (2013)
  • Author(s): Yang SS, Fang YW, Tseng MH, Chu PY, Yu IS, Wu HC, Lin SW, Chau T, Uchida S, Sasaki S, Lin YF, Sytwu HK, Lin SH.
  • Journal Title: J Am Soc Nephrol. Volume: 24 Pages: 1587-97
  • DOI: 10.1681/asn.2012070742
  • Peer Reviewed
• [Journal Article] Immunolocalization and translocation of aquaporin-5 water channel in sweat glands (2013)
  • Author(s): Inoue R, Sohara E, Rai T, Satoh T, Yokozeki H, Sasaki S, Uchida S.
  • Journal Title: J Dermatol Sci Volume: 70 Pages: 26-33
  • DOI: 10.1016/j.jdermsci.2013.01.013
  • Peer Reviewed
• [Journal Article] Impaired KLHL3-Mediated Ubiquitination of WNK4 Causes Human Hypertension. (2013)
  • Author(s): Wakabayashi M
  • Journal Title: Cell Reports. Volume: 28 Pages: 858-68
  • DOI: 10.1016/j.celrep.2013.02.024
  • Peer Reviewed
• [Journal Article] Nomura N, Kamiya K, Ikeda K, Yui N, Chiga M, Sohara E, Rai T, Sakaki S, Uchida S Treatment with 17-allylamino-17-demethoxygeldanamycin ameliorated symptoms of Bartter syndrome type IV caused by mutated Bsnd in mice. (2013)
  • Author(s): Nomura N, Kamiya K, Ikeda K, Yui N, Chiga M, Sohara E, Rai T, Sasaki S, Uchida S.
  • Journal Title: Biochem Biophys Res Commun Volume: 441 Pages: 544-49
  • DOI: 10.1016/j.bbrc.2013.10.129
  • Peer Reviewed
• [Presentation] Impaired KLHL3-Mediated Ubiquitination of WNK4 Causes Human Hypertension. (2014)
  • Author(s): Uchida S.
  • Organizer: The 47th Annual Meeting of American Society of Nephrology
  • Place of Presentation: フィラデルフィア、米国
  • Year and Date: 2014-11-13 – 2014-11-16
  • Invited
• [Presentation] Clinical Significance of Urinary Thiazide-Sensitive Na-Cl Cotransporter (NCC) Measurement by Newly Development Enzyme-Linked Immunosorbent Assays. (2013)
  • Author(s): Isobe K, Mori T, Sohara E, Rai T, Sasaki S, Uchida S.
  • Organizer: The 46th Annual meeting of American Society of Nephrology
  • Place of Presentation: アトランタ, アメリカ合衆国
• [Presentation] Impaired KLHL3-mediated ubiquitination of WNK4 activates OSR1 and SPAK kinases-NaCl cotransporter (NCC) signaling and causes hypertension. (2013)
  • Author(s): Wakabayashi M, Mori T, Isobe K, Sohara E, Susa K, Araki Y, Chiga M, Kikuchi E, Nomura N, Mori Y, Rai T, Sasaki S, Uchida S.
  • Organizer: The 46th Annual meeting of American Society of Nephrology
  • Place of Presentation: アトランタ, アメリカ合衆国
• [Presentation] KLHL2 interacts with and ubiquitinates WNK kinases. (2013)
  • Author(s): Takahashi D, Mori T, Wakabayashi M, Mori Y, Susa K, Zeniya M, Sohara E, Rai T, Sasaki S, Uchida S.
  • Organizer: The 46th Annual meeting of American Society of Nephrology
  • Place of Presentation: アトランタ, アメリカ合衆国
• [Presentation] WNK3 Regulates Blood Pressure Through the Regulation of Vascular OSR1/SPAK-NKCC1 Phosphorylation Cascade. (2013)
  • Author(s): Zeniya M, Sohara E, Kita S, Iwamoto T, Susa K, Mori T, Oi K, Chiga M, Takahashi D, Yang SS, Lin SH, Rai T, Sasaki S, Uchida S.
  • Organizer: The 46th Annual meeting of American Society of Nephrology
  • Place of Presentation: アトランタ, アメリカ合衆国
• [Presentation] Chemical library screening for WNK signaling inhibitors by using fluorescent correlation spectroscopy. (2013)
  • Author(s): Mori T, Eriko K, Sohara E, Rai T, Sasaki S, Uchida S.
  • Organizer: The 46th Annual meeting of American Society of Nephrology
  • Place of Presentation: アトランタ, アメリカ合衆国
• [Remarks] 東京医科歯科大学腎臓内科学
  • URL: http://www.tmd.ac.jp/grad/kid/kid-J.htm