Development of novel hypertensive drugs based on inhibition of protein interaction

• Project/Area Number: 24659412
• 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: RAI Tatemitsu 東京医科歯科大学, 医歯(薬)学総合研究科, 准教授 (80334431)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2012: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• Keywords: WNKキナーゼ / OSR1 / SPAK / SLC12A輸送体 / NCC / NKCC1 / NKCC2 / 蛍光相関分光法 / Slc12a輸送体 / ELISA

Outline of Final Research Achievements

The WNK (with-no-lysine kinase) phosphorylation signal cascade plays an important role in blood pressure control through regulation of NaCl reabsorption and vasoconstriction. Therefore, agents that can modulate this signal cascade could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we organized two high-throughput drug-screening systems to find novel specific inhibitors of the WNK - OSR1 / SPAK - Slc12a cascade. First, fluorescent correlation spectroscopy was employed to detect inhibition of the binding of WNK and SPAK. Next, a new ELISA-based screening system was developed to find drugs that inhibit SPAK activity. As a result of screening over 20,000 compounds by these two methods, we discovered several compounds that could inhibit the WNK cascade activation, namely the activation of Slc12a transporters in vitro and in mice. These compounds could be promising seeds of new types of antihypertensive drugs.

Research Products

• [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 Issue: 9 Pages: 2129-2138
  • DOI: 10.1681/asn.2014070639
  • Peer Reviewed
• [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
  • Peer Reviewed
• [Journal Article] WNK4 is the major WNK kinase 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) Issue: 3 Pages: 195-205
  • DOI: 10.1042/bsr20140047
  • Peer Reviewed / Open Access
• [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) Issue: 19 Pages: 5052-60
  • DOI: 10.1093/hmg/ddu217
  • Peer Reviewed
• [Journal Article] Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters (2014)
  • 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 Issue: 7 Pages: 1525-1536
  • DOI: 10.1681/asn.2014060560
  • 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) Issue: 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 Issue: 3 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) Issue: 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) Issue: 3 Pages: 457-62
  • DOI: 10.1016/j.bbrc.2013.06.104
  • Peer Reviewed
• [Journal Article] Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension. (2013)
  • Author(s): Wakabayashi M
  • Journal Title: Cell Rep. Volume: 28 Issue: 3 Pages: 858-868
  • DOI: 10.1016/j.celrep.2013.02.024
  • Peer Reviewed
• [Journal Article] Effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels. (2012)
  • Author(s): Susa K
  • Journal Title: Clin Exp Nephrol. Volume: 16(4) Pages: 530-8
  • Peer Reviewed
• [Presentation] KLHL2 Mediates Angiotensin II-WNK3 Signaling Involved in the Regulation of Vascular Tonus. (2014)
  • Author(s): Zeniya M, Takahashi D, Mori Y, Mori T, Ando F, Nomura N, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2014
  • Place of Presentation: Philadelphia, USA
  • Year and Date: 2014-11-13 – 2014-11-15
• [Presentation] Molecular Pathogenesis of PHAII in KLHL3R528H/+ Knock-In Mice (2014)
  • Author(s): Susa K, Sohara E, Tatemitsu Rai T, Zeniya M, Mori Y, Takayasu Mori T, Chiga M, Takahashi D, Inoue Y, Isobe K, Takeda N, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2014
  • Place of Presentation: Philadelphia, USA
  • Year and Date: 2014-11-13 – 2014-11-15
• [Presentation] Phosphorylation of KLHL3 in the Kelch-Repeat Regulates Its Binding Ability to WNK4 (2014)
  • Author(s): Yoshizaki Y, Sohara E, Mori T, Mori Y, Wakabayashi M, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2014
  • Place of Presentation: Philadelphia, USA
  • Year and Date: 2014-11-13 – 2014-11-15
• [Presentation] p62-Mediated Selective Autophagy Is Involved in KLHL3-Dependent WNK4 Degradation (2014)
  • Author(s): Mori Y, Wakabayashi M, Takayasu Mori T, Zeniya M, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2014
  • Place of Presentation: Philadelphia, USA
  • Year and Date: 2014-11-13 – 2014-11-15
• [Presentation] Impaired degradation of WNK1 and WNK4 kinases causes PHAII in Mutant KLHL3 knock-in mice (2014)
  • Author(s): Sohara E, Susa K, Rai T, Zeniya M, Mori Y, Sasaki S, Uchida S
  • Organizer: The 14th Asian Pacific Congress of Nephrology
  • Place of Presentation: Tokyo, Japan
  • Year and Date: 2014-05-14 – 2014-05-17
• [Presentation] Chemical library screening for WNK signaling inhibitors by using fluorescent correlation spectroscopy (2013)
  • Author(s): Mori T, Kikuchi E, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
• [Presentation] Impaired KHLH3-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: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
• [Presentation] Analyses of KHLH3 mutants that cause Pseudohypoaldosteronism type II (2013)
  • Author(s): Mori Y, Wakabayashi M, Mori T, Araki Y, Sohara E, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
• [Presentation] KHLH2 interacts with and ubiquitinates WNK4 kinases (2013)
  • Author(s): Takahashi D, Mori T, Wakabayashi M, Mori Y, Susa K, Zeniya M, Rai T, Sohara E, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
• [Presentation] WNK3 regulates blood pressure through the regulation of vascular OSR1/SPAK-NKCC1 phosphorylation cascade (2013)
  • Author(s): Zeniya M, Sohara E, Oi K, Chiga M, Susa K, Mori T, Takahashi D, Rai T, Sasaki S, Uchida S
  • Organizer: American Society of Nephrology Kidney Week 2013
  • Place of Presentation: Atlanta, U.S.A.
• [Presentation] Chemical Library Screening for Direct SPAK Inhibitors by a Newly Devoloped ELISA. (2012)
  • Author(s): Kikuchi E
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, USA
• [Presentation] Dietary Salt Intake and Angiotensin II Regulates WNK-SPAK-NKCC1 Phosphorylation Cascade in Mouse Aorta. (2012)
  • Author(s): Zeniya M
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, USA
• [Presentation] Generation and Analysis of WT-WNK4 Transgenic Mice Reveal the Physiological Role of WNK4. (2012)
  • Author(s): Mori T
  • Organizer: American Society of Nephrology Kidney Week 2012
  • Place of Presentation: San Diego, USA