(12) United States Patent (10) Patent No.: US 9,345,245 B2 Cutler Et Al
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USOO9345245B2 (12) United States Patent (10) Patent No.: US 9,345,245 B2 Cutler et al. (45) Date of Patent: May 24, 2016 (54) SYNTHETIC COMPOUNDS FOR Cutler et al., “Abscisic Acid. Emergence of a Core Signaling Net VEGETATIVE ABA RESPONSES work”. Annual Review of Plant Biology, vol. 61, pp. 651-679 (2010). Iyer et al., "Adaptations of the helix-grip fold for ligand binding and (71) Applicant: THE REGENTS OF THE catalysis in the START domain superfamily”. Protens: Structure, UNIVERSITY OF CALIFORNLA, Function, and Bioinformaticis, vol.43, No. 2, pp. 134-144 (2001). Oakland, CA (US) Melcher et al., “Thirsty plants and beyond: structural mechanisms of abscisic acid perception and signaling'. Current Opinion in Struc (72) Inventors: Sean R. Cutler, Riverside, CA (US); tural Biology, vol. 20, No. 6, pp. 722-729 (2010). Masanori Okamoto, Riverside, CA Notman, “Organic compound comes to the aid of thirsty plants'. (US) Royal Society of Chemistry (May 1, 2009) http://www.rsc.org/ chemistry world/News/2009/May/01050901.asp (downloaded on (73) Assignee: THE REGENTS OF THE Jun. 29, 2015). UNIVERSITY OF CALIFORNLA, Parket al., Abscisic Acid Inhibits Type 2C Protein Phosphatases via Oakland, CA (US) the PYR/PYL Family of START Proteins, Science, vol. 324, No. 5930, pp. 1068-1071 (2009). (*) Notice: Subject to any disclaimer, the term of this Ponting etal. “START: a lipid-binding domain in StAR, HD-ZIP and patent is extended or adjusted under 35 signalling proteins”. Trends Biochem, vol. 24. No. 4, pp. 130-132 U.S.C. 154(b) by 0 days. (1999). Radauer, “The Bet v 1 fold: an ancient, versatile scaffold for binding (21) Appl. No.: 14/385,695 of large, hydrophobic ligands'. BMC Evol. Biol., vol. 8, pp. 286 (2008). (22) PCT Filed: Mar. 15, 2013 Rademacher W. Maisch R, Liessegang J, Jung J (1987) Water con Sumption and yield formation in crop plants under the influence of (86). 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(57) ABSTRACT 6,127,382 A 10, 2000 Beard et al. 2011/023O350 A1 9/2011 Frackenpohl et al. 2011/0271408 A1 11/2011 Cutler et al. The present invention provides agonist compounds that active 2013,0045952 A1 2/2013 Xu et al. ABA receptors, and agricultural formulations comprising the agonist compounds. The agricultural formulations are useful FOREIGN PATENT DOCUMENTS for inducing ABA responses in plant vegetative tissues, reducing abiotic stress in plants, and inhibiting germination WO 2010/093954 A2 8, 2010 of plant seeds. The compounds are also useful for inducing WO 2011, 1397.98 A2 11/2011 expression of ABA-responsive genes in cells that express OTHER PUBLICATIONS endogenous or heterologous ABA receptors. Bartel et al., “Analyzing protein-protein interactions using two-hy brid system'. 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Fujii et al., “In vitro Reconstitution of an ABA Signaling Pathway”. Agrochemicals, vol. 49, No. 7, Jul. 2010, pp. 484-485. Nature, vol. 462, No. 7273, pp. 660-664 (2009). Zhou, et al., “Synthesis and Plant Growth Regulation Activity of Leon-KloosterZiel et al., “Isolation and characterization of abscisic Pyrabactin”, Agrochemicals, vol. 49, No. 7, Jul. 2010, pp. 484-485. acid-deficient Arabidopsis mutants at two new loci', Plant J. vol. 10, Patent Examination Report No. 1, Australian Pat. Applin. No. No. 4, pp. 655-661 (1996). 201324.0193, dated Feb. 9, 2016, 4 pages. U.S. Patent May 24, 2016 Sheet 1 of 27 US 9,345,245 B2 H O H O O Nss Ng7 CC w O - COOH - O- ) 11 C 10 (+)-ABA) Pyrabactin (PyrA) NO.1 3B r NO.24H (-)- ABA LC66C6 No. 1 11E NO.25E FIG. 1A S Š S3 U.S. Patent May 24, 2016 Sheet 2 of 27 US 9,345,245 B2 / ORSRO H Y-X °C)2. O Pyrabactin Quinabactin O / HN 1.O O2 Analog3 FIG. 2A U.S. Patent May 24, 2016 Sheet 3 of 27 US 9,345,245 B2 DMSO Pyrabactin ABA Quinabactin 120 . ZZZZZZZZZ,R LLg ÇNQQ U.S. Patent May 24, 2016 Sheet 4 of 27 US 9,345,245 B2 DMSO Analog1 Analog4 E. Analog5 ABA O Analog2 <= C)CO<!--CNCOEDO !!!!!!!!!!!!!!!!!!!!!!!!!! FIG. 2C U.S. Patent May 24, 2016 Sheet 5 Of 27 US 9,345,245 B2 PYR1 PYL1 40 2 O O 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000 Chemical concentration (nM) Chemical Concentration (nM) PYL5 O 2000 4000 6000 8000 10000 O 2000 4000 6000 8000 10000 Chemical concentration (nM) Chemical Concentration (nM) PYL9 PYL10 120 1 O O O 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000 Chemical Concentration (nM) Chemical Concentration (nM) FIG. 3A U.S. Patent May 24, 2016 Sheet 6 of 27 US 9,345,245 B2 0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000 Chemical Concentration (nM) Chemical concentration (nM) PYL6 PYL8 i 0 2000 4000 6000 8000 10000 O 2000 4000 6000 8000 10000 Chemical concentration (nM) Chemical concentration (nM) SS -O- ABA 2. 2 & - Quinabactin n - 7 - Pyrabactin 0 2000 4000 6000 8000 10000 Chemical Concentration (nM) FIG. 3A (Continued) U.S. Patent May 24, 2016 Sheet 7 Of 27 US 9,345,245 B2 878,"501-J |HÅd /09 999 (Wu)090|| U.S. Patent May 24, 2016 Sheet 8 of 27 US 9,345,245 B2 '. 6NYN ABA O O1 OH O Pyrabactin O No M OY OCYN O Quinabactin FIG. 4A ABA Pyrabactin Guinabactin PYR1 307 656 103 PYL1 301 1197 250 DimeriC PYL2 151 >10,000 267 PYL3 70 >10,000 724 PYL4 68 >10,000 > 1 OOOO PYL5 27 5174 649 PYL6 29 >10,000 > 1 OOOO PYL11 64 >10,000 > 1 OOOO PYL12 nd nd no MOnOmeriC PYL7 nd nd no PYL9 60 >10,000 > 1 OOOO PYL8 57 >10,000 > 1 OOOO PYL10 121 1892 > 1 OOOO FIG. 4B U.S. Patent May 24, 2016 Sheet 10 of 27 US 9,345,245 B2 Wild type abi 1 quadruple FIG. Wildtype 2. quadruple ELI 5 10 20 40 5 10 20 40 5 10 20 40 §ž Pyra (+)ABA No.lo: FIG. O 5 10 20 5 to 2040 M Pyra LC66C6 (+)-ABA FIG.