US008148089B2 (12) United States Patent (10) Patent No.: US 8,148,089 B2 Anderson et al. (45) Date of Patent: Apr. 3, 2012 (54) ALPHA-SYNUCLEIN KINASE FOREIGN PATENT DOCUMENTS (75) Inventors: John P. Anderson, San Francisco, CA WO WO 2006/073734 A2 T 2006 WO WO 2006/1248.92 A2 11/2006 (US); Kelly Banducci, Pleasanton, CA WO WO 2004/069175 A2 8, 2007 (US); Guriobal S. Basi, Palo Alto, CA WO WO 2007/089862 A2 8, 2007 (US); David Chereau, San Mateo, CA WO WO 2009/103010 A2 8, 2009 (US); Tamie J. Chilcote, San Francisco, CA (US); Normand L. Frigon, Jr., OTHER PUBLICATIONS Millbrae, CA (US); Jason Goldstein, U.S. Appl. No. 61/053,632, filed May 15, 2008, Anderson et al. Burlingame, CA (US); Irene Griswold, Anderson et al., “Phosphorylation of Ser-129 is the Dominant Patho San Francisco, CA (US) logical Modification of Alpha-Synclein in Familial and Sporadic Lewy Body Disease.” Journal of Biological Chemistry, 281 (73) Assignee: Elan Pharma International Limited, (40):29739-29752 (2006). Dublin (IE) Chen et al., “Alpha-Synuclein Phosphorylation Controls Neurotoxic ity and Inclusion Formation in a Drosophila Model of Parkinson (*) Notice: Subject to any disclaimer, the term of this Disease.” Nature Neuroscience, 8(5):657-663 (2005). patent is extended or adjusted under 35 Ellis et al., “Alpha-Synuclein is Phosphorylated by Members of the SCR Family of Protein-Tyrosine Kinases,” Journal of Biological U.S.C. 154(b) by 167 days. Chemistry, 276 (6):3879-3884 (2001). Inglis et al., “Polo-like kinase 2 (PLK2) phosphorylates alpha (21) Appl. No.: 12/702,201 Synuclein at serine 129 in the central nervous system.” J. Biol. Chem. Papers in Press, pp. 1-11 (2008). Harris, “Neuronal polo-like kinase in Alzheimer Disease indicates (22) Filed: Feb. 8, 2010 cell cycle changes.” Neurobiology of Aging, 21:837-841 (2000). Johnson et al., “Pharmacological and Functional Comparison of the Polo-like Kinase Family: Insight into Inhibitor and Substrate Speci (65) Prior Publication Data ficity.” Biochemistry, 46:9551-9563 (2007). Naoto et al., “Serine 129 phosohorylation of alpha-synuclein induces US 2010/0143946A1 Jun. 10, 2010 unfolded protein response-mediated cell death.” Journal of Biologi cal Chemistry, 28.3(34):23179-23188 (2008) Biosis Preview, Related U.S. Application Data Abstract only. (60) Division of application No. 12/030,849, filed on Feb. Okochi et al., "Constitutive Physophorylation of The Parkinson's 13, 2008, which is a continuation-in-part of Disease Associated Alpha-Synuclein.” Journal of Biological Chem application No. 1 1/669,093, filed on Jan. 30, 2007, istry, 275(1):390-397 (2000). PCT Written Opinion of Oct. 28, 2008 for application PCT/US2007/ now Pat. No. 7,553,639. OO2685. (60) Provisional application No. 60/764,000, filed on Jan. PCT Search Report of Sep. 26, 2008 for application PCT/US2007/ 31, 2006. OO2685. PCT Search Report of Sep. 16, 2009 for application PCT/US2009/ (51) Int. Cl. O34135. GOIN33/53 (2006.01) PCT Written Opinion of Sep. 16, 2009 for application PCT/US2009/ O34135. (52) U.S. Cl. ........................................................ 435/7.1 Pronin et al., “Syncleins Are a Novel Class of Substrates for G (58) Field of Classification Search .................... 435/7.1 Protein-Coupled Receptor Kinases” Journal of Biological Chemis See application file for complete search history. try, 275(34):26515-26522 (2000). Steegmaier et al., “BI 2536, a Potent and Selective Inhibitor of (56) References Cited Polo-like Kinase 1, Inhibits Tumor Growth in Vivo. Current Biol ogy, 17:316-322 (2007). U.S. PATENT DOCUMENTS Supplemental European Search Report of Sep. 1, 2009 for European 6,861,422 B2 3/2005 Hoffmann et al. Application 07762749.5. 7,553,639 B2 6, 2009 Chilcote et al. 2002/015 1464 A1 10, 2002 Wolozin et al. (Continued) 2004/0176380 A1 9, 2004 Hoffmann et al. 2005, OO14761 A1 1/2005 Hoffmann et al. 2006, OO25411 A1 2/2006 Hoffmann et al. Primary Examiner — Karen Carlson 2006/0057652 A1 3, 2006 Green et al. 2006, OO74088 A1 4/2006 Munzert et al. (74) Attorney, Agent, or Firm — Alston & Bird LLP 2006, OO795.03 A1 4/2006 Schwede et al. 2006/0223833 A1 10, 2006 Schulze et al. 2007/0010565 A1 1/2007 Prien et al. (57) ABSTRACT 2007/0010566 A1 1/2007 Prien et al. 2007, OO15759 A1 1/2007 Schulze et al. The invention provides agents and methods for treatment of 2007/0O37862 A1 2/2007 Siemeister et al. diseases associated with Lewy body diseases (LBDs) in the 2007/O135387 A1 6, 2007 Michaelides et al. 2007/01791.77 A1 8/2007 Brenchley et al. brain of apatient. Preferred agents include inhibitors of PLK2 2007/0203143 A1 8/2007 Sheppard et al. kinase. 2008/0300206 A1 12/2008 Anderson et al. 2009/0304664 A1* 12/2009 Lindquist et al. ............ 424/94.5 2011/02O7796 A1 8/2011 Anderson et al. 12 Claims, 13 Drawing Sheets US 8,148,089 B2 Page 2 OTHER PUBLICATIONS U.S. Appl. No. 1 1/669,093, Notice of Allowance mailed Dec. 16, Takahashietal., “Phosphorylation of Alpha-Synuclein Characteristic 2009. of Synucleinopathy Lesions is Recapitulated in Alpha-Synuclein U.S. Appl. No. 12/030,849, Restriction Requirement mailed May 8, Transgenic Drosphila,” Neuroscience Letters, 336:155-158 (2003). 2009. Winkles et al., “Differential regulation of polo-like kinase 1,2,3, and U.S. Appl. No. 12/030,849. Non-final Office Action mailed Mar. 5, 4 gene expression in mammalian cells and tissues.” Oncogene, 2010. 24:260-266 (2005). Zhou et al., “A Human Single-Chain Fv Intrabody Blocks Aberrant U.S. Appl. No. 12/030,849, Final Rejection mailed Apr. 13, 2011. Cellular Effects of Overexpressed Alpha-Synuclein.” Molecular U.S. Appl. No. 12/030,849, Advisory Action mailed Aug. 1, 2011. Therapy, 10(6):1023-1031 (2004). U.S. Appl. No. 12/865,857. Restriction Requirement mailed Nov. 23, U.S. Appl. No. 1 1/669,093, Office Action mailed Sep. 25, 2008. 2011. U.S. Appl. No. 1 1/669,093, Notice of Allowance mailed Mar. 10, 2009. * cited by examiner U.S. Patent Apr. 3, 2012 Sheet 1 of 13 US 8,148,089 B2 Normalized to Molar Ratio (1:200 kinase:AS) g 3. f molar molar 1:200 (Kinase:AS) 1:200 (Kinase; AS) g i MAP2K GRK6 MET PK2 MAPK3PRKG2 total AS FIG 1A Normalized to Molar Ratio (1:200 kinase:AS) molar molar 1:200 (Kinase; AS) 1:200 (Kinase:AS) CK CK FAK6 ARK CAMKPIKG2 MAP2K GRK6 MET FK2 MAPK3FRKG2 i in in Inn in in in i in in in in in pSer 129 AS. FIG. 1B U.S. Patent Apr. 3, 2012 Sheet 2 of 13 US 8,148,089 B2 Normalized to Molar Ratio (1:200 kinase; AS) g S 2 l molar molar 1:200 (Kinase; AS) 1:200 (Kinase:AS) g CK CKI PAK6 ARK CAMKPHKG2 in in Kinase pSer87 AS FIG. 1C U.S. Patent Apr. 3, 2012 Sheet 3 of 13 US 8,148,089 B2 GRKAnalysis Normalized to Molar Ratio (1:200 kinase:AS) molar o 1:200 (Kinase:AS) N d s . se f % s s i. total AS FIG. 1D GRKAnalysis Normalized to Molar Ratio (1:200 kinase:AS) rinolar 1:200 (Kinase:AS) s i i i i Phospho S129 AS F.G. 1E U.S. Patent Apr. 3, 2012 Sheet 4 of 13 US 8,148,089 B2 GRKAnalysis Normalized to Molar Ratio (1:200 kinase:AS) molar N 1:200 (kinase:AS) f O C. s s : : i ; i ; Phospho S87 AS FIG. 1F U.S. Patent Apr. 3, 2012 Sheet 5 of 13 US 8,148,089 B2 Normalized to Activity Units (nmol phosphate into uM synthetic peptide) i CK CK FAK6 ARK CAMKPHKG2 i MAP2K GRK6 CAMKIIS ME MAPK13RKG2 total AS FIG. 2A Normalized to Activity Units (nmol phosphate into uM synthetic peptide) MAP2K GRK6 CAMKIIS ME MARK3RRKG2 i CK CK PAKG ARK CAMKFKG2 pSer 129 AS FIG. 2B U.S. Patent Apr. 3, 2012 Sheet 6 of 13 US 8,148,089 B2 Normalized to Molar Ratio (1:200 kinase:AS) molar molar 1:200 (Kinase; AS) 1:200 (Kinase:AS) CK CKI PAK6 ARK CAMKIPHKG2 MAP2K GRK6 MET PK2 MAPK3PRKG2 total AS FIG. 3A Normalized to Molar Ratio (1:200 kinase. AS) rinolar molar t:200 (Kinase:AS) 1:200 (Kinase:AS) CK CKI PAKS ARK CAMKPHKG2 MAP2K GRK6 MET PLK2 MAPK13RKG2 Kinase i in in in in in i retirer in in lir in pSer129 AS FIG. 3B U.S. Patent Apr. 3, 2012 Sheet 7 of 13 US 8,148,089 B2 Normalized to Molar Ratio (1:200 kinase:AS) g d Y 2 rnolar molar g 1:200 (Kinase; AS) 1:200 (Kinase:AS) s a. CK CK. PAK6 ARK CAMKPHKG2 MAP2K GRK3 MET PLK2 MAPK13PRKG2 in in i? i? in Kirlase in in in in in pSer87 AS FG. 3C U.S. Patent Apr. 3, 2012 Sheet 8 of 13 US 8,148,089 B2 GRKAnalysis Normalized to Molar Ratio (1:200 kinase:AS) molar d 1:200 (Kinase:AS) N (d c sh. a. s s i. total AS FIG. 4A GRKAnalysis Normalized to Molar Ratio (1:200 kinase:AS) molar 1:200 (Kinase:AS) Phospho S129 AS FIG. 4B U.S. Patent Apr. 3, 2012 Sheet 9 of 13 US 8,148,089 B2 3 3 25 2 1 1 O cDNA Transfected into Cells FIG. 5 U.S. Patent Apr. 3, 2012 Sheet 10 of 13 US 8,148,089 B2 cd ueonuAS - O - Oudsoul lueOued U.S.