Europäisches Patentamt *EP001281771A2* (19) European Patent Office Office européen des brevets (11) EP 1 281 771 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.7: C12Q 1/44, C07K 14/00, 05.02.2003 Bulletin 2003/06 C12N 5/00, C12N 15/00, G01N 33/50 (21) Application number: 02254973.7 (22) Date of filing: 16.07.2002 (84) Designated Contracting States: • Lebel, Lorraine Ann AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Groton, Connecticut 06340 (US) IE IT LI LU MC NL PT SE SK TR • Menniti, Frank Samuel Designated Extension States: Groton, Connecticut 06340 (US) AL LT LV MK RO SI • Strick, Christine Ann Groton, Connecticut 06340 (US) (30) Priority: 31.07.2001 US 308978 P (74) Representative: Hayles, James Richard et al (71) Applicant: Pfizer Products Inc. UK Patent Department, Groton, CT 06340-5146 (US) Pfizer Limited, Ramsgate Road (72) Inventors: Sandwich, Kent CT13 9NJ (GB) • James, Larry Carlton Groton, Connecticut 06340 (US) (54) Phosphodiesterase 10A cell-based assay and sequences (57) The invention features a method of screening EC200 and the cGMP EC200, wherein the agent is iden- for an agent that inhibits intracellular phosphodiesterase tified as a PDE10A inhibitor if the ratio of cAMP EC200/ 10A activity, comprising administering an agent to stri- cGMP EC200 is comparable to the ratio produced by ad- atal medium spiny neurons and submaximally activating ministration of papaverine under the same assay con- adenylate cyclase, administering an agent to striatal ditions. Also featured are rat PDE10A polynucleotide medium spiny neurons and submaximally activating and polypeptide sequences. guanylate cyclase, measuring cAMP generation and cGMP generation in the cells, and calculating the cAMP EP 1 281 771 A2 Printed by Jouve, 75001 PARIS (FR) EP 1 281 771 A2 Description Field of the Invention 5 [0001] The present invention provides methods for identifying agents that modulate PDE10A activity and the poly- nucleotide and polypeptide sequences for rat PDE10A. Background 10 [0002] Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP (cyclic adenosine 3'5'-monophosphate) and cGMP (cyclic guanine 3'5'-monophosphate) and play a pivotal regulatory role in a wide variety of signal transduction pathways (Beavo, Physiol. Rev. 75: 725-48, 1995). For example, PDEs mediate processes involved in vision (McLaughlin et al., Nat. Genet. 4: 130-34, 1993), olfaction (Yan et al., Proc. Natl. Acad. Sci. USA 92: 9677-81, 1995), platelet aggregation (Dickinson et al., Biochem. J. 323: 371-77, 1997), aldosterone 15 synthesis (MacFarland et al., J. Biol. Chem. 266: 136-42, 1991), insulin secretion (Zhao et al., J. Clin. Invest. 102: 869-73, 1998), T cell activation (Li et al., Science 283: 848-51, 1999), and smooth muscle relaxation (Boolell et al., Int. J. Impot. Res. 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-71, 1998). [0003] PDEs form a superfamily of enzymes that are subdivided into 11 major families (Beavo, Physiol. Rev. 75: 725-48, 1995; Beavo et al., Mol. Pharmacol. 46: 399-05, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-96, 20 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-77, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: 751-56, 1998; Soderling et al., J. Biol. Chem. 273: 15553-58, 1998; Fisher et al., J. Biol. Chem. 273: 15559-64, 1998; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-76, 1999; and Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000). [0004] Each PDE family is distinguished functionally by unique enzymatic characteristics and pharmacological pro- 25 files. In addition, each family exhibits distinct tissue, cellular, and subcellular expression patterns (Beavo et al., Mol. Pharmacol. 46: 399-405, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-96, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-77, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: 751-56, 1998; Soderling et al., J. Biol. Chem. 273: 15553-58, 1998; Fisher et al., J. Biol. Chem. 273: 15559-64, 1998; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-76, 1999; Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-07, 2000; Boolell 30 et al., Int. J. Impot. Res. 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-71, 1998; Houslay, Semin. Cell Dev. Biol. 9: 161-67, 1998; and Torphy et al., Pulm. Pharmacol. Ther. 12: 131-35, 1999). Accordingly, by administering a compound that selectively regulates the activity of one family or subfamily of PDE enzymes, it is possible to regulate cAMP and/ or cGMP signal transduction pathways in a cell- or tissue-specific manner. [0005] PDE10 is identified as a unique PDE based on primary amino acid sequence and distinct enzymatic activity. 35 Homology screening of EST databases revealed PDE10A as the first member of the PDE10 family of phosphodieste- rases (Fujishige et al., J. Biol. Chem. 274: 18438-18445, 1999; Loughney et al., Gene 234:109-117, 1999). The human, rat, and murine homologues have been cloned and N-terminal splice variants have been identified for both the rat and human genes (Kotera et al., Biochem. Biophys. Res. Comm. 261: 551-557, 1999; Fujishige et al., Eur. J. Biochem. 266: 1118-1127, 1999; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-7076, 1999); there is a high degree of 40 homology across species. PDE10A hydrolyzes cAMP and cGMP to AMP and GMP,respectively. The affinity of PDE10A for cAMP (Km = 0.05 µM) is higher than for cGMP (Km =3µM). However, the approximately 5-fold greater Vmax for cGMP over cAMP has led to the suggestion that PDE10A is a unique cAMP-inhibited cGMPase (Fujishige et al., J. Biol. Chem. 274:18438-18445, 1999). [0006] PDE10A is uniquely localized in mammals relative to other PDE families. Messenger RNA for PDE10A is 45 highly expressed only in testis and brain (Lanfear and Robas, EP 0967284; Fujishige et al., Eur. J. Biochem. 266: 1118-1127, 1999; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-7076, 1999; Loughney et al., Gene 234:109-117, 1999). Initial studies indicated that, within the brain, expression is highest in the striatum (caudate and putamen), nucleus accumbens, and olfactory tubercle (Lanfear and Robas, supra). Accordingly, PDE10A selective modulation could be used to modulate levels of cyclic nucleotides in these brain areas. 50 Summary of the Invention [0007] The present invention provides methods for identifying agents that selectively modulate PDE10A activity and the polynucleotide and polypeptide sequences for rat PDE10A. 55 [0008] In one aspect, the invention features a method of screening for an agent that inhibits intracellular phosphodi- esterase 10A activity comprising administering the agent to striatal medium spiny neurons and submaximally activating adenylate cyclase, administering the agent to striatal medium spiny neurons and submaximally activating guanylate cyclase, measuring cAMP generation and cGMP generation, respectively, and calculating the cAMP EC200 and the 2 EP 1 281 771 A2 cGMP EC200, respectively, wherein the agent is identified as a PDE10A inhibitor if the ratio of cAMP EC200/ cGMP EC200 is comparable to the ratio produced by administration of papaverine under the same assay conditions. [0009] Preferably, the striatal medium spiny neurons are prepared as primary cultured neurons, adenylate cyclase is activated by forskolin, guanylate cyclase is activated by sodium nitroprusside, and the cAMP EC200/cGMP EC200 5 ratio ranges from 1.75-5.25, more preferably, from 3.0-4.0. Preferably, the concentration of cAMP and cGMP is meas- ured by scintillation proximity assay. It is preferred that the neurons used to assess cAMP and cGMP are in separate samples. In addition, it is preferred that the agent is first identified in vitro as a PDE10A selective inhibitor. Alternatively, the agent is further identified as a PDE10A selective inhibitor by in vitro assay. [0010] In another aspect, the invention features an isolated or purified polypeptide comprising the amino acid se- 10 quence of SEQ ID NO: 2. [0011] In a related aspect, the invention features an isolated or purified polynucleotide comprising a nucleic acid sequence encoding the polypeptide of SEQ ID NO: 2 and/or the coding sequence of SEQ ID NO: 1. [0012] The invention also features a vector comprising the coding sequence of SEQ ID NO: 1, and a host cell ex- pressing the coding sequence of SEQ ID NO: 1. 15 [0013] In addition, the invention provides a method of identifying an agent that modulates PDE10A activity, comprising contacting the agent with a rat PDE10A polypeptide comprising SEQ ID NO: 2 and measuring the activity of the PDE10A polypeptide, wherein a difference between the PDE10A polypeptide activity in the presence of the agent and in the absence of the agent is indicative that the agent modulates PDE10A activity. [0014] Also featured by the invention is a method of identifying an agent that modulates PDE10A activity, comprising 20 contacting the agent with a host cell expressing the coding sequence of SEQ ID NO: 1 and measuring the activity of the PDE10A polypeptide expressed by SEQ ID NO: 1, wherein a difference between the PDE10A polypeptide activity in the presence of the agent and in the absence of the agent is indicative that the agent modulates PDE10A activity. [0015] Those skilled in the art will fully understand the terms used herein in the description and the appendant claims to describe the present invention. Nonetheless, unless otherwise provided herein, the following terms are as described 25 immediately below. [0016] An "agent that increases PDE10A activity" refers to a molecule which intensifies or mimics the biological activity of a PDE10A polypeptide.