US 2003O1484.49A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0148449 A1 Kuliopulos et al. (43) Pub. Date: Aug. 7, 2003 (54) G PROTEIN COUPLED RECEPTOR Related U.S. Application Data AGONSTS AND ANTAGONSTS AND METHODS OF ACTIVATING AND (63) Continuation-in-part of application No. 09/841,091, INHIBITING G PROTEIN COUPLED filed on Apr. 23, 2001. RECEPTORS USING THE SAME (60) Provisional application No. 60/198.993, filed on Apr. (76) Inventors: Athan Kuliopulos, Winchester, MA 21, 2000. (US); Lidija Covic, Somerville, MA (US) Publication Classification Correspondence Address: (51) Int. Cl." .......................... A61K 38/17; C12P 21/02; Ingrid A. Beattie, Ph.D., J.D. C12N 5/06; CO7K 14/705; Mintz, Levin, Cohn, Ferris, A61K 31/20 Glovsky and Popeo, P.C. (52) U.S. Cl. ..................... 435/69.1; 435/320.1; 435/325; One Financial Center 530/350; 514/12; 514/558 Boston, MA 02111 (US) (57) ABSTRACT (21) Appl. No.: 10/251,703 The invention relates generally to G protein coupled recep tors and in particular to agonists and antagonists of G protein (22) Filed: Sep. 20, 2002 receptors and methods of using the Same. Patent Application Publication US 2003/0148449 A1 Patent Application Publication US 2003/0148449 A1 Z 0 Ca2+ (ARBITARY UNITS) Patent Application Publication Aug. 7, 2003. Sheet 3 of 28 US 2003/0148449 A1 Plpol-19 Plpol-19 w SFLRN SFLLRN SFLLRN ! P p Fig. 1C Plpol-13 Pi3-9 Y - - Plpol-19Q Pipal-19E y - Pir 19 Plpol-2 Y - - ----- SFL RN Y Plpol-7 (D 0 Y 4. - t PALMECACD Y H- m - --- F C CD as 100 200 AGGREGATION S Fig. 1D Patent Application Publication Aug. 7, 2003 Sheet 4 of 28 US 2003/0148449 A1 PLATELETS 100 OY s Fluor-i3 Z t; Fluor-Pal-i3 s s O it Tim In 10 02 O3 LOG FLUORESCENCE FIBROBLASTS OO Y A. s Fluor-i} Z 5 Fluor-Pa-i3 s Y O I - I T O 02 OG FLUORESCENCE Y - / Fig. 1E Patent Application Publication Aug. 7, 2003 Sheet 5 of 28 US 2003/0148449 A1 PAR SEQ ID NO: 1 PAR2 SEQ ID NO: 7 PAR4 SEQ ID NO: 9 Plpol-19 SEQ ID NO: 1 Plpal-l3 SEQ LD NO: 2 P. pal-7 SEQ ID NO:3 P. pal-l2 SEQ ID NO: 4 Pl pal-l9Q SEQ ID NO: 5 Pl pal-l9E SEQ ID NO: 6 P2 pol-2 SEQ LD NO: 7 P2 pol-2F SEQ ID NO:8 C - PALMITATE Fig. 2A PAR-ROt 100 s 80 - - A - Plpol-13 5 -O- Plpol-12 O ... O ... Plpol-7 () 60 O Y OL 9 40 20 08 06 lo ?pal-peptidel (M) Fig. 2B Patent Application Publication Aug. 7, 2003. Sheet 6 of 28 US 2003/0148449 A1 100 -- A - Plpol-19 PAR-ROt -A-. Plpol-19Q ---O--- Plpol-19E 8-O- MASTOPARAN - X: Plpol-19/ 60 Rot (alone 4O 2O O - titmi t-tim t t t im it trim t t tim 08 10° o peptide) (M) Fig. 2C PAR2-COS/ CS - O €d 27 OU P2pal-2F 5 2 O O Y OL Ol f C P2pal-2) mini i tTg it its tim 0-0 08 106 0 (pal-peptidel (M) Fig. 2D Patent Application Publication Aug. 7, 2003. Sheet 7 of 28 US 2003/0148449 A1 E. Pipal-l3 O EXTRACELULAR AGONS Pipal-19 O EXTRACEULAR Patent Application Publication Aug. 7, 2003. Sheet 8 of 28 US 2003/0148449 A1 P2pal-2F 6 P2pal-2 m EXTRACELULAR G5 AGONS S2 3 4. 3 O 32 C OO 5 O P Pol-19 (M) Fig. 3A Patent Application Publication Aug. 7, 2003. Sheet 9 of 28 US 2003/0148449 A1 OO 5 O O -4 O -2 100 10 -8 THROMBIN) (M) Fig. 3B O WT OO S309P O A377 5 O O-7 O -5 SFL RN) (M) Fig. 3C Patent Application Publication Aug. 7, 2003. Sheet 10 of 28 US 2003/0148449 A1 vs. SFLLRN SFLLRN 2 D a. J.- SFRN E5. A AYPGKF 5. 2 Plpol-2 y AYPGKF - O A 5 2OOS P4pal-l5 PAR4 PAR Fig. 4A SFLLRN AYPGKF 8Y +Plpol-12 +P4paf-l5 < : 200s 5. | (-) (-) 20 Fig. 4B Patent Application Publication Aug. 7, 2003 Sheet 11 of 28 US 2003/0148449 A1 5 O OO 0-8 07 06 O-5 pal-peptide) (M) Fig. 4C 5 O O O O 8 07 06 05 04 pol-peptide) (M) Fig. 4D Patent Application Publication Aug. 7, 2003. Sheet 12 of 28 US 2003/0148449 A1 ?NI??C?C?GTSNIVÈLENBd-TTHO Etz'6|- NWTOTTBOVNIXEA ONW79||| Patent Application Publication Aug. 7, 2003 Sheet 13 of 28 US 2003/0148449 A1 FIUOr-Pol-i3 FIUOr-i3 OO -- - - - - -- C-NTACT CELLS SC-SONICATED CELS 8O (v) Z z 60 5 in SONICATE, 2ND PRONAS t -LL 40 s SONICATE, 2ND PRONASE t 2O - ass PRONASE - - - - -- - - - - - -- C SC C SC EXP if 2 3 5 6 7 8 Patent Application Publication Aug. 7, 2003. Sheet 14 of 28 US 2003/0148449 A1 I:ON(IIÒGIS z:ONCIIÒGIS Iz:0N(IIÒGIS ZZ:ON(IIÒ?S £z:ONCIIOAS ?Z:ON(IIÒGIS 6°:ON(IIÒ?S yTerraepae----------------------------------------------------------------------------SNOECIWIKISS!!TWRI §TASISTESybrae,SSEJAHI--------------------------------------------------------------------------§SSXAX{ d'OOT£| §TA?IYDI,IN:SSNSHIYIN:HSSSÐISTÖHTIGTDRidd'!).IX?IXDOCISCIEXXÔSSLLSåDI---(EDTOESODI?S™ELECHI??ZTETI JITHASYD!------------------------------------------------------------------------------S^{N(AVISSSTICH yAay?ay?:SA?RTHA(GIS------------------------------------------------------------SCÉDEIG?S??M?TI:I !T!!!!!!----------------------------------------------------------------------------------3)S(TILH Patent Application Publication Aug. 7, 2003 Sheet 15 of 28 US 2003/0148449 A1 MAX ACTIVATION t t t t it r t t t Tim t t t t its 08 O7 06 O Fig. 6B (AGONIST) (M) MAX AINATION CCKB 3OONM 8 CCK-8 Ol A. C Z 4 O Plpol-19 C) 9 a A. O l Pipal-l3 O - - Tri- rtm tit- SS; 08 O7 06 05 Fig. 6C AGONIST) (M) Patent Application Publication Aug. 7, 2003 Sheet 16 of 28 US 2003/0148449 A1 - MAX ACTIVATION SS AGONIST) (M) Fig. 6D Patent Application Publication Aug. 7, 2003. Sheet 17 of 28 US 2003/0148449 A1 r OO 8O 5. O-MSH ais se 5 40 MC4pal-l4 g 2O O 0-0 08 06 O' AGONIST) (M) Fig. 7 Patent Application Publication Aug. 7, 2003 Sheet 18 of 28 US 2003/0148449 A1 LBS-1 INTERFERENCE OF INTRAMOLECULAR PARACTIVATION THROMBINACTIVATION OF PARI LBS- INTERMOLECULAR 1 LGAND LBS-2 LBS-2 c s - LBS-PE INTERMOLECULAR u- LIGAND LBS-2 c LBS-2 sir s GDP Fig. 8B Patent Application Publication Aug. 7, 2003. Sheet 19 of 28 US 2003/0148449 A1 CC o-c-, o O O) s O O O ll- 2 ors - CD f < C) f CS. eP s itzr O n- C2 \ > CD g - G E3 9/ - l Patent Application Publication Aug. 7, 2003 Sheet 20 of 28 US 2003/0148449 A1 3 nMT SFLRN No a t 2 BS 330 () 200' 400 600 800 O 200 A00 600 TIME (SEC) TIME (SEC) Fig. 9C Fig. 9D (O- LBS C 4. O LBS-PE 2 O -- - O 40 80 2O (PEPTIDE) (uM) Fig. 9E Patent Application Publication Aug. 7, 2003 Sheet 21 of 28 US 2003/0148449 A1 FIG. OA Patent Application Publication Aug. 7, 2003 Sheet 22 of 28 US 2003/0148449 A1 Patent Application Publication Aug. 7, 2003 Sheet 24 of 28 US 2003/0148449 A1 FIG. 2 P<0.002 - PCO.001 3. b >600 - IV-de?ivery 1 500 100 Cld s S 400 2 Fluor-i3 C) s E 300- -- C Fluor-Pal-i3 O 50 d)0. O o 200 g s i roo O O d H 100 (5. C. r. Y O l Lil 0 (n=17) (n=16) (n=16) 10 10? c\ ^ Log fluorescence d ex ove de 1200 50 - S.8 100000 P<0.003 S S 800 Oup wd : 600 2 3 2s w s 400 D E 9 200 O y O - so o a' epilAYPGKF - H s ses ov ob. P4pal-10) - - 0.3 1 3 micromoles/L Patent Application Publication Aug. 7, 2003. Sheet 25 of 28 US 2003/0148449 A1 Figure 13. Chare 1 Channel 2 Charine Cannel 4 Channe Charlet 2 Channel 3 Charle 4 300 nMY12pal-24 3 MSFLLRN O. in 1oo- O----- : - - - - - - 100 : s : : O. : 300 nM Y12pa-18. ...go 10----- H -i- - - . .so : ; ; ; 20. r: . so 20 . ; . - - - m r - - - - - -]. +10 MY12pal-24 7. 3 MSFLLRN . : apyrase - - - - W - 8 +3,MY12pal-2440 cri +1 uM Y12pal-2 3MSFLLRN" - - - - - - -- . lo 100.-- : . -- 200 alo CO 80 oroo 20 140 2.00 a.oO 6:00 8:00 100 12: 4:00 & 8 ' ' -- time (minsee) irre (Tinsec) Y12pal-18 Pal-YVRTRGVGKVPRKKVNVF-NH2 SEQ ID NO:33 Y12pal-24 Pal-KELYRSYVRTRGVGKVPRKKVNVF-NH2 SEQ ID NO:34 Patent Application Publication Aug. 7, 2003. Sheet 26 of 28 US 2003/0148449 A1 Figure 14. A. B GLP-1R1 nM GLP-1 GLP-1R vs. 1M pepducin vs. 10 Mi pepducin 500 12000 s s 400 S W GS 8000 Sl2 300 Sl2 as 200 is 4000: CU 100 s SS O O SS m s Q m CSY C s C s G1pa-15 Pal-ANLMSKTDIKCRLAF-NH2 SEQ ID NO:35 G1pa-23 Pal-SIWSKLKANLMSKTDIKCRLAF-NH2 SEQ LD NO:36 Patent Application Publication Aug. 7, 2003 Sheet 27 of 28 US 2003/0148449 A1 Fidure 15. 140 120 60 40 20 Patent Application Publication Aug. 7, 2003 Sheet 28 of 28 US 2003/0148449 A1 Figure 16. trypsin) (M) US 2003/0148449 A1 Aug. 7, 2003 G PROTEIN COUPLED RECEPTORAGONSTS SUMMARY OF THE INVENTION AND ANTAGONSTS AND METHODS OF ACTIVATING AND INHIBITING G PROTEIN 0008.