- Email: [email protected]
7,7-Difluoroprostacyclin derivative, AFP-07, a highly selective and potent agonist for the prostacyclin receptor
7,7=Difluoroprostacyclin Derivative, AFP-07, a Highly Selective and Potent Agonist for the Prostacyclin Receptor Chang-Sheng Chang*, Manabu Negishi*, Takashi Nakano#, Yoshitomi Morizawa#, Yasushi Matsumurd and Atsushi Ichikawa”
*Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan; §Research Center, Asahi Glass Co. Ltd., Yokohama 221, Japan
Recently, we cloned cDNAs for the prostacyclin receptor (IP) and the four mouse PGE receptor subtypes, EPl, EP2, EP3 and EP4, and established Chinese hamster ovary cells that stably express each receptor. We examined the agonist potency and selectivity of AFP-07, a 7,7difluoroprostacyclin derivative, compared with widely used stable prostacyclin analogue, iloprost, using the cells expressing each cloned receptor. AFP-07 strongly displaced the rH] iloprost binding to the IP receptor-expressing cell membranes, the half maximal concentration for the displacement being 3 nM, which was one order lower than that of iloprost. AFP-07 concentration-dependently stimulated CAMP formation in the IP-expressing cells, the half-maximal concentration for the stimulation being 10 PM, which was one order lower than that of iloprost. On the other hand, AFP-07 showed lower affinity for EPl, EP2, EP3 and EP4 than PGEB but iloprost had the same affinity as PGE, for the EPl. These results demonstrate that AFP-07 is a potent and highly selective agonist for the IP receptor. 0 1997 by Elsevier Science Inc.
To
whom correspondence should be addressed. Atsushi Ichikawa, Department of: Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Sakyo-ku 606, Japan, Tel: +81-75-753-4527, Fax: +81-75-753-4557, E-mail: [email protected] Abbreviations used are: PG, prostaglandin; EP, PGE receptor; IP, prostacyclin receptor; CHO, Chinese hamster ovary.
Prostaglaudins 53:83-90, 1997 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0090-6980/97/$17.00 PII SOO90-6980(97)00003-B
IF-Selective Agonist, AFP-07: Chang et al.
Keywords: prostacyclin receptor; PGE receptor; EPl; EP2; EP3; EP4
Introduction Prostacyclin (PGI,) is an unstable prostanoid which, in blood vessels, is largely produced by endothelial cells’. It inhibits platelet aggregation and vasodilatation in pulmonary vascular beds. Other actions of PGI, are suggested to be regulation of renal blood flow, renin release and glomerular filtration rate in the kidney cortex and stimulation of secretion in the stomach and large intestine. The actions of PGIz are presumed to be mediated by a cell surface receptor?. Since PGI, is very labile, a variety of chemically stable PGI analogues has been developed4. Among them, iloprost, a member of carbacyclin, is a potent IP agonist, which is well characterized and widely used, but it also showed potent EPl agonist activity 516.Thus, there is an urgent need to develop highly specific and potent ligands for IP receptor. Recently, we cloned cDNAs for the mouse IP receptor and four PGE receptor subtypes, EPl, EP2, EP3 and EP4, and established Chinese hamster ovary (CHO) cells stably expressing each receptor ’.I2. We developed several fluoroprostacyclin derivatives for IP receptor, and examined the potency and selectivity for these receptors using the cells specifically expressing each receptor. We report here that a 7,7difluoroprostacyclin derivative, AFP-07, is a potent and highly selective IP agonist. Materials and Methods Materials [5,6,8,1 1,12,14,15-3H]PGE, (191 Ci/mmol), [aH] iloprost (14.1 Ci/mmol), iloprost and the 1251-labeled CAMP assay system were obtained from Amersham Corp. PG& was purchased from Funakoshi Pharmaceuticals (Tokyo, Japan). All other chemicals were reagent grade. Establishment of CHO cell line stably expressing each prostanoid receptor has been described previously ’-12. Receptor density of each cell membrane is as follows: IP, 1.2 pmol/mg; EPl, 0.95 pmol/mg; EP2, 1.5 pmol/mg; EP3, 2.3 pmol/mg; and EP4,0.85 pmol/mg. Mock-transfected CHO cells only expressed EP4 receptor among these prostanoid receptors, but the expression level was very low (0.005 pmol/mg). 13H] PGE, or rH] Iloprost Binding Assay For the assay, the membrane fraction (250,000 x g pellet) was prepared
from CHO cells stably expressing the mouse IP receptor or each mouse PGE receptor subtype, as described previously13. The standard assay Prostaglandins 199753,
Febnwy
IP-Selective Agonist, AFP-07: Chang et al. mixture comprised either 20 nM [3H] iloprost (28.2 nCi) or 4 nM [3H] PGEz (76.4 nCi), and 80 ug of the membrane prepared from each receptor-expressing CHO cell type in 100 ul of 10 mM Mes-NaOH, pH 6.0, containing 1 mM EDTA and 10 mM MgCl, (buffer A). After incubation for 1 h at 37”C, the reaction was terminated by the addition of ice-cold buffer A, after which the mixture was rapidly filtered through a Whatman GF/C glass filter. The filter was then washed with ice-cold buffer A and the radioactivity on the filter was measured by scintillation counting. Nonspecific binding was determined using a l,OOO-foldexcess of the respective unlabeled PG in the incubation mixture. The specific binding was calculated by subtracting the nonspecific binding from the total binding. Measurement of CAMP Formation Cyclic AMP levels in CHO cells expressing the IP receptor were determined as reported previously’. Cells cultured in 24-well plates (5 x lo5 cells/well) were washed with 0.5 ml of Hepes-buffered saline containing 140 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl,, 1.2 mM MgClz, 1.2 mM KI-I,P04, 11 mM glucose and 15 mM Hepes (pH 7.4), and preincubated for 5 min. Reactions were started by the addition of agonists along with 0.1 mM Ro-20-1724. After incubation for 10 min at 37”C, the reactions were terminated by the addition of 10% trichloroacetic acid. The CAMP content of the cells was determined by radioimmunoassaying with an Amersham [ ‘251]cAMPassay system. Results and Discussion We developed three types of 7-fluoroprostacyclin derivatives. Fig. 1 shows chemical structures of a 7-monofluoroprostacyclin derivative (AFP-03)‘4, and two 7,7-difluoroprostacyclin derivatives (AFP-06 and AFP-07)15.To compare the binding affinities of these derivatives and iloprost for the IP receptor, we examined the effects of these derivatives and iloprost on specific [3H] iloprost binding to the membrane of CHO cells expressing the IP receptor. As shown in Fig. 2, specific [3H]iloprost binding to the IP receptor was inhibited by these ligands in the order of AFP-07 > AFP-06 > iloprost > AFP-03. From Fig. 2, we obtained Ki values for these ligands using the Cheng-Prussoff equation, and the values are 0.561 (AFP-07), 3.74 @P-06), 9.35 (iloprost), and 16.8 nM (AFP-03). The value for iloprost was 16-fold higher than that for AFP-07. Thus, AFP-07 showed stronger affinity than iloprost. We further compared these ligands, as to agonist activity for the IP receptor, which is coupled to the stimulation of adenylate cyclase. Fig. 3 shows the concentration dependencies of the effects of the fluoropro-
Prostaglandins 199753, February
85
IP-Selective Agonist, AFP-07: Chang et al. COONa
I f
9L 6”
6”
Prostacyclin
&
t
AFP-03
+OONa
AFP-06
AFP-07
FIGURE1. Chemical st~ctures of prostacyciinand three 74uoroprostacyclii derivatives, AFP03, AFP-06 and AFP-07.
stacyclin derivatives and iloprost on the CAMP formation in the IP receptor-expressing cells. To ensure that the change in the CAMP level of the cells is not influenced by alterations in CAMP phosphodiesterase activity, we added a phosphodiesterase inhibitor, Ro-20-1724, to the medium. The ligands concentration-dependently stimulated the increase in the CAMP level of the IP receptor-expressing cells in the order of AFP-07 z AFP-06 > iloprost r AFP-03. The order of agonist potency of these ligands (Fig. 3) was consistent with that of their binding affinities (Fig. 2). However, the ECm values of the response for these ligands are about 100 times less than their Ki values, suggesting that the receptor-effector coupling is more efficient than the ligand-receptor binding. Such variations in coupling efficacy of the PGIz receptor was observed in platelets of various species6. The 7-monofluoroprostacyclin derivative is less potent agonist, and 7,7difluoroprostacyclin derivatives are much more potent. Addition of two fluorides to PGI, structure at C-7 would increase the agonist potency. In addition, AFP-07 is more potent than AFP-06, and they are different only in structure of the w-side chain, indicating that structure of *side chain is also responsible for IP agonist activity.
Prostaglandins 199753,
February
IP-Selective Agonist, AFP-07: Chang et al. 120 u c z JJ
ITi= ?Lo QZ o. z -0 3 -8 0;E .-0 : m
100 80 60 40 20 0 0
10
9
8
Ligands
7
6
5
(-log M)
FIGURE2. Effects of AFP-03, AFP-06, AFP-07 and iloprost on [‘HI iloprost binding to the membrane of IP receptor-expressing CHO ceils. Membrane of IP receptor-expressing CHO cells was incubated with 20 nM [‘HI iloprost and the indicated concentrations of AFP-3 (4, AFP-6 (B), AFP-7 (0) or iloprost (0). Specific 13H] iloprost binding was determined as described under “Materials and Methods”, and was 90%. Maximum binding in the control was 0.7 pmol/mg for [%t] iloprost. The results shown are the means f S. E. for three independent experiments.
Among them, AFP-07 is the most potent IP agonist, its potency being one order of magnitude stronger than that of iloprost. To evaluate the selectivity of AFP-07 and iloprost for the IP receptor and four PGE receptor subtypes, we examined the effects of AFP-07 and iloprost on specific [3H] PGEz binding to CHO cell membranes expressing the four PGE receptor subtypes, EPl, EP2, EP3 and EP4, and compared their binding affinities with those for PGEz. As shown in Fig. 4, specific [3H] PGE, binding to the EPl receptor subtype was inhibited by the ligands in the order of PGE, = iloprost > AFP-07. Iloprost showed the same affinity as PGEz, supporting the notion that iloprost is a potent EP 1 agonist 5*6.Specific 13H]PGE, binding to EP2 was inhibited in the order of PGE, > AFP-07 = iloprost. AFP-07 and iloprost showed the same affinity. Specific [3H]PGEz binding to EP3 was inhibited by these ligands in the order of PGEz > iloprost > AFP-07. The affinity for AFP-07 was lower than that for iloprost. Specific [ 3H] PGE, binding to EP4 was inhibited by these ligands in the order of PGEz > AFP-07 > iloprost. The affinity for AFP-07 was higher than that for iloprost. As judged from these results, AFP-07 shows the weaker affinity than PGE, in all four PGE receptor subtypes, indicating that AFP-07 is a highly selective IP agonist. In contrast, iloprost is not selective to IP receptor; it also binds to the EPl receptor as well as the IP receptor. Prostaglandins 199753, February
87
IP-Selective Agonist, AFP-07: Chang et al.
041
’
0
13
I
I
I
I
I
I
12
11
10
9
8
7
Ligands
I
(-log WI)
FPXJRE3. Efksta of AFP-03, AFF-00, AFP-07 and ibpmst on &fblP W in the IP remptorfwqtorrmra~~~ominat37ocwith AfWB 0, m-07 (*I or Bopmt (0), in the oonbnt woo mwmmdmhmibadunder of 100 @& Re-20-1724. The &lP ‘e and M&hods”. The mutts shown am the mcOm f S. E. for three irMepmdent experkmh.
The potencies of agonists and antagonists for IP receptor have so far been carried out mainly on platelet aggregation and other systems in which the actions of PG12 can be evaluated readily, such as smooth muscle systems3. However, many tissues, including various platelets and smooth muscles, express IP receptor and multiple EP receptors, and in many cases ligands cross-react with multiple receptors. Thus, evaluation of the potency of an agonist or antagonist for a single type of receptor is very difficult. We have isolated cDNA clones encoding the IP receptor and the four PGE receptor subtypes, EPl, EP2, EP3 and EP4, and established cell lines expressing each receptor. Assay systems involving these cell lines, as shown in this work, allow clear evaluation of the potency of an agonist or antagonist for a single receptor. Here we developed an IP-selective agonist, APP-07, and evaluated its agonist potency and its selectivity, using these established cell lines, and demonstrated that APP-07 is a potent and highly selective agonist for IP. This assay system will facilitate the development of more selective agonists and antagonists for regulating receptors.
Prosta~mdins
199753,
Febrnatry
IP-Selective Agonist, AFP-07: Chang et al.
-I
0
Ligands
9
(-log
7
5
M)
FIGURE4. Effects of AFP-07 and iloprost on [‘HI PGEz binding to EPI, EP2, EP3 and EP4. The membrane fractions prepared from the indicated receptor-expressing CHO ceils were incubated with 4 nM [5H] PGE2 in the presence of various concentrations of PGE2 (0) AFP-07 (0) or iloprost (A). All values were corrected for nonspecific binding and are expressed as percentages of the respective controls, as described under “Materials and Methods”. The percentages of specific binding to EPl, EP2, EP3 and EP4 are 62%, 82%, 95%, and 74%, respectively. Maximum binding in the controls was 0.1 (EPl), 0.2 (EP2), 0.5 (EP3), and 0.2 (EP4) pmol/mg for [JH] PGE*, respectively. The results shown are the means f S. E. for three independent experiments.
References Samuelsson, B., Goldyne, M., Grandstrom, E., Hamberg, M., Hammarstrom, S., and Malmsten, C. Prostaglandins and thromboxanes. Annu Rev Biochem 47: 997. 1978. Negishi, M., Sugimoto, Y., and Ichikawa, A. Prostanoid receptors and their biological actions. Prog Lipid Res 32: 417. 1993. Coleman, R.A., Kennedy, I., Humphrey, P.P.A., Bunce, K., and Lumley, P. Prostanoids and their receptors. in Comprehensive Medicinal Chemistry (Hansch, C., Sammes, P.G., Taylor, J.B., and Emmett, J.C. eds) 3: 643. 1989. Pergamon Press, Oxford
Prostaglandins 199753, February
89
IP-Selective Agonist, AFP-07: Chang et al. 4. Coleman, R.A., Smith, W.L., and Narumiya, S. VIII. International union of pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 46: 205. 1994 5. Dong, Y.J., Jones, R.L., and Wilson, N.H. Prostaglandin E receptor subtypes in smooth muscle: agonist activities of stable prostacyclin analogues. Br J Pharmacol87: 97.1986. 6. Armstrong, R.A., Lawrence, R.A., and Jones, R.L., Wilson, N.H., and Collier, A. Functional and ligand binding studies suggest heterogeneity of platelet prostacyclin receptors. Br J Pharmacol97: 657. 1989. 7. Namba, T., Oida, H., and Sugimoto, Y. et al. cDNA cloning of a mouse prostacyclin receptor. J Biol Chem 269: 9986. 1994. 8. Watabe, A., Sugimoto, Y., and Honda, A. et al. Cloning and expression of cDNA for a mouse EPl subtype of prostaglandin E receptor. J Biol Chem 268: 20175. 1993. 9. Katsuyama, M., Nishigaki, N., and Sugimoto, Y. et al. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis. FEBS Lett 372: 151. 1995. 10. Sugimoto, Y., Namba, T., and Honda, A. et al. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J Biol Chem 267: 6463.1992. 11. Honda, A., Sugimoto, Y., and Namba, T. et al. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype. J Biol Chem 268: 7759. 1993. 12. Nishigaki, N., Negishi, M., and Honda, A. et al. Identification of prostaglandin E receptor ‘EP2’cloned from mastocytoma cells as EP4 subtype. FEBS Lett 364: 339. 1995. 13. Negishi, M., Sugimoto, Y., Irie, A., Narumiya, S., and Ichikawa, A. Two isoforms of prostaglandin E receptor EP3 subtype. J Biol Chem 268: 95 17. 1993. 14. Matsumura, Y., Shimada, T., and Nakayama, T. et al. Synthesis of 7-fluoro2,4-methylene- 17,20dimethylprosacyclins. Novel stable prostacyclin analogs as potent anti-angina1 agents. Tetrahedron 52: 8771. 1995. 15. Matsumura, Y., Nakano, T., Makino, M., and Morizawa, Y. Novel difluoroprostacyclin analogs with remarkable stability and potent activities. AFMC International Medicinal Chemistry Symposium 95, Tokyo, September 1995. Editor: Dr. P. Halushka
90
Received:
10-23-95
Accepted:
1-19-96
Prostaglandins 199753, F&mwy
*Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan; §Research Center, Asahi Glass Co. Ltd., Yokohama 221, Japan
Recently, we cloned cDNAs for the prostacyclin receptor (IP) and the four mouse PGE receptor subtypes, EPl, EP2, EP3 and EP4, and established Chinese hamster ovary cells that stably express each receptor. We examined the agonist potency and selectivity of AFP-07, a 7,7difluoroprostacyclin derivative, compared with widely used stable prostacyclin analogue, iloprost, using the cells expressing each cloned receptor. AFP-07 strongly displaced the rH] iloprost binding to the IP receptor-expressing cell membranes, the half maximal concentration for the displacement being 3 nM, which was one order lower than that of iloprost. AFP-07 concentration-dependently stimulated CAMP formation in the IP-expressing cells, the half-maximal concentration for the stimulation being 10 PM, which was one order lower than that of iloprost. On the other hand, AFP-07 showed lower affinity for EPl, EP2, EP3 and EP4 than PGEB but iloprost had the same affinity as PGE, for the EPl. These results demonstrate that AFP-07 is a potent and highly selective agonist for the IP receptor. 0 1997 by Elsevier Science Inc.
To
whom correspondence should be addressed. Atsushi Ichikawa, Department of: Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Sakyo-ku 606, Japan, Tel: +81-75-753-4527, Fax: +81-75-753-4557, E-mail: [email protected] Abbreviations used are: PG, prostaglandin; EP, PGE receptor; IP, prostacyclin receptor; CHO, Chinese hamster ovary.
Prostaglaudins 53:83-90, 1997 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0090-6980/97/$17.00 PII SOO90-6980(97)00003-B
IF-Selective Agonist, AFP-07: Chang et al.
Keywords: prostacyclin receptor; PGE receptor; EPl; EP2; EP3; EP4
Introduction Prostacyclin (PGI,) is an unstable prostanoid which, in blood vessels, is largely produced by endothelial cells’. It inhibits platelet aggregation and vasodilatation in pulmonary vascular beds. Other actions of PGI, are suggested to be regulation of renal blood flow, renin release and glomerular filtration rate in the kidney cortex and stimulation of secretion in the stomach and large intestine. The actions of PGIz are presumed to be mediated by a cell surface receptor?. Since PGI, is very labile, a variety of chemically stable PGI analogues has been developed4. Among them, iloprost, a member of carbacyclin, is a potent IP agonist, which is well characterized and widely used, but it also showed potent EPl agonist activity 516.Thus, there is an urgent need to develop highly specific and potent ligands for IP receptor. Recently, we cloned cDNAs for the mouse IP receptor and four PGE receptor subtypes, EPl, EP2, EP3 and EP4, and established Chinese hamster ovary (CHO) cells stably expressing each receptor ’.I2. We developed several fluoroprostacyclin derivatives for IP receptor, and examined the potency and selectivity for these receptors using the cells specifically expressing each receptor. We report here that a 7,7difluoroprostacyclin derivative, AFP-07, is a potent and highly selective IP agonist. Materials and Methods Materials [5,6,8,1 1,12,14,15-3H]PGE, (191 Ci/mmol), [aH] iloprost (14.1 Ci/mmol), iloprost and the 1251-labeled CAMP assay system were obtained from Amersham Corp. PG& was purchased from Funakoshi Pharmaceuticals (Tokyo, Japan). All other chemicals were reagent grade. Establishment of CHO cell line stably expressing each prostanoid receptor has been described previously ’-12. Receptor density of each cell membrane is as follows: IP, 1.2 pmol/mg; EPl, 0.95 pmol/mg; EP2, 1.5 pmol/mg; EP3, 2.3 pmol/mg; and EP4,0.85 pmol/mg. Mock-transfected CHO cells only expressed EP4 receptor among these prostanoid receptors, but the expression level was very low (0.005 pmol/mg). 13H] PGE, or rH] Iloprost Binding Assay For the assay, the membrane fraction (250,000 x g pellet) was prepared
from CHO cells stably expressing the mouse IP receptor or each mouse PGE receptor subtype, as described previously13. The standard assay Prostaglandins 199753,
Febnwy
IP-Selective Agonist, AFP-07: Chang et al. mixture comprised either 20 nM [3H] iloprost (28.2 nCi) or 4 nM [3H] PGEz (76.4 nCi), and 80 ug of the membrane prepared from each receptor-expressing CHO cell type in 100 ul of 10 mM Mes-NaOH, pH 6.0, containing 1 mM EDTA and 10 mM MgCl, (buffer A). After incubation for 1 h at 37”C, the reaction was terminated by the addition of ice-cold buffer A, after which the mixture was rapidly filtered through a Whatman GF/C glass filter. The filter was then washed with ice-cold buffer A and the radioactivity on the filter was measured by scintillation counting. Nonspecific binding was determined using a l,OOO-foldexcess of the respective unlabeled PG in the incubation mixture. The specific binding was calculated by subtracting the nonspecific binding from the total binding. Measurement of CAMP Formation Cyclic AMP levels in CHO cells expressing the IP receptor were determined as reported previously’. Cells cultured in 24-well plates (5 x lo5 cells/well) were washed with 0.5 ml of Hepes-buffered saline containing 140 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl,, 1.2 mM MgClz, 1.2 mM KI-I,P04, 11 mM glucose and 15 mM Hepes (pH 7.4), and preincubated for 5 min. Reactions were started by the addition of agonists along with 0.1 mM Ro-20-1724. After incubation for 10 min at 37”C, the reactions were terminated by the addition of 10% trichloroacetic acid. The CAMP content of the cells was determined by radioimmunoassaying with an Amersham [ ‘251]cAMPassay system. Results and Discussion We developed three types of 7-fluoroprostacyclin derivatives. Fig. 1 shows chemical structures of a 7-monofluoroprostacyclin derivative (AFP-03)‘4, and two 7,7-difluoroprostacyclin derivatives (AFP-06 and AFP-07)15.To compare the binding affinities of these derivatives and iloprost for the IP receptor, we examined the effects of these derivatives and iloprost on specific [3H] iloprost binding to the membrane of CHO cells expressing the IP receptor. As shown in Fig. 2, specific [3H]iloprost binding to the IP receptor was inhibited by these ligands in the order of AFP-07 > AFP-06 > iloprost > AFP-03. From Fig. 2, we obtained Ki values for these ligands using the Cheng-Prussoff equation, and the values are 0.561 (AFP-07), 3.74 @P-06), 9.35 (iloprost), and 16.8 nM (AFP-03). The value for iloprost was 16-fold higher than that for AFP-07. Thus, AFP-07 showed stronger affinity than iloprost. We further compared these ligands, as to agonist activity for the IP receptor, which is coupled to the stimulation of adenylate cyclase. Fig. 3 shows the concentration dependencies of the effects of the fluoropro-
Prostaglandins 199753, February
85
IP-Selective Agonist, AFP-07: Chang et al. COONa
I f
9L 6”
6”
Prostacyclin
&
t
AFP-03
+OONa
AFP-06
AFP-07
FIGURE1. Chemical st~ctures of prostacyciinand three 74uoroprostacyclii derivatives, AFP03, AFP-06 and AFP-07.
stacyclin derivatives and iloprost on the CAMP formation in the IP receptor-expressing cells. To ensure that the change in the CAMP level of the cells is not influenced by alterations in CAMP phosphodiesterase activity, we added a phosphodiesterase inhibitor, Ro-20-1724, to the medium. The ligands concentration-dependently stimulated the increase in the CAMP level of the IP receptor-expressing cells in the order of AFP-07 z AFP-06 > iloprost r AFP-03. The order of agonist potency of these ligands (Fig. 3) was consistent with that of their binding affinities (Fig. 2). However, the ECm values of the response for these ligands are about 100 times less than their Ki values, suggesting that the receptor-effector coupling is more efficient than the ligand-receptor binding. Such variations in coupling efficacy of the PGIz receptor was observed in platelets of various species6. The 7-monofluoroprostacyclin derivative is less potent agonist, and 7,7difluoroprostacyclin derivatives are much more potent. Addition of two fluorides to PGI, structure at C-7 would increase the agonist potency. In addition, AFP-07 is more potent than AFP-06, and they are different only in structure of the w-side chain, indicating that structure of *side chain is also responsible for IP agonist activity.
Prostaglandins 199753,
February
IP-Selective Agonist, AFP-07: Chang et al. 120 u c z JJ
ITi= ?Lo QZ o. z -0 3 -8 0;E .-0 : m
100 80 60 40 20 0 0
10
9
8
Ligands
7
6
5
(-log M)
FIGURE2. Effects of AFP-03, AFP-06, AFP-07 and iloprost on [‘HI iloprost binding to the membrane of IP receptor-expressing CHO ceils. Membrane of IP receptor-expressing CHO cells was incubated with 20 nM [‘HI iloprost and the indicated concentrations of AFP-3 (4, AFP-6 (B), AFP-7 (0) or iloprost (0). Specific 13H] iloprost binding was determined as described under “Materials and Methods”, and was 90%. Maximum binding in the control was 0.7 pmol/mg for [%t] iloprost. The results shown are the means f S. E. for three independent experiments.
Among them, AFP-07 is the most potent IP agonist, its potency being one order of magnitude stronger than that of iloprost. To evaluate the selectivity of AFP-07 and iloprost for the IP receptor and four PGE receptor subtypes, we examined the effects of AFP-07 and iloprost on specific [3H] PGEz binding to CHO cell membranes expressing the four PGE receptor subtypes, EPl, EP2, EP3 and EP4, and compared their binding affinities with those for PGEz. As shown in Fig. 4, specific [3H] PGE, binding to the EPl receptor subtype was inhibited by the ligands in the order of PGE, = iloprost > AFP-07. Iloprost showed the same affinity as PGEz, supporting the notion that iloprost is a potent EP 1 agonist 5*6.Specific 13H]PGE, binding to EP2 was inhibited in the order of PGE, > AFP-07 = iloprost. AFP-07 and iloprost showed the same affinity. Specific [3H]PGEz binding to EP3 was inhibited by these ligands in the order of PGEz > iloprost > AFP-07. The affinity for AFP-07 was lower than that for iloprost. Specific [ 3H] PGE, binding to EP4 was inhibited by these ligands in the order of PGEz > AFP-07 > iloprost. The affinity for AFP-07 was higher than that for iloprost. As judged from these results, AFP-07 shows the weaker affinity than PGE, in all four PGE receptor subtypes, indicating that AFP-07 is a highly selective IP agonist. In contrast, iloprost is not selective to IP receptor; it also binds to the EPl receptor as well as the IP receptor. Prostaglandins 199753, February
87
IP-Selective Agonist, AFP-07: Chang et al.
041
’
0
13
I
I
I
I
I
I
12
11
10
9
8
7
Ligands
I
(-log WI)
FPXJRE3. Efksta of AFP-03, AFF-00, AFP-07 and ibpmst on &fblP W in the IP remptorfwqtorrmra~~~ominat37ocwith AfWB 0, m-07 (*I or Bopmt (0), in the oonbnt woo mwmmdmhmibadunder of 100 @& Re-20-1724. The &lP ‘e and M&hods”. The mutts shown am the mcOm f S. E. for three irMepmdent experkmh.
The potencies of agonists and antagonists for IP receptor have so far been carried out mainly on platelet aggregation and other systems in which the actions of PG12 can be evaluated readily, such as smooth muscle systems3. However, many tissues, including various platelets and smooth muscles, express IP receptor and multiple EP receptors, and in many cases ligands cross-react with multiple receptors. Thus, evaluation of the potency of an agonist or antagonist for a single type of receptor is very difficult. We have isolated cDNA clones encoding the IP receptor and the four PGE receptor subtypes, EPl, EP2, EP3 and EP4, and established cell lines expressing each receptor. Assay systems involving these cell lines, as shown in this work, allow clear evaluation of the potency of an agonist or antagonist for a single receptor. Here we developed an IP-selective agonist, APP-07, and evaluated its agonist potency and its selectivity, using these established cell lines, and demonstrated that APP-07 is a potent and highly selective agonist for IP. This assay system will facilitate the development of more selective agonists and antagonists for regulating receptors.
Prosta~mdins
199753,
Febrnatry
IP-Selective Agonist, AFP-07: Chang et al.
-I
0
Ligands
9
(-log
7
5
M)
FIGURE4. Effects of AFP-07 and iloprost on [‘HI PGEz binding to EPI, EP2, EP3 and EP4. The membrane fractions prepared from the indicated receptor-expressing CHO ceils were incubated with 4 nM [5H] PGE2 in the presence of various concentrations of PGE2 (0) AFP-07 (0) or iloprost (A). All values were corrected for nonspecific binding and are expressed as percentages of the respective controls, as described under “Materials and Methods”. The percentages of specific binding to EPl, EP2, EP3 and EP4 are 62%, 82%, 95%, and 74%, respectively. Maximum binding in the controls was 0.1 (EPl), 0.2 (EP2), 0.5 (EP3), and 0.2 (EP4) pmol/mg for [JH] PGE*, respectively. The results shown are the means f S. E. for three independent experiments.
References Samuelsson, B., Goldyne, M., Grandstrom, E., Hamberg, M., Hammarstrom, S., and Malmsten, C. Prostaglandins and thromboxanes. Annu Rev Biochem 47: 997. 1978. Negishi, M., Sugimoto, Y., and Ichikawa, A. Prostanoid receptors and their biological actions. Prog Lipid Res 32: 417. 1993. Coleman, R.A., Kennedy, I., Humphrey, P.P.A., Bunce, K., and Lumley, P. Prostanoids and their receptors. in Comprehensive Medicinal Chemistry (Hansch, C., Sammes, P.G., Taylor, J.B., and Emmett, J.C. eds) 3: 643. 1989. Pergamon Press, Oxford
Prostaglandins 199753, February
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IP-Selective Agonist, AFP-07: Chang et al. 4. Coleman, R.A., Smith, W.L., and Narumiya, S. VIII. International union of pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 46: 205. 1994 5. Dong, Y.J., Jones, R.L., and Wilson, N.H. Prostaglandin E receptor subtypes in smooth muscle: agonist activities of stable prostacyclin analogues. Br J Pharmacol87: 97.1986. 6. Armstrong, R.A., Lawrence, R.A., and Jones, R.L., Wilson, N.H., and Collier, A. Functional and ligand binding studies suggest heterogeneity of platelet prostacyclin receptors. Br J Pharmacol97: 657. 1989. 7. Namba, T., Oida, H., and Sugimoto, Y. et al. cDNA cloning of a mouse prostacyclin receptor. J Biol Chem 269: 9986. 1994. 8. Watabe, A., Sugimoto, Y., and Honda, A. et al. Cloning and expression of cDNA for a mouse EPl subtype of prostaglandin E receptor. J Biol Chem 268: 20175. 1993. 9. Katsuyama, M., Nishigaki, N., and Sugimoto, Y. et al. The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and northern blot analysis. FEBS Lett 372: 151. 1995. 10. Sugimoto, Y., Namba, T., and Honda, A. et al. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J Biol Chem 267: 6463.1992. 11. Honda, A., Sugimoto, Y., and Namba, T. et al. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype. J Biol Chem 268: 7759. 1993. 12. Nishigaki, N., Negishi, M., and Honda, A. et al. Identification of prostaglandin E receptor ‘EP2’cloned from mastocytoma cells as EP4 subtype. FEBS Lett 364: 339. 1995. 13. Negishi, M., Sugimoto, Y., Irie, A., Narumiya, S., and Ichikawa, A. Two isoforms of prostaglandin E receptor EP3 subtype. J Biol Chem 268: 95 17. 1993. 14. Matsumura, Y., Shimada, T., and Nakayama, T. et al. Synthesis of 7-fluoro2,4-methylene- 17,20dimethylprosacyclins. Novel stable prostacyclin analogs as potent anti-angina1 agents. Tetrahedron 52: 8771. 1995. 15. Matsumura, Y., Nakano, T., Makino, M., and Morizawa, Y. Novel difluoroprostacyclin analogs with remarkable stability and potent activities. AFMC International Medicinal Chemistry Symposium 95, Tokyo, September 1995. Editor: Dr. P. Halushka
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Received:
10-23-95
Accepted:
1-19-96
Prostaglandins 199753, F&mwy