Characterization of subtype selection properties of R-(-)-DM-phencynonate hydrochloride and its racemate on muscarinic receptors

Abstract

Abstract: In order to compare the potential selectivity of R-(-)-DM-phencynonate hydrochloride with its racemate (±)-DM-phencynonate hydrochloride on acetylcholine muscarinic receptor subtypes, the five human acetylcholine muscarinic receptor subtypes (M1–M5) (CHO-hm1-5R) were cloned and expressed in Chinese hamster ovary (CHO-K1) cell line. The specific mRNAs of the five acetylcholine muscarinic receptor subtypes were detected by the reverse transcription-polymerase chain reaction (RT-PCR) method, demonstrating the definite expression of muscarinic receptor subtype genes (CHO-hm1-5R). The affinity and saturability of different muscarinic receptor subtypes to [³H] N-methylscopolamine ([³H]-NMS) were obtained by radioligand binding assay. Equilibrium binding assay revealed that the maximum binding capacity of [³H]-NMS (Bmax value) to CHO-hm1-5R were 40.22±3.23, 24.53±4.11, 29.65±2.65, 25.41±2.46, 32.78±4.81 pmol/mg·protein, respectively. Kd values of [³H]-NMS to muscarinic receptors M1 to M5 were 0.97±0.22, 1.16±0.14, 0.99±0.06, 0.56±0.08, 1.12±0.06 nM, respectively. R-(-)-DM-phencynonate hydrochloride was found to block the M4 receptor with a much higher potency (pD2 = 7.48) than those displayed on M1 (pD2 = 6.20), M2(pD2 = 5.99), M3(pD2 = 5.99) and M5(pD2 = 6.70) subtypes. However, for (±)-DM-phencynonate hydrochloride, no significant subtype receptor selectivity was found. Both (±)-DM- and R-(-)-DM-phencynonate hydrochloride showed allosteric effects on muscarinic receptors, the Hill coefficient (nH) of five receptor subtypes was less than 1, respectively. The results revealed that R-(-)-DM-phencynonate hydrochloride showed selectivity torwards M4 subtype, and there were allosteric effects for both R-(-)-DM-phencynonate hydrochloride and (±)-DM-phencynonate hydrochloride on muscarinic receptors.

Key words: Optical isomers, Optical isomers, Muscarinic acetylcholine receptors, Muscarinic acetylcholine receptors, Subtype receptor selectivity, Subtype receptor selectivity, Radioligand binding assay, Radioligand binding assay

CLC Number:

R962.2

Supporting:

Li-Yun Wang, Hong-Liang Sun, Nan Mou, Bo-Hua Zhong, Ke-Liang Liu, Jian-Quan Zheng^* . Characterization of subtype selection properties of R-(-)-DM-phencynonate hydrochloride and its racemate on muscarinic receptors [J]. .

References

[1] Felder, C.C.; Bymaster, F.P.; Ward, J.; Delapp, N. J. Med. Chem. 2000, 43, 4333-4353.

[2] Eglen, R.M.; Choppin, A.; Watson, N.T. Trends Pharmacol. Sci. 2001, 22, 409-414.

[3] Wess, J. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 423-450.

[4] Dörje, F.; Levey, A.I.; Brann, M.R. Mol. Pharmacol. 1991, 40, 459-462.

[5] Caufield, M.P. Pharmacol. Ther. 1993, 58, 319-379.

[6] Wess, J. Crit. Rev. Neurobiol. 1996, 10, 69-99.

[7] Abrams, P.; Andersson, K.E.; Buccafusco, J.J.; Chapple, C.; de Groat, W.C.; Fryer, A.D.; Kay, G.; Laties, A.; Nathanson, N.M.; Pasricha, P.J.; Wein, A.J. Br. J. Pharmacol. 2006, 148, 565-578.

[8] Volpicelli, L.A.; Levey, A.I. Prog. Brain Res. 2004, 145, 59-66.

[9] Shash, R.R.; Midgley, J.M.; Branch, S.K. Adv. Drug React. Toxicol. Rev. 1998, 17, 145-190.

[10] Caldwell, J. Modern Drug Discov. 1999, 2, 51-60.

[11] Deng, Y.J.; Zhang, Y.M. Chin. J. New Drugs. 2001, 10, 45-34.

[12] Wang, L.Y.; Zheng, J.Q.; Wang, Y.; Zhong, B.H.; Ruan, J.X.; Liu, K.L.; Acta Pharmacol. Sin. 2005, 26, 1187-1192.

[13] Caulfield, M.P.; Birdsall, N.J.M. Pharmacol. Rev. 1998, 50, 279-290.

[14] Wei, J.; Walton, E.A.; Milici, A.; Buccafusco, J.J. J. Neurochem. 1994, 63, 815-821.

[15] Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. J. Biol. Chem. 1951, 193, 265-275.

[16] Cheng, Y.; Prusoff, W.H. Biochem. Pharmacol. 1973, 22, 3099-3108.

[17] Buckley, N.J.; Bonner, T.I.; Buckley, C.M.; Brann, M.R. Mol. Pharmacol. 1989, 35, 469-476.

[18] Pavia, J.; Marquez, E.; Laukkonen, S.; Martos, F.; Gomez, A.; Sanchez, de la.; Cuesta, F. Methods Find Exp. Clin Pharmacol. 1991, 13, 653-660.

[19] Giraldo, E.; Martos, F.; Gomez, A.; Garcia, A.; Vigano, M.A.; Ladinsky, H.; Sanchez, de La.; Cuesta, F. Life Sci. 1988, 43, 1507-1515.

[20] Khattar, S.K.; Bora, R.S.; Priyadarsiny, P.; Gupta, D.; Khanna, A.; Narayanan, K.L.; Babu, V.; Chugh, A.; Saini, K.S. Biotechnol. lett. 2006, 28, 121-129.

[21] Wang, S.Z.; El-fakahany, E.E. J. Pharmacol. Exp. Ther. 1993, 266, 237-243.

[22] Andrew, S.; Felix, B.; David, F. Aust. Prescr. 2004, 27, 47-49.

[23] Christopoulos, A.; Lanzafame, A.; Mitchelson, F. Clin. Exp. Pharmacol. Physiol. 1998, 25(3-4), 185-194.

[24] Lazareno, S.; Gharagozloo, P.; Kuonen, D.; Popham, A.; Birdsall, N.J. Mol. Pharmacol. 1998, 53, 573-589.

[25] Jakubík, J.; Bacáková, L.; El-fakahany, E.E.; Tucek, S. J. Pharmacol. Exp. Ther. 1995, 274, 1077-1083.