Photoaffinity Labeling of the Angiotensin 11 Receptor (17) N. C. Chaturvedi, W. K. Park, R. R. Smeby, and F. M. Bumpus, J. Med. Chem., 13, 177 (1970). (18) R. Schwyzer,Ergeb. Physiol. Biol. Chem. Exp., 5 3 , l (1963). (19) S. Y. Lin, H. Ellis, B. Weisblum, and T. L. Goodfriend, Biochem. Pharmacol., 19, 651 (1970). (20) S. W. Dietrich, E. C. Jorgensen, P. A. Kollman, and S. 98, 8310 (1976). Rothenberg, J. Am. Chem. SOC.,
Journal of Medicinal Chemistry, 1979, Vol. 22, No. 9 1047 (21) E. C. Jorgensen, G.C. Windridge, W. Patton, and T. C. Lee, J . Med. Chem., 12, 733 (1969). (22) E. C. Jorgensen, G. C. Windridge, and T. C. Lee, J. Med. Chem., 13, 352 (1970). (23) E. C. Jorgensen and R. A. Wiley, J . Pharm. Sci., 52, 122 (1963). (24) E. Schnabel, Justus Liebigs Ann. Chem., 702, 188 (1967).
Photoaffinity Labeling of the Angiotensin I1 Receptor. 3. Receptor Inactivation with Photolabile Hormone Analogues Emanuel H. F. Escher* and Gagtan Guillemettel Department de Pharmacologie, Facultd de Me‘decine, Universitc? de Sherbrooke, Sherbrooke, Que‘bec, J l H 5N4, Canada, Received February 13, 1979 It has been shown that the receptor of angiotensin I1 (AT) in rabbit aorta strips, rat aorta, and rat stomach can ([ Sar’]AT) be blocked specifically and irreversibly by several photolabile analogues of Sar-Arg-Val-Tyr-Val-His-Pro-Phe with irradiation. The effectiveness of a photolabel with light of wavelength 365 nm depends on the labeling amino L-4’-diazoniumphenylalanine, or L-4’-azidophenylalanine)and on its position in the acid (L-4’-nitrophenylalanine, peptide (replacing Tyr4and/or Phe8). The (4’-azido)Phe-containing analogues are all good to fair photoinactivators. and Their decreasing order of effectiveness is as follows: [Sar’,(4’-azido)Phe8]AT,[Sar’,(4’-a~ido)Phe~,~]AT, [Sar’,(4’-azido)Phe4]AT. The (4’-nitro)Phe analogues show the opposite relation: the good ligand [Sar1,(4’exhibits good, specific photoinactivation. nitro)Phe8]ATis almost ineffective, but the nonligand [Sar1,(4-nitro)Phe4]AT This can be explained by the existence of a different photolysis pathway for (4’-nitro)Phe: this analogue probably undergoes a multiphoton decay with a long-lived first excited state. A peptide in this state may differ in its pharmacological properties from the ground state and become a ligand. The isolation and purification of peptide hormone reradiation, the peptides exhibit great differences in bioceptors are the goals of continuing research efforts. Up logical activity, which are summarized in Table I. t o now, all the successful isolation^^-^ were of receptors The expected pharmacological effect of photoaffinity which retained hormone-binding ability after solubilization labeling the AT receptor was either permanent stimulation of the cell membrane. Unfortunately, several peptide or permanent block of the AT response, giving further hormone receptors lose their binding ability upon total information about the receptors’ kinetics and phenomena solubilization and have, thus, resisted isolation. For exsuch as tachyphylaxis. If the photolabeling process does ample, the homogenization, solubilization, and subsequent not significantly alter the receptor conformation, the isolation of the angiotensin I1 (AT) receptor has often been following possible consequences can be considered: a attempted. Preparations from adrenals have always lost permanently activated response would suggest an occuAT affinity upon addition of detergent.5 A preparation pation mechanism where the receptor is locked in a “on” from rabbit aorta retained some affinity in the presence position. This has been observed with a photoaffinity of low detergent concentration, but no isolation was labeling experiment on the gastrin receptor.’* Conversely, achieved.6 Affinity labeling studies7 were similarly una permanent block could, under certain conditions, support successful. the rate model.15 In this theory, transition between In a previous paper,8 we described the aim of this reunoccupied and occupied receptor is the response releasing search and the synthesis and biological activities of several principle. AT analogues designed for photoaffinity labeling of the From earlier results with chymotrypsin” and other AT receptor. In a later paper,g these biological activities experiments,16 it was known that photoactivated labels were discussed in more detail and new conclusions were which are not in contact with the “receptor” can rearrange drawn about structure-activity relationships of AT. We and react subsequently by nucleophile attack on any part now report the influence of photolabile peptide analogues of the proteins in the cell membrane. The work with on several AT-sensitive tissues in the presence of ultrachymotrypsin showed that this undesirable side reaction violet light. In a preliminary report, we have already can be eliminated by the addition of the scavenger Lpresented the first example of irreversible and specific (4’-amino)Phe to the photolysis solution. It was also shown inactivation of the AT response on rabbit aorta strip with that the chymotrypsin “receptor” (tosyl-hole) can be one of our peptides.l0 protected from photolabeling by a specific reversible inThe investigated peptides (see Table I) contained either hibitor and that this protection was strictly competitive. ~-(4’-nitro)Phe,~-(4’-azido)Phe,or ~-(4’-diazonium)Phein Digestion of photolabeled chymotrypsin showed that the positions 4 and/or 8 of Sar-Arg-Val-Tyr-Val-His-Pro-Pheincorporation of the previously competitive photolabel was ([Sar’] AT). The azido compounds are well-known phohighly specific for the chymotrypsin “re~eptor”.’~ Another toaffinity labels,”J2 but the nitro compounds have also possible side reaction which has been established’O is proven useful.” The diazonium compounds are potential photosensitized or photooxidized inactivation due to irphotolabels known to be photosensitive and to yield radiation of preformed photoproducts. Experiments with radicals upon photoly~is.’~ In the absence of photolysing prephotolyzed peptides showed that all our analogues 0022-26231791 1822-lO41$Ol.OO J O
0 1979 American Chemical Society
Escher, Guillernette
1048 Journal of Medicinal Chemistry, 1979, Vol. 22, No. 9
Table I. Photoinactivation of the Angiotensin I1 Response of Rabbit Aorta Stripsa photolabeling expts biol act. in the dark' compet. 1st 2nd concn, no. peptide aE pD, re1 aff, % pM Na response protect.b treatment treatment 10018 92. 10 85i8 1.0 9.23 100 0.2 1001 1 0 0 [Sar' ]AT [Sar' ,( 4'-NO ,)Phe4]AT [Sar',(4'-NO,)Phes]AT [Sar I , ( 4'-NO,)Phe4%8] AT
0 0.46 0.5
