Proc. Natl. Acad. Sci. USAVol. 84, pp. 8095-8099, November 1987Medical Sciences

Interaction of tachykinins with their receptors studied with cyclicanalogues of substance P and neurokinin B

(conformation restriction/rat brain synaptosomes/guinea pig ileum/vascular preparations)

OLIVIER PLOUX*, SOLANGE LAVIELLE*t, GERARD CHASSAING*, SYLVIANE JULIEN*, ANDREE MARQUET*,PEDRO D'ORLEANS-JUSTEt, STEPHANE DIONO, DOMENICO REGOLIt, JEAN-CLAUDE BEAUJOUAN§,LENA BERGSTROM§, YVETTE TORRENS§, AND JACQUES GLOWINSKI§*Laboratoire de Chimie Organique Biologique, Centre National de la Recherche Scientifique Unitd Associde 493, Universitd P. et M. Curie, 4, place Jussieu,75252 Paris Cedex 05, France; tDepartement de Pharmacologie, Facultd de MWdecine, Universitd de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada; and§Chaire de Neuropharmacologie, Institut National de la Santd et de la Recherche Mddicale Unitd 114, College de France, 11, place M. Berthelot, 75230Paris Cedex 05, France

Communicated by Jean-Marie Lehn, June 29, 1987 (received for review April 14, 1986)

ABSTRACT The activities of two groups of cyclic agonistsof substance P (SP) have been studied. The disulfide bridgeconstraints have been designed on the basis of conformationalstudies on SP and physalaemin indicating an a-helical structurefor the core of these two tachykinins (group I) and a folding ofthe C-terminal carboxamide towards the side chains of theglutamines 5 and 6 (group II). Only peptides simulating thea-helix present substantial potencies. [Cys3,6JSP is as active asSP in inhibiting '25I-labeled Bolton and Hunter SP-specificbinding on rat brain synaptosomes and on dog carotid bioas-say, two assays specific for the neurokinin 1 receptor. More-over, [Cys3,6]SP is as potent as neurokinin B in inhibiting125I-labeled Bolton and Hunter eledoisin-specific binding on ratcortical synaptosomes as well as in stimulating rat portal vein,two tests specific for the neurokinin 3 receptor. Interestingly,in contrast to neurokinin B, [Cys3,6JSP is a weak agonist of theneurokinin 2 receptor subtype, as evidenced by its bindingpotency in inhibiting 3H-labeled neurokinin A-specific bindingon rat duodenum and in inducing the contractions of the rabbitpulmonary artery, a neurokinin 2-type bioassay. To increasethe specificity of the cyclic analogue [Cys3"6]SP positions 8 and9 were modified. [Cys3'6,Tyr8,Ala9SP is slightly less selectivethan SP for the neurokinin 1 receptor subtype. [Cys2,5]neuro-kinin B constitutes a selective cyclic agonist for the neurokinin3 receptor. The very weak potencies of the peptides from groupII indicate that a certain degree of flexibility in the C-terminalmoiety is required. Collectively, these results suggest that theneurokinin 1 and neurokinin 3 tachykinin receptors mayrecognize a similar three-dimensional structure of the core ofthe tachykinins. Different orientations of the common C-terminal tripeptide may be related to the selectivity for thedifferent receptor subtypes.

Conformational analyses of tachykinins (Table 1) were firstdone on the substance P(5-11) fragment because an earlierreport indicated a crucial role of the C-terminal hexapeptidesof substance P (SP) and eledoisin (ELE) for spasmogenicactivity on the guinea pig ileum (1). Conformational energycalculations have been conducted using both SP and SP(5-11) (2-4). The most stable predicted conformations exhibit aturn around either the Gly-9 and Phe-8 or Phe-7 and Phe-8residues leading to a spatial proximity of the C-terminalmethionine and of the glutamine side chains (2-7). However,we have demonstrated the existence in the rat brain (8, 9) andspinal cord (10) of a specific binding site for SP that requiresthe whole sequence ofSP for full binding potency. This resultwas further confirmed by several groups (11-13). This ob-

servation led us to study the conformational behavior of theundecapeptide in different solvents using NMR and CDspectroscopies. The main features of the conformationalmodel deduced from this study were the flexibility of theN-terminal Arg-Pro-Lys, the a-helical structure of the SPcore, Pro-Gln-Gln-Phe-Phe, and, in agreement with confor-mational energy calculations, the folding of the C-terminalcarboxamide towards the primary amides of both glutamines(14). Physalaemin, a nonmammalian tachykinin, that pos-sesses a pharmacological profile similar to that of SP (15) wasshown to adopt a very close spatial structure in methanol(16).The aim of this study was to compare the biological and

binding potencies of two groups of SP cyclic analoguessimulating two different three-dimensional features of SP.Analogues of group I were designed to mimic the a-helicalstructure from Pro4 through the Phe8 moiety (14), whereas thecompounds of the second group were synthesized to bringcloser the C-terminal methionine amide and the glutamineside-chains (2-7, 14). Because some of these analogues couldeventually recognize the tachykinin receptors of the neuro-kinin 2 (NK2) and neurokinin 3 (NK3) types in addition tothose of neurokinin 1 (NK1), the potency of all synthesizedanalogues was first tested on the guinea pig ileum (gpI), whichhas been shown to possess the three subtypes of tachykininreceptors (17-19). The selectivities of the analogues werethen determined using binding and pharmacological testsspecific for the SP, neurokinin B (NKB), and neurokinin A(NKA) receptor types. Binding studies were done withappropriate ligands and tissue preparations that allow for thediscrimination of the three different tachykinin binding sitesdiscovered in mammals-i.e., 125I-labeled Bolton and HunterSP (125I-BHSP)- and 125I-labeled Bolton and Hunter eledoisin(125I-BHELE)-specific bindings on rat brain synaptosomes(9, 20) and 3H-labeled NKA-specific binding on membranesfrom the rat duodenum (41). Indeed, pharmacological inves-tigations have established that SP, NKB, and NKA are thepreferred endogenous ligands for these respective bindingsites (19, 21). Finally, the potencies of these cyclic analogueswere evaluated in three vascular preparations, the dogcarotid artery (dCA), the rat portal vein (rPV), and the rabbitpulmonary artery (rPA) (15), which provide sensitive andselective biological assays for NK1, NK3, and NK2 recep-

Abbreviations: SP, substance P; 125I-BHSP, 125I-labeled Bolton andHunter SP; 125I-BHELE, 125I-labeled Bolton and Hunter eledoisin;gpl, guinea pig ileum; dCA, dog carotid artery; rPV, rat portal vein;rPA, rabbit pulmonary artery; NKA, neurokinin A; NKB, neuro-kinin B; NK1, NK2, NK3, tachykinin receptors 1, 2, and 3 named inaccordance with the recommendation of the committee from "Sub-stance P and Neurokinins" symposium (July, 1986; Montreal).tTo whom reprint requests should be addressed.

8095

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8096 Medical Sciences: Ploux et al.

Table 1. Amino acid sequences of the most commonly studied tachykinins

Tachykinin Sequence

SP Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2PHY pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2NKB Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2ELE pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2NKA His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2

PHY, physalaemin; ELE, eledoisin.

tors, respectively, as evidenced by comparing the affinities oftachykinins and of their synthetic analogues (22, 23).

MATERIALS AND METHODSPeptides Synthesis. SP, NKB, NKA, and the cyclic ana-

logues of SP were synthesized by solid-phase methodologyand purified by gel filtration and by ion-exchange chroma-tography (24). The disulfide bridge cyclizations were doneeither by classical high-dilution ferricyanide oxidation (25) orby a new procedure involving a polymeric support (26). Thephysical parameters of the cyclic compounds, whose syn-theses have not been previously described, are listed in Table2. As an example, the synthesis of [Cys3'6,Tyr8]SP is de-scribed in detail.Methylbenzhydrylamine resin (0.40 mmol/g of resin; 2.25

g) was used as the solid support. N-a-tert-butyloxycarbonyl-protected amino acids were used throughout the synthesis.Side-chain functional group protection consisted of benzylfor tyrosine, p-methoxybenzyl for cysteine and tosyl forarginine. All amino acids from the C-terminal pentapeptidewere activated by the dicyclohexylcarbodiimide-1-hydroxy-benzotriazole in dimethylformamide/CH2Cl2, 1:5 (27). Thesubsequent residues were coupled in dimethylformamide.N-a-Boc-Gln5 was introduced as its p-nitrophenyl ester.Coupling efficiency was monitored with the Kaiser test (28).After removal of the last N-a-Boc protecting group, one halfof the'peptidyl resin was treated with 1.5 ml of anisole, 0.25ml of dimethyl sulfide, and 10 ml of HF per g of resin for 30min at -20°C and 30 min at 0°C. The peptide solution wasdiluted to 400 ml with degassed water and added dropwise toa ferricyanide solution to form the disulfide bridge as de-scribed by Rivier et al. (25). After oxidation, the solution waschromatographed on an anion-exchange resin, AG 3-X4(acetate form), to remove the ferri- and ferrocyanide anionsand was lyophilized to yield 1.26 g of crude material, whichwas purified by batch. Starting from 335 mg of crude peptide,103 mg was recovered from filtration on Sephadex G-25F(25% acetic acid), which was purified to homogeneity on

Table 2. Characterization of the SP cyclic analoguesTLC Rft in

PeeYieldt solution HPLC§Peptides* % A B % Rt [a]WO

[Cys3'6]SP 16 0.24 0.70 24.0 22.7 -10.4[Cys3'6,Tyr8]SP 16 0.38 0.69 21.6 24.2 -13.2[Cys3'6,Val8]SP 16 0.22 0.70 21.6 15.5 -17.0[Cys3'6,Tyr8,Ala]SP 4 0.19 0.69 21.6 29.0 -12.5

*Results obtained from amino acid analyses were in accordance withexpected values.

tYield of the final product based on the amino substitution ofmethylbenzhydrylamine resin.tA, 1-butanol/acetic acid/water, 4:1:5 (vol/vol); B, 1-butanol/pyridine/acetic acid/water, 5:5:1:4 (vol/vol).§,uBondapak C18, isocratic 1.5 ml/min, % CH3CN in 0.25 Mtriethylammonium phosphate buffer, pH 3.0. R., retention time inmin.

9cl, 10% acetic acid.

carboxymethylcellulose exchange resin (1.5 cm x 15 cmcolumn) using a linear gradient [400 ml of 0.01 M NH4OAc,pH4.5/CH3CN, 1:1 (vol/vol) and 400 ml of0.3M NH4OAc, pH6.5] to yield 27 mg. Total yield after cleavage of all thepeptidyl-resin and purification, as described, was 110 mg(16% starting from the amino substitution of the methylbenz-hydrylamine resin).

Binding Assays on Rat Brain Synaptosomes. The radiolig-ands 1251-BHSP and 125I-BHELE were obtained by acylationof SP and eledoisin, respectively, with 125I-Bolton andHunter reagent (2000 Ci/mmol, mono-iododerivative fromAmersham; 1 Ci = 37 GBq). Crude synaptosomal fractionsfrom rat brain minus the cerebral cortex and from rat cerebralcortex for the 1251-BHSP and I251-BHELE binding assayswere used, respectively. Synaptosomes were prepared asdescribed previously (9, 20), the final pellet being'resuspend-ed in a Krebs-Ringer phosphate buffer containing bovineserum albumin (0.4 mg/ml), bacitracin (30 ,ug/ml), andglucose (1 mg/ml). Finally, synaptosomes were incubated inEppendorf tubes at 20°C in 200 gl of buffer containing 40 pMof 125I-BHSP (5 min) or 125I-BHELE (15 min) and increasingconcentrations of peptides (9, 20).3H-NKA Binding on Membranes from Rat Duodenum. A

membrane suspension of rat duodenal smooth muscle wasprepared according to described procedure (17). Twentymicroliters of the membrane suspension (300 ug of protein)was incubated with 3H-NKA (0.9 nM, specific activity 75Ci/mmol) with or without unlabeled ligand in a final volumeof 200,ul for 25 min at 20°C. Centrifugations and'washingswere done as described for 125I-BHSP and I251-BHELEbinding assays (9, 20). The pellet-bound radioactivity wascounted in a LKB-Wallace (Rockville, MD) liquid spectrom-eter.

Bioassays. Peptides were tested on tissue taken from guineapigs (male, Charles River Breeding Laboratories, 400-450 g),rats (male, Sprague-Dawley, 250-300 g), rabbits (of eithersex, New Zealand, 1.0-1.5 kg) and dogs (German Shepherd,15-20 kg). All animals were killed by stunning and exsan-guination, except for the dogs, which were anesthetized withsodium pentobarbital (50 mg/kg i.p.) and killed by hemor-rhage.The terminal portion of the gpl was used to obtain strips of

the longitudinal smooth muscle. A 1-cm-long strip of gpI wassuspended 'in a 12.5-ml organ bath containing oxygenatedTyrode's solution at 37°C under a tension of 0.5 g. The dogcarotid arteries were rapidly dissected with care to preservethe endothelium (15). The rat portal-mesenteric veins werecleared in situ of surrounding tissues and then cut longitudi-nally to make segments (29). Helicoidal rings of rabbitpulmonary arteries were prepared from tissues in which theendothelium had been removed by inserting a thin glasscoated with filter paper in the lumen of the artery (30).Preparations were suspended in 10-ml organ baths containingoxygenated (95% 02/5% C02) Krebs solution at 37°C, undera tension of 2 g for the dCA, 0.5 g for the'rPV and 1 g for rPA.Relaxations ofthe dCA, previously contracted with noradren-aline (0.02 -,M), as well as the contractions of the rPV andrPA in response to tachykinin-related peptides were recordedisometrically. In the gpI assay, concentration-response

Proc. Natl. Acad. Sci. USA 84 (1987)

Proc. Natl. Acad. Sci. USA 84 (1987) 8097

curves were obtained using either individual dose assay(10-min resting period between two additions) or a cumula-tive dose assay (time between additions and washing was <40s and time between two consecutive dose response curveswas >12 min) (24). Because the dCA and rPA respond totachykinins with stable relaxations and contractions, cumu-lative concentration-response curves were measured with allpeptides to evaluate the maximum effects and the averageconcentration producing 50% maximum effect (EC50). In therPV, the effect of peptides was measured by applying indi-vidual concentrations and leaving at least 15 min between twoapplications.

RESULTS

Correlation Between the Binding and the Biological Assays.In preliminary experiments, excellent correlations were ob-served between the respective affinities of tachykinins and ofseveral noncyclic analogues on 125I-BHSP-, 125I-BHSenk-tide-{N-a-([1251]desamino-3-iodotyrosl)-[Asp5,6,N-methyl-Phe8]SP(5-11)} (42), and 125I-labeled Bolton and HunterNKA-specific bindings and their potencies on dCA, rPV, and

-log EC5*(M)

Is

3

I-i

4J

50-J00n

C

7

S

rPA bioassays, respectively (31). With the active cyclicanalogues similar correlations were obtained using 125[-BHSP, 1251-BHELE, 3H-NKA and the same three bioassays,as shown in Fig. 1.

Binding Potencies and Biological Activities of TachykininCyclic Analogues from Group I. As indicated in Table 3, thecyclic analogues having a -bridgehead in position 7 or 8[D-Cys4,Cys7]SP, [D-Cys5,Cys8]SP were weak agonists onNK1, NK3, and NK2 specific assays. In contrast the cycliza-tion involving residues 3 and 6 of SP yielded active peptides,the L-Cys3 L-Cys6 being more potent than the D-CQS3_L-CYS6cyclic analogue. [Cys3,6]SP was found to be almost equi-potent with SP and NKB, when tested, respectively, on NK1(125I-BHSP-specific binding and dCA bioassay) and NK3(125I-BHELE-specific binding and rPV bioassay) specificassays. [Cys3'6]SP presented a weak potency comparablewith that of SP on NK2-specific tests (i.e., 3H-NKA-specificbinding site on rat duodenum and rPA bioassay).

Modifications of Xaa in the common C-terminal pentapep-tide Phe-Xaa-Gly-Leu-Met-NH2 by tyrosine or valine affect-ed the potency or the selectivity of the cyclic analogue. Thus,[Cys3'6,Tyr8]SP was slightly more potent than [Cys3'6]SP, but

SPS.

7

1

03

AI

5 6 7 8

BINDING AFFINITY

9 10- log ICso(M)

FIG. 1. Correlations between the biological activities and the binding affinities.*, 1251-BHSP/dCA, NK1 receptor; A, '25I-BHELE/rPV, NK3receptor; +, 3H-NKA/rPA, NK2 receptor. Active tachykinin analogues were numbered from 1 to 10. 1, [Cys3,6]SP; 2, [Cys3 6,Tyr8]SP; 3,[Cys36,Val8]SP; 4, [CyS3.6,Tyr8,AIa9]SP; 5, [D-Cys3,Cys6]SP; 6, [D-Cys4,Cys7]SP; 7, [Cys59]SP; 8, [Hcy59]SP; 9, [Cys5"1]SP; and 10,[Hcy5"l]SP. Hcy, homocysteine.

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Table 3. Binding affinities and biological activities of the tachykinin cyclic analogues

NK1 receptor NK3 receptor NK2 receptor

gpI 125IBHSP dCA 125I-BHELE rPV 3H-NKA rPAPeptides EC5o, nM IC5o, nM EC~o, nM IC50, nM EC50, nM IC50, nM EC50, nM

UnmodifiedSP 2 0.64 0.10 130 1500 206 740NKB 2.2 2200 1.30 5.1 21 33 35NKA 5.3 140 0.40 100 355 7.4 6.0

Cyclic NKB[Cys2'5]NKB 2.1 3400 0.90 6.9 7.6 1300 660

Group I cyclic analogues[Cys36]SP 2.5 1.3 0.18 6.4 44 5200 676[Cys36,Tyr8]SP 2.2 0.42 0.06 3.0 32 5000 955[Cys36,Val8]SP 2.8 1.6 0.49 1.6 10 63 72[Cys3.6,Tyr8,Ala9]SP 1.6 0.67 0.04 41 209 4550 630[D-Cys3,Cys6]SP 4.0 20 0.22 72 355 2900 Inac[D-Cys4,Cys7]SP 100 1700 34 >104 >104 >104 >104[D-Cys5,Cys8]SP 70 200 ND 1400 ND >104 ND

Group II cyclic analogue[Cys5.9]SP 100 920 39 >104 Inac >104 Inac[Hcy5'9]SP 100 530 35 >104 Inac -104 Inac[D-Cys5,Hcy10]SP 2000 1300 ND >104 ND -104 ND[Cys5s11]Sp 400 360 26 >104 lnac >104 Inac[Hcy5.'1]Sp 330 270 16 >104 Inac -104 Inac

Binding data represent results obtained from 2 to 10 independent experiments run in triplicate. The radioactive ligand and increasingconcentrations of the analogues to be tested, from 10-11 M to 10-5 M, were mixed and incubated with rat brain synaptosomes (125I-BHSP and125I-BHELE radioreceptor assays) or with rat duodenal membranes (3H-NKA binding assay). Peptide concentrations required to inhibit thespecific binding of the radioligand to either synaptosomes or membranes by 50%o (IC50) were estimated from the Hill plots of the titration curves.SDs were not >68%, 75%, and 50%o of the means for '25I-BHSP, 125I-BHELE, and 3H-NKA experiments, respectively. Nonspecific binding,determined in the presence of 10-6 M of the corresponding unlabeled ligand, represented 25%, 11%, and 25% of the total binding in the threedifferent assays, respectively. Each peptide was tested in at least six different preparations; increasing concentrations of peptides were testedover a range of 3 or 4 logarithmic units. Preparations that showed large variations of sensitivity were discarded. In general, the EC50 values ofeach peptide in various experiments did not show variations >10%. Hcy, homocysteine; ND, not determined; Inac, inactive.

the NK1/NK3 and NK1/NK2 respective selectivities weresimilar. The potency of [Cys3'6,Val8]SP for the NK1 and NK3receptor was only slightly affected when compared with thatof [Cys3'6]SP, whereas that for the NK2 receptors wasdrastically increased, leading to the less selective potentcyclic analogue. The introduction of an alanine into position9 led to the most selective cyclic peptide for the NK1receptor. [Cys3,6,Tyr8,Ala9]SP showed comparable NK1/NK3 and NK1/NK2 selectivities to those observed with theendogenous SP.

Binding Potencies and Biological Activities of TachykininCyclic Analogues from Group II. The cyclizations involvingpositions 5 and 9, 5 and 10, or 5 and 11, designed in order tomimic the spatial proximity of the three primary amides, ledto analogues with reduced activity on NK1-specific assaysand no activity on NK3 and NK2 specific assays (Table 3).

DISCUSSIONThe topography of a binding site of a ligand can be viewed asthe imprint of the three-dimensional structure of the sub-strate. The conformation of the peptide deduced from con-formational analyses (NMR, CD spectroscopies) and/orfrom energy calculations should provide insights about thestructural requirements of the binding protein. However,small peptides are flexible and normally exist in aqueoussolution as an equilibrium mixture of conformers. Conse-quently, the question arises as to whether the conformationobserved in certain solvents or obtained from semi-empiricalcalculations has any relevance to the conformation thatinteracts with the binding site. The design of constrainedcyclic analogues of the substrate that simulate predictedpredominant conformers is a decisive contribution for over-coming this problem. When active, such rigidified analoguesprovide a more accurate approach of the bioactive confor-

mation (32) and are valuable pharmacological probes be-cause, generally, they are more resistant to proteolyticdegradation (33).Among all the compounds made in our laboratory, the

cyclization of SP involving positions 3 and 6 was the only oneleading to potent agonists of tachykinins. This type ofconstraint was selected on the basis of our conformationalanalyses on SP (14) and physalaemin (16), predicting ana-helical conformation for the core of the two peptides. Onepoint of particular interest is that [Cys3'6]SP had very closepotencies, in the nanomolar range, for the NK1 and NK3receptors, whereas SP is a very poor substrate for the NK3receptor. Moreover, NKB has a rather high activity for theNK2 receptor, whereas [Cys3,6]SP is less potent by a factorof 50-150 than NKB.To increase the specificity of the cyclic analogue [Cys3'6]-

SP for the NK1 receptor, a few modifications were made. Theintroduction of a tyrosine in position 8, suggested by thehigher potency of [Tyr8]SP when compared with SP (9),yielded the most potent cyclic analogue. However, [Cys3'6,Tyr8ISP exhibited a selectivity similar to that of [Cys3'6]SP.Because [Ala9]SP is more specific for the NK1 receptor thanSP itself (34, 35), Gly-9 was substituted by Ala-9 yielding[Cys3'6,Tyr8,Ala9]SP. This cyclic analogue was more selec-tive than [Cys3'6,Tyr8]SP for the NK1 receptor, being almostas selective as SP for this receptor subtype. The replacementof the aromatic residue phenylalanine or tyrosine in position8 by the aliphatic side chain of valine, which is present in theNKB and NKA sequences, provided the least selective cycliccompound. [Cys3,6,ValS]SP possessed only -0.1 the potencyofNKA for the NK2 receptor, suggesting a crucial role ofthevaline residue for the recognition of this tachykinin receptorsubtype.As previously reported (36), reintroduction of the amino

acid side chains belonging to the sequence of NKB, leading

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Proc. Natl. Acad. Sci. USA 84 (1987) 8099

to [Cys2'5]NKB, increases the peptide specificity towards theNK3 receptor compared with the NK1 receptor. The avail-ability of NKA-specific assays enabled us to establish, in thepresent study, that in contrast to NKB, [Cys2,5]NKB has avery low potency, in the micromolar range, for the NK2receptor (Table 3). Thus, [Cys2'5]NKB constitutes a selectivecyclic agonist for the NK3 receptor.

Finally, in agreement with results obtained by other groups(37-40), the cyclizations simulating the folding of the C-terminal tripeptide toward the two glutamines led to veryweak agonists of SP. Furthermore, the corresponding com-pounds were found to be completely inactive on NK3 andNK2 assays. As we have recently proposed, on the basis ofthe high potencies of [Met5]SP and [Met6]SP for the NK1receptor (24), Gly-Leu-Met-NH2 must undergo a conforma-tional change when interacting with the binding site, andtherefore a certain degree of flexibility in the C-terminalsequence is required.The constrained agonists of SP synthesized in this study

demonstrate the potential utility of cyclic peptides in mod-eling the interactions between a peptide and its binding site.All together, these results suggest that (i) the a-helicalconformation of the core of SP observed in methanol may berelevant to its bioactive conformation and (ii) the nonhomol-ogous N-terminal sequences of SP and NKB might adopt asimilar three-dimensional structure when bound to theirrespective receptors. Finally, the N-terminal moieties of thetwo tachykinins possibly influence the orientation of Gly-Leu-Met-NH2. And, as previously suggested on the basis ofthe results obtained with linear analogues of SP (34, 35), thiscommon C-terminal tripeptide might fit differently into theNK1 and NK3 tachykinin receptors.

We thank Mr. G. McCort for helpful suggestions in editing thismanuscript, Dr. J.-L. Morgat (Commissariat A l'Energie Atomique,Saclay) for the amino acid analysis and Miss E. Pacini for typing themanuscript. This work was supported by grants from the CentreNational de la Recherche Scientifique (ATP 823) and from Rh6ne-Poulenc Sante. Work done in the Canadian laboratory was supportedby a grant of the Medical Research Council of Canada. D.R. is aCareer Investigator of the Medical Research Council of Canada.

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Medical Sciences: Ploux et al.