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Selective agonists for substance P and neurokinin receptors
Neuropepride~ 0 Longman
(1987) 10, 43-54 Ltd 1987
Group UK
SELECTIVE AGONISTS
FOR SUBSTANCE
P AND NEUROKININ
RECEPTORS
G. Drapeau, P. D'Orleans-Juste, S. Dion, N.-E. Rhaleb, N.-E. Rouissi and Medical School, University of Department of Pharmacology, D. Regoli. (reprint requests to DR). Sherbrooke, Sherbrooke, Canada JlH 5N4. ABSTRACT analogues and fragments have been prepared in an A series of neurokinin attempt to identify selective agonists for NK-P, NK-A and NK-B receptors. have been tested on the dog carotid artery (NK-P receptor The compounds artery (NK-A) and the rat portal vein the rabbit pulmonary system), analogues of the three neurokinins have C-terminal substituted (NK-B). is improved by the indication that NK-P receptor selectivity provided selectivity for NK-A is oxydation of methionine to MettO ), while favoured by replacing Met with N 1e. Selec ivity for NK-P receptors is improved by the replacement of Gl$‘ with Sar. Se+ectivity and further is markedly increased when Val is replaced receptors affinity for NK-B Tte ;es;iul.zn;ft$ with MePhe in both the fragment NKB ( 10) andlr;;P 4that a) [Sar ,Met(02) present study indicate for the NK-P receptors of the dog carotid artery: b) select've agonist and selective stimulant of receptors for [MePhe']NKB is a very polgnt neurokinin B and c) [Nle ]NKA (4-10) is a promising compound, showing further modifications are however some selectivity for NK-A receptor: needed to improve its affinity. INTRODUCTION Substance P and related neurokinins are a group of neuropeptides widely distributed in the central and peripheral nervous system. Once released and possibly some chemical stimuli (l), neurokinins act on by nervous specific receptors to exert various biological actions. Even before the discovery of neurokinin A and neurokinin B, growing evidence, indicating the presence of multiple receptors for these peptides, was emerging from pharmacological studies performed with substance P homologues (2), fragments (3) and analogues (4). With the discovery of substance K (5) and neuromedin K (6), later renamed neurokinin A (NKA) and neurokinin B (NKB) (7), further evidence in support of the existence of three different receptors for these peptides were brought using as a criterion the order of potency of agonists in both pharmacological (8, 9) and biochemical (10, 11) experiments. It has however been rapidly realized that the naturally occurring neurokinins, as well as their fragments and even some homologues
43
tachykinins are not selective enough for discriminating between one and receptor the other Even using selective pharmacological type. to contain almost exclusively a single receptor preparations that appear type (8), the natural peptides show little selectivity. In the present study, an attempt has been made to identify chemical that will be suitable modification for improving the selectivity of neurokinin agonists for one or another receptor type. Furthermore, we have tried to identify the shortest peptide sequences that maintain full activity and selectivity, in order to eliminate some of the effects that the N-terminal part of the natural molecules, are attributed to particularly that of substance P. Results obtained with new compounds prepared in our laboratory are compared with those of selective agonists for neurokinin receptors reported by other workers (12, 13, 14).
MATERIALS
AND METHODS
Peptides. [Sar']SP was purchased from Peninsula Laboratories: all other were prepared in our laboratory using solid phase methodology as peptides described previously (17). In brief, methylbenzhydrylamine resin was used peptides with a C-terminal amide group, while C-terminal free to obtain acid peptides were obtained with chloromethylated resin. N-Boc protection acid derivatives with the following side chain was used for the amino tosy1 protecting groups: for Arg and His, cyclohexyl for Asp, for Lys and benzyl for Ser and Thr. Met(O) and 2-chloro-benzyloxycarbonyl (Sigma) were treated with di-t-butyl dicarbonate in order to Met(0 ) and Boc-Met(02) affor 2 Boc-Met(O) used to obtain respectively, peptides C-terminal methionine sulfoxide and methionine sulfone. containing SP-OMe according to Watson et al. (12). Couplings were performed was prepared using the symmetric anhydride method, except for Boc-Arg(Tos) and Boc-Gln the dicyclohexylcarbodiimide/l-hydroxybenzowhich w re activated using Coupling were monitored using the qualitative Kaiser (15) method. triaYfe removal of the last N-BOC protecting group, the resin was After test. The peptides were cleaved from the resin with dried overnight in vacua. (10 ml/g resin) in the presence of 10% anisole and dimethyl liquid HF was purified by gel sulfide (0.25 ml/g resin). The crude product The Sephadex LH20 filtration on (50% CH CN in 0.2 M CH3 COOH). further purified by prepara & rve HPLC, with an acetonitrile products were in 0.05% TFA. Purety of the final products was controlled by TLC gradient and by analytical HPLC. Amino acid analyses solvent systems in several gave the expected composition, although N-methyl amino acids could not be Some of the compounds in which methionine has been oxydated or detected. replaced with Nle and Ala have been described before (4, 16, 17). The peptides were tested as agonists and some of them assays. Biological on three pharmacological preparations described by also as antagonists (8) as selective for one or the other neurokinin. The dog Regoli et al. artery with intact endothelium as described by D'Orlkans-Juste et carotid sensitive to substance P and provides a good selective al. (18) is very the rabbit the substance P receptor, NK-P; preparation for studying
44
artery without endothelium, prepared according to pulmonary D'Orleans-Juste et al. (19) is a preparation that responds by contraction and contains to neurokinin A the NK-A receptor the third type: sensitive to neurokinin preparation, B is the rat portal vein, which is considered selective for the NK-B receptor (20) type. The tissues were suspended in 10 ml organ baths, filled with oxygenated Krebs' solution at of tension were recorded isometrically on a Grass 37OC and the changes parameters of pD2 system. The pharmacological (apparent affinity of agonists) was used according to Ariens (21) to characterize and compare the various compounds. Relative affinities of agonists were calculated and displaced in % of those of each neurokinin in the respective selective preparation. All peptides were prepared in concentrated solutions (1-2 mg/ml) in distilled water and kept at -2OOC until use. Noradrenaline solutions, used to contract the dog carotid artery, were added with 5% of ascorbic acid in order to prevent the oxydation. NKB was dissolved in 10% sulfolane. RESULTS Neurokinin
analogues modified
at the C-terminal
position
In a first series of experiments, several substitutions were made in the C-terminal In particular, the thioether positions of SP, NKA and NKB. function of Methionine was oxydated to sulfoxide or sulfone; the residue was also replaced with either the isosteric Nle or with Ala. The compounds were tested on three selective pharmacological preparations, the artery, the rabbit pulmonary artery and the rat portal vein, dog carotid to evaluate their activities respectively on NK-P, NK-A and NK-B receptors. The compounds activities are expressed in terms of apparent affinity (pD2) and in percent of the most potent of the affinity neurokinin in each preparation (relative activity), according to Regoli et The results, summarized in Table al. (8, 9). 1, indicate that the oxydation of the thioether function of Methionine in substance P favours the interaction with the NK-P of the dog carotid artery. In receptor fact, the sulfoxide derivative of SP (compound no 2 of Table 1) maintains full activity in the D.C.A. and R.P.V., but is practically inactive on the R.P.A. The sulfone derivative is even more active than SP on the D.C.A., while loosing and particularly on the R.P.V. a;f'tPityofon the R.P.A. Replacement of SP with the isosteric Nle bringt about variable decreases of fffinity which are however more evident in the R.P.A. and R.P.V.: [Nle ISP maintains almost 50% of the SP-activity on the D.C.A. (17)11 As reported before for the D.C.A. and other tissues (3, 4) [Ala ]SP shows little activity in all preparations. AS favourable change appears to be the replacement of Met 160rbyNKG;e.the [iyz"]NKA maintains a good affinity on the R.P.A. and R.P.V., but i-&$;n&o;t s~Z?wstimes less potent than NKA on the D.C.A. Therefore, some selectivity 16or the NK-A receptor system. On the contrary, the oxydation of Met of NKA to sulfoxide or sulfone, bringt about marked decreases of affinities in the three tissues.
45
MetlO
Replacement of all activities.
by
Ala
(compound
10 of Table 1) reduces markedly
TABLE 1 ACTIVITIES OF NEUROKININS MODIFIED AT THE C-TERMINAL END ON SELECTIVE PHARMACOLOGICAL PREPARATIONS.
PEPTIDE
PREPARATION R.P.A
D.C.A pD2
1
10.00 10.06 10.1 9.7 7.6
Substance P
6 7 8 9 10
Neurokinin A
9.4
[Met(O)';&-NKA [Met$C$) ]-NKA [Nle ]-NKA [Ala"]-NKA
7.6 8.5 8.0 6.0
11 12 13 14 15
Neurokinin B [Met(O)lNKB [Met\OR)lG]-NKB [Nle O]-NKB [AlalO]-NKB
pD2:
R.A:
R.A
pD2
100 114 126 46 0.4
6.1 5.7 5.6 inact.
R.A
4.0 0.01 0.4 0.3
25 0.4 2.8 1.1 0.01
8.2 6.8 7.5 8.0 6.1
100 3.4 17 62 0.8
8.0 1.2 2.8 8.3
7.4 6.7 6.2 6.4 5.4
17 3.2 1.0 1.6 0.2
8.9
8.1 8.4 8.9
R.P.V pD2
R.A
5.8 5.6 inact. inact. inact.
1.4 0.8
6.4 5.7 6.6 6.5
6.0 1.1 8.5 6.5 1.0
7.7 100 1.2 5.7 9.3 6.6 4.0 6.3 inact.
apparent affinity, calculated from the middle point (50 % of the maximum effect) of at least 6 concentration response curves. pD is 2 expressed by the -log of the molar concentration of compound. relative affinity indicated in the percent of the affinity of the the most active neurokinin in each preparation.
10 are accompanied by some of Met all substitutions In the NKB series, in fact, all analogues shown decrease of affinity for the NX-B receptors: in Table 1 are at least 10 times leas potent than NKB in the R.P.V. (see compounds 12, 13, 14 and 15 of Table 1). They maintain some activity in the other two preparations and therefore they do not show any selectivity for NK-B receptors. Selective NK-P receptor agonists A
combination
of several modifications
46
were made in the sequence of SP to
The results obtain potent and selective agonists for the NK-P receptor. are summarized in Table 2, where the activities of SP are first compared with those of some fragments and of compounds that have been shown to be selective stimulants of the receptors for substance P (8, 19, 23). TABLE 2 ACTIVITIES OF SUBSTANCE P FRAGMENTS AND ANALOGUES SELECTIVE PHARMACOLOGICAL PREPARATIONS.
ON
PREPARATION
PEPTIDE
R.A
pD2
R.A
pD2
R.A
10.00 9.67 9.56
100 47 36
6.1 6.2 6.0
100 126 78
5.8 5.9 6.1
100 112 174
9.60 9.70
40 50
10.16 10.38
145 240
5.5
10.45 10.21 10.35
282 162 224
inact. inact.
9.92
83
pD2
1 2 3
Substance P SP(4-11) [pGld]-SP(6-11)
4 5
SP-OMe [pGlu6,Prog]-SP(6-11)
6 7
[Pro']-SP [Sarg]-SP
8 [Sarg,Met(02)11]-SP 9 [Pro9 Met(0 )ll]-SP 10 [B-A1L4,Sar3,Met(O2)"] -SP(4-11) 11 [pGlu6,Sarg,Met(02)11]
R.P.V
R.P.A
D.C.A
inact. 5.2
12
inact. 5.6
24 0.1
inact. 5.8 98 inact. inact.
0.1 4.60
58
3.0
0.1 5.46
39
-SP(6-11)
R.A.: pD2:
relative affinity, expressed the three preparations. as in Table 1.
in percent of that of substance
P in
The results of Table 2 indicate that C-terminal fragments of SP such as SP (4-11) and SP (6-11) are less selective than the natural undecapeptide, because they are less potent than SP on the D.C.A., but maintain fairly high activities on the R.P.A. and particularly on the R.P.V. SP methyl ester (23) is selective for the NK-P receptors, but h%s on4y 40% of the SP-activity on the D.C.A. The hexapeptide [pGlu ,Pro ]SP (6-ll), which has been recommended as a selective agonist for SP receptors by Laufer et al. (241, is still fairly active on the R.P.A. and R.P.V.: furthermore, the compound has 50% of the SP-activity on the D.C.A. [Pro9]SP, a compound recently described by Ploux et al. (25), is more potent than SP on the NK-P receptor system and shows some selectivity
47
because it is inactive on the R.P.V. The compound maintains however9some activity on the NK-A system of the R.P.A. Another compound, [Sar ]SP, described several years ago by Sandberg et al. (131, is almost 2.5 times more potent t$an SP on the D.C.A. and is practically inactive on the [Sar ]SP maintains however fu14 activity on the R.P.V. and is R.P.A.; not selective [Safl]SP shows a pharmacological therefore enough. rather different from spectrum [Met(02) ISP, the compound (Table 1) which is completely inactive on the R.P.V. on these findings, the two modifications in positions 9 and 11 of SP Based combined, in order to e~~~tna~~t~~e,aEtivit~~~r~~~~~~oR~~~~~pwi~~ wer 5 and in the R.P.V. Sar activator of NK-P receptors of thz D.C.A. (3.5 times iore potent a potent and is inactive on the other preparations. than two SP) is therefore to be considered a pot$nt and [Sar',Met(O ) 'l]SP igonist of the NK-P receptor. selective Replacementgof Gly 91 Sar to be more favourable than by Pro, since [Pro ,Met(O ) appears 1-f ctiveon :;; ,N:-; alth [Sar ugh ,MetrO:;)ifISP receptors, is only half as ac $ ive as 8
. . ..
its selectivity, that Despite [Sar',Met(O ) 'l1S.P is an undecapeptide contains the N-terminal Arg and Lys w rch are responsible for the release of histamine (26) and some other effects of SP (28). We therefore decided and test shorter sequences, containing the same modifications. to prepare As shown in Table 2, the octapeptide (compound 10) is potent on the D.C.A. and quite selective, while the shorter hexapeptide (compound 11) regains on the R.P.A. and especially on the R.P.V. Further studies some activity to improve affinity and selectivity of hexapeptide sequences are needed for the NK-P receptor.
2.
Selective NK-B receptor agonists experiments were performed in an attempt to identify chemical Further that will favour occupation and activation of NK-B receptors. The changes results of this study are summarized in Table 3, where the activities of neurokinin B in the three pharmacological preparations are compared with As shown those of analogues of two heptapeptides (NKB (4-10) and DiMeC7). (8), the shortening of the NKB sequence by the before by Regoli et al. of three residues at the N-terminal (as in NKB (4-lo)), give a elimination that is 10 fold less active than NKB on the R.P.V., but is almost compound NKB (4-10) active as NKB on the D.C.A. and more potent on the R.P.A. as the minimum active sequence of NKB and it was therefore is however Such selected for further design of NK-B selective agonists {see below). a design was based on the observation that DiMeC7, the compound reported et al. (13), several years ago, although weak on the R.P.V., by Sandberg shows some selectivity on the NK-B receptor (9, 23) system. shown by the results of Table 3, DiMeC7 maintains less than 5% of the on the D.C.A. and 6% on the R.P.V., but is inactive on the NKB activity These findings lead us to utilize into the receptor of the R.P.A. NK-A heptapeptide NKB (4-10) the chemical modifications which are present in 4, 5 and 6 of Table 3. When We therefore prepared compounds DiMeC7. AS
48
tested on the three preparations, it was apparent that the replacement of GAY Shy Sar favours the occupation and activation of NK-P receptors, since [Sar ]NKB (4-10) is potent in the D.C.A., but rather weak on the R.P.A. and R.P.V.
TABLE 3 BIOLOGICAL
ACTIVITIES
OF NEIJROKININ B PEPTIDES.
R.P.V
R.P.A
D.C.A pD2
R.A
pD2
R.A
pD2
R.A.
8.90 8.70
100 63
7.45 7.95
100 316
7.68 6.67
100 10
7.50 7.61 9.26 7.09 7.21
4.0 5.1 229 1.5 2
inact. inact. 6.72 5.50 6.31
8 [Pro7]-NKB(4-lo) 9 [Phe7]-NKB(4-10) 10 [MeVa17]-NKB(4-10)
6.20 8.63 7.64
0.2 54 5.5
11 [MePhe7]-NKB 12 Senktide
7.15 6.39
1.8 0.3
1
Neurokinin
2
NKB(4-10)
B
3
DiMeC7
4 5 6 7
[MePhe7,Sar8]-NKB(4-10) [Sar8]-NKB(4-10) [MePhe7]-NKB(4-10) [Sar7]-NKB(4-10)
as in table 1 relative activity expressed
19 1.1 7.2
6.35 7.03 6.44 7.58 5.86
4.7 22 6 79 1.5
5.65 6.31 5.65
1.6 7 1.6
5.85 5.86 7.68
1.5 1.5 100
5.24 5.46
0.6 0.7
a.30 7.61
417 85
in percent of that of NKB.
The c mbin tion of the two substitutions in positions 7 and 8, as in FI [MePhe' ,Sar INKB (4-lo), gives a compound slightly better then DiMeC7, since it is more active (by 0.7 log units) on the R.P.V., it is inactive on the R.P.A. and it has practically the same activity as DiMeC7 on the D.C.A. These results suggest that the most favourab e changes for NK-B receptor selectivity might be the substitution of Val + with MePhe. This assump ion was confirmed by the biological activities obtained with 5 [MePhe INKB (4-101, which is almost as active as NKB on the R.P.V., bu 5 maintains only 1% of NKB activities in the other two preparations. Val of NKB (4-10) could also be favourably replaced with MeVal: in fact, compound 10 of Table 3 wa found to be as active as NKB on the R.P.V. and $ to was equivalent [MePhe ]NKB (4-10) in the other two prepa ations. 5 Compound 10 is therefore as selective as compound 6. When Val of NKB
49
(4-10) was replaced with other residues, such as Phe, Pro or Sar, the affinities of the resulting compounds (no 8, 9 and 10 of Table 3) on the system of the R.P.V. NX-B were markedly decreased. Some of these compounds (no 8 and 10) showed fairly good affinities in the D.C.A. and R.P.A. Selectivity for NK-B receptor system appears therefore to be favoured by the presence of a N-methyl residue in the center position of NKB (4-10). The validity of this statement is supported by the sequence the comparison of he biological activities7of NKB (4-10) with those of 5 its analogue [MePhe INKB (4-10) and [MeVal INKB (4-lo), in Table 3 (see also Drapeau et al., 1987) (22). To
improve the agonist affinity for NK-B receptors, a decapeptide further MePhe in position analogue of NKB, containing 7, was prepared and As shown in Table 3, this compound is more active than NKB tested. (more than 4 folds) on the R.P.V., but is 150 and 250 fimes less potent in the D.C.A. and R.P.A. than NKB respectively [MePhe INKB is the most agonist for NK-B receptors that we have tested: active it is soluble in water and appears to be promising for physiological and pharmacological studies. the present work was under way, Wormser et al. (14) published the While of the sequence SP (5-ll), containing a N-methyl senktide, an analogue residue in the center position. This compound was also prepared and tested in our laboratory and the results of the biological activities are in Table 3. Senktide appears to be quite selective for the NK-B presented on the R.P.V. recept r, but its affinity is 5 fol less than that of 4 Senktide is more [MePhe'jNKB. active than [MePhe ]NKB on the NK-A receptor system of the R.P.A. Selective
NK-A receptor agonists
Table 4 summarizes
some preliminary
results obtained with NKA-related
TABLE 4 ACTIVITIES OF NEUROKININ A, ITS FRAGMENTS AND ANALOGUES ON SELECTIVE PHARMACOLOGICAL PREPARATIONS. D.C.A.
1 2 3 4
Neurokinin A NKA(4-10) [NlelO]-NRA [Nlelo]-NKA(4-10)
R.P.A
R.P.V.
pD2
R.A.
pD2
R.A.
pD2
R.A.
9.40 8.62 8.04 7.00
100 16.6 4.4 0.4
8.20 8.50 8.00 7.90
100 191 60 49
6.45 6.80 6.50
100 224 112 cl.0
pD : as in Table 1 R.i : relative activity expressed in percent of that of NKA.
50
peptides, prepared with the purpqse of improving agonists affinity and selectivity for the receptors of neurokinin A. As shown before (see Ttble by 1 and the papef0 by Drapeau et al. (17)), the substitution of Met Nle as in by a marked decrease of affinity [Nle INKA, is accompanied with minor changes in the (by approximately 25 folds) in the D.C.A., R.P.A. observations and R.P.V. Because of previous (8, 9) that the C-terminal NKA (4-10) is more potent than NKA on the R.P.A. heptapeptide and in other preparations (for instance the human isolated bronchus (27)), C-terminal with Nle to obtain Met was replaced the sequence the 10 [Nle INKA (4-10). The results in Table 4 confirm previous presented findings that NKA (4-10) is more selective than NKA, since it is twice as but is 6 folds less potent on the D.C.A. ~~~~"]NK"~ ~~"10~" i~heev~~p.~o~e selective , since it maintains almost 50% of activity on the R.P.A., but is practically inactive on the R.P.V. and is two and a half orders of magnitude less active than NKA on the D.C.A. CONCLUSIONS Selective agonists for NK-P receptors have been obtained by reducing the con ormational freedom of the undecapeptide SP with the replacement of 4 have been further improved by Selectivity and potency Gly by ";;;ll oxydizing Histamine releasing ability and metabolic to Met(02). degradation have eliminated or reduced by using been the same modifications in the much [ B_Ala4,sar9,;;~;~e~!~;;p S~4_~~;ll~;pe~~~ho~~ ;;os;ng affinity. potent and selective NK-P agonist: $he shorter hexapeptide sequence is however not selective enough. The chemical essential to improve change selectivity of NKB-related pep$ides for the NK-B receptor has been found to be the replacement of Val by a N-methyl residue, preferably7MePhe. High selectivity for NK-B recept rs has shown by [MePhe INKB (4-10) and especially by been [MePheq]NKB. This compound is selective for NK-B, it is extremely active, and it is soluble 'n water. If adequately protected from metabolic degradation, [MePhe']NKB could provide an excellent ligand for NK-B receptors studies. An attempt has been made to improve selectivity o NKA-related peptides f0 for the NK-A receptors system. Replacement of Met by Nle confers some selectivity as does the reduction 18 f the decapeptide chain by three residues from the N-terminal. [Nle INKA (4-10) represents a first step towards the identification of a selective NK-A receptor agonist. ACKNOWLEDGEMENTS The authors acknowledge the secretarial work of Mrs. C. Theberge and the technical assistance of Mr. M. Boussougou and Mr. R. Laprise. This work was performed with the financial assistance of the Medical Research Council of Canada (M.R.C.C.) and the Quebec Heart Foundation. D.R. is a Career Investigator of the M.R.C.C.
51
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Pharmacology,
Vol. 1, Academic
Press,
22. Drapeau, G., D'Orleans-Juste, P., Dion, S., Rhaleb, N.-E. and Regoli, Eur. J. D. (1987). Specific agonists for neurokinin B receptors. Pharmacol. (in press). 23. Dion, S., D'Orleans-Juste, P., Rhaleb, N.-E., Drapeau, G., Rovero, P. and Regoli, D. (1987). Neurokinin fragments and analogues as selective activators of receptors. Substance P and Neurokinins, Montreal'86. J.-L. Henry, R. Couture, A.C. Cuello, G. Pelletier, R. Quirion and D. Regoli Eds. Springer-Verlag, New York. (in press). 24. Lau er, R., Gilon, C. Chorev, M. and Selinger, Z. (1986). [~Glu', 4 Pro ]SP (6-ll), a selective agonist for the substance P receptor subtype. J. Med. Chem. 29: 1284-1288. 25. Ploux, O., Lavielle, S., Chassaing, G., Julien, S., Marquet, A., Beaujouan, J.-C., Torrens, Y., Bergstrom, L. and Glowinsky, J. (1987). Conformationally restricted analogues of substance P. Substance P and Neurokinins, Montreal'86. J.-L. Henry, R. Couture, A.C. Cuello, G. Pelletier, R. Quirion and D. Regoli Eds. Springer-Verlag, New York. (in press). 26. Devillier, P., Regoli, D., Asseraf, A., Descours, B., Marsac, J. and Renoux, M. (1986). Histamine release and local responses of rat and human skin to substance P and other mammalian tachykinins. Pharmacology 32: 340-347. 27. Advenier, C., Naline, E., Drapeau, G. and Regoli, D. (1987). Comparison Of neurokinins activities in guinea pig trachea and human bronchus. Eur. J. Pharmacol. (in press).
53
28. Pelleymounter, M.-A., Fisher, Q., Schlesinger, K., Hall, M., Dearmey, P. and Stewart, J. (1986). The effect of substance P and its fragments on passive avoidance retention and brain monoamine activity. Behavioural Brain Res. 21: 119-127.
Received Accepted
5 May 1987 19 May 1987
54
(1987) 10, 43-54 Ltd 1987
Group UK
SELECTIVE AGONISTS
FOR SUBSTANCE
P AND NEUROKININ
RECEPTORS
G. Drapeau, P. D'Orleans-Juste, S. Dion, N.-E. Rhaleb, N.-E. Rouissi and Medical School, University of Department of Pharmacology, D. Regoli. (reprint requests to DR). Sherbrooke, Sherbrooke, Canada JlH 5N4. ABSTRACT analogues and fragments have been prepared in an A series of neurokinin attempt to identify selective agonists for NK-P, NK-A and NK-B receptors. have been tested on the dog carotid artery (NK-P receptor The compounds artery (NK-A) and the rat portal vein the rabbit pulmonary system), analogues of the three neurokinins have C-terminal substituted (NK-B). is improved by the indication that NK-P receptor selectivity provided selectivity for NK-A is oxydation of methionine to MettO ), while favoured by replacing Met with N 1e. Selec ivity for NK-P receptors is improved by the replacement of Gl$‘ with Sar. Se+ectivity and further is markedly increased when Val is replaced receptors affinity for NK-B Tte ;es;iul.zn;ft$ with MePhe in both the fragment NKB ( 10) andlr;;P 4that a) [Sar ,Met(02) present study indicate for the NK-P receptors of the dog carotid artery: b) select've agonist and selective stimulant of receptors for [MePhe']NKB is a very polgnt neurokinin B and c) [Nle ]NKA (4-10) is a promising compound, showing further modifications are however some selectivity for NK-A receptor: needed to improve its affinity. INTRODUCTION Substance P and related neurokinins are a group of neuropeptides widely distributed in the central and peripheral nervous system. Once released and possibly some chemical stimuli (l), neurokinins act on by nervous specific receptors to exert various biological actions. Even before the discovery of neurokinin A and neurokinin B, growing evidence, indicating the presence of multiple receptors for these peptides, was emerging from pharmacological studies performed with substance P homologues (2), fragments (3) and analogues (4). With the discovery of substance K (5) and neuromedin K (6), later renamed neurokinin A (NKA) and neurokinin B (NKB) (7), further evidence in support of the existence of three different receptors for these peptides were brought using as a criterion the order of potency of agonists in both pharmacological (8, 9) and biochemical (10, 11) experiments. It has however been rapidly realized that the naturally occurring neurokinins, as well as their fragments and even some homologues
43
tachykinins are not selective enough for discriminating between one and receptor the other Even using selective pharmacological type. to contain almost exclusively a single receptor preparations that appear type (8), the natural peptides show little selectivity. In the present study, an attempt has been made to identify chemical that will be suitable modification for improving the selectivity of neurokinin agonists for one or another receptor type. Furthermore, we have tried to identify the shortest peptide sequences that maintain full activity and selectivity, in order to eliminate some of the effects that the N-terminal part of the natural molecules, are attributed to particularly that of substance P. Results obtained with new compounds prepared in our laboratory are compared with those of selective agonists for neurokinin receptors reported by other workers (12, 13, 14).
MATERIALS
AND METHODS
Peptides. [Sar']SP was purchased from Peninsula Laboratories: all other were prepared in our laboratory using solid phase methodology as peptides described previously (17). In brief, methylbenzhydrylamine resin was used peptides with a C-terminal amide group, while C-terminal free to obtain acid peptides were obtained with chloromethylated resin. N-Boc protection acid derivatives with the following side chain was used for the amino tosy1 protecting groups: for Arg and His, cyclohexyl for Asp, for Lys and benzyl for Ser and Thr. Met(O) and 2-chloro-benzyloxycarbonyl (Sigma) were treated with di-t-butyl dicarbonate in order to Met(0 ) and Boc-Met(02) affor 2 Boc-Met(O) used to obtain respectively, peptides C-terminal methionine sulfoxide and methionine sulfone. containing SP-OMe according to Watson et al. (12). Couplings were performed was prepared using the symmetric anhydride method, except for Boc-Arg(Tos) and Boc-Gln the dicyclohexylcarbodiimide/l-hydroxybenzowhich w re activated using Coupling were monitored using the qualitative Kaiser (15) method. triaYfe removal of the last N-BOC protecting group, the resin was After test. The peptides were cleaved from the resin with dried overnight in vacua. (10 ml/g resin) in the presence of 10% anisole and dimethyl liquid HF was purified by gel sulfide (0.25 ml/g resin). The crude product The Sephadex LH20 filtration on (50% CH CN in 0.2 M CH3 COOH). further purified by prepara & rve HPLC, with an acetonitrile products were in 0.05% TFA. Purety of the final products was controlled by TLC gradient and by analytical HPLC. Amino acid analyses solvent systems in several gave the expected composition, although N-methyl amino acids could not be Some of the compounds in which methionine has been oxydated or detected. replaced with Nle and Ala have been described before (4, 16, 17). The peptides were tested as agonists and some of them assays. Biological on three pharmacological preparations described by also as antagonists (8) as selective for one or the other neurokinin. The dog Regoli et al. artery with intact endothelium as described by D'Orlkans-Juste et carotid sensitive to substance P and provides a good selective al. (18) is very the rabbit the substance P receptor, NK-P; preparation for studying
44
artery without endothelium, prepared according to pulmonary D'Orleans-Juste et al. (19) is a preparation that responds by contraction and contains to neurokinin A the NK-A receptor the third type: sensitive to neurokinin preparation, B is the rat portal vein, which is considered selective for the NK-B receptor (20) type. The tissues were suspended in 10 ml organ baths, filled with oxygenated Krebs' solution at of tension were recorded isometrically on a Grass 37OC and the changes parameters of pD2 system. The pharmacological (apparent affinity of agonists) was used according to Ariens (21) to characterize and compare the various compounds. Relative affinities of agonists were calculated and displaced in % of those of each neurokinin in the respective selective preparation. All peptides were prepared in concentrated solutions (1-2 mg/ml) in distilled water and kept at -2OOC until use. Noradrenaline solutions, used to contract the dog carotid artery, were added with 5% of ascorbic acid in order to prevent the oxydation. NKB was dissolved in 10% sulfolane. RESULTS Neurokinin
analogues modified
at the C-terminal
position
In a first series of experiments, several substitutions were made in the C-terminal In particular, the thioether positions of SP, NKA and NKB. function of Methionine was oxydated to sulfoxide or sulfone; the residue was also replaced with either the isosteric Nle or with Ala. The compounds were tested on three selective pharmacological preparations, the artery, the rabbit pulmonary artery and the rat portal vein, dog carotid to evaluate their activities respectively on NK-P, NK-A and NK-B receptors. The compounds activities are expressed in terms of apparent affinity (pD2) and in percent of the most potent of the affinity neurokinin in each preparation (relative activity), according to Regoli et The results, summarized in Table al. (8, 9). 1, indicate that the oxydation of the thioether function of Methionine in substance P favours the interaction with the NK-P of the dog carotid artery. In receptor fact, the sulfoxide derivative of SP (compound no 2 of Table 1) maintains full activity in the D.C.A. and R.P.V., but is practically inactive on the R.P.A. The sulfone derivative is even more active than SP on the D.C.A., while loosing and particularly on the R.P.V. a;f'tPityofon the R.P.A. Replacement of SP with the isosteric Nle bringt about variable decreases of fffinity which are however more evident in the R.P.A. and R.P.V.: [Nle ISP maintains almost 50% of the SP-activity on the D.C.A. (17)11 As reported before for the D.C.A. and other tissues (3, 4) [Ala ]SP shows little activity in all preparations. AS favourable change appears to be the replacement of Met 160rbyNKG;e.the [iyz"]NKA maintains a good affinity on the R.P.A. and R.P.V., but i-&$;n&o;t s~Z?wstimes less potent than NKA on the D.C.A. Therefore, some selectivity 16or the NK-A receptor system. On the contrary, the oxydation of Met of NKA to sulfoxide or sulfone, bringt about marked decreases of affinities in the three tissues.
45
MetlO
Replacement of all activities.
by
Ala
(compound
10 of Table 1) reduces markedly
TABLE 1 ACTIVITIES OF NEUROKININS MODIFIED AT THE C-TERMINAL END ON SELECTIVE PHARMACOLOGICAL PREPARATIONS.
PEPTIDE
PREPARATION R.P.A
D.C.A pD2
1
10.00 10.06 10.1 9.7 7.6
Substance P
6 7 8 9 10
Neurokinin A
9.4
[Met(O)';&-NKA [Met$C$) ]-NKA [Nle ]-NKA [Ala"]-NKA
7.6 8.5 8.0 6.0
11 12 13 14 15
Neurokinin B [Met(O)lNKB [Met\OR)lG]-NKB [Nle O]-NKB [AlalO]-NKB
pD2:
R.A:
R.A
pD2
100 114 126 46 0.4
6.1 5.7 5.6 inact.
R.A
4.0 0.01 0.4 0.3
25 0.4 2.8 1.1 0.01
8.2 6.8 7.5 8.0 6.1
100 3.4 17 62 0.8
8.0 1.2 2.8 8.3
7.4 6.7 6.2 6.4 5.4
17 3.2 1.0 1.6 0.2
8.9
8.1 8.4 8.9
R.P.V pD2
R.A
5.8 5.6 inact. inact. inact.
1.4 0.8
6.4 5.7 6.6 6.5
6.0 1.1 8.5 6.5 1.0
7.7 100 1.2 5.7 9.3 6.6 4.0 6.3 inact.
apparent affinity, calculated from the middle point (50 % of the maximum effect) of at least 6 concentration response curves. pD is 2 expressed by the -log of the molar concentration of compound. relative affinity indicated in the percent of the affinity of the the most active neurokinin in each preparation.
10 are accompanied by some of Met all substitutions In the NKB series, in fact, all analogues shown decrease of affinity for the NX-B receptors: in Table 1 are at least 10 times leas potent than NKB in the R.P.V. (see compounds 12, 13, 14 and 15 of Table 1). They maintain some activity in the other two preparations and therefore they do not show any selectivity for NK-B receptors. Selective NK-P receptor agonists A
combination
of several modifications
46
were made in the sequence of SP to
The results obtain potent and selective agonists for the NK-P receptor. are summarized in Table 2, where the activities of SP are first compared with those of some fragments and of compounds that have been shown to be selective stimulants of the receptors for substance P (8, 19, 23). TABLE 2 ACTIVITIES OF SUBSTANCE P FRAGMENTS AND ANALOGUES SELECTIVE PHARMACOLOGICAL PREPARATIONS.
ON
PREPARATION
PEPTIDE
R.A
pD2
R.A
pD2
R.A
10.00 9.67 9.56
100 47 36
6.1 6.2 6.0
100 126 78
5.8 5.9 6.1
100 112 174
9.60 9.70
40 50
10.16 10.38
145 240
5.5
10.45 10.21 10.35
282 162 224
inact. inact.
9.92
83
pD2
1 2 3
Substance P SP(4-11) [pGld]-SP(6-11)
4 5
SP-OMe [pGlu6,Prog]-SP(6-11)
6 7
[Pro']-SP [Sarg]-SP
8 [Sarg,Met(02)11]-SP 9 [Pro9 Met(0 )ll]-SP 10 [B-A1L4,Sar3,Met(O2)"] -SP(4-11) 11 [pGlu6,Sarg,Met(02)11]
R.P.V
R.P.A
D.C.A
inact. 5.2
12
inact. 5.6
24 0.1
inact. 5.8 98 inact. inact.
0.1 4.60
58
3.0
0.1 5.46
39
-SP(6-11)
R.A.: pD2:
relative affinity, expressed the three preparations. as in Table 1.
in percent of that of substance
P in
The results of Table 2 indicate that C-terminal fragments of SP such as SP (4-11) and SP (6-11) are less selective than the natural undecapeptide, because they are less potent than SP on the D.C.A., but maintain fairly high activities on the R.P.A. and particularly on the R.P.V. SP methyl ester (23) is selective for the NK-P receptors, but h%s on4y 40% of the SP-activity on the D.C.A. The hexapeptide [pGlu ,Pro ]SP (6-ll), which has been recommended as a selective agonist for SP receptors by Laufer et al. (241, is still fairly active on the R.P.A. and R.P.V.: furthermore, the compound has 50% of the SP-activity on the D.C.A. [Pro9]SP, a compound recently described by Ploux et al. (25), is more potent than SP on the NK-P receptor system and shows some selectivity
47
because it is inactive on the R.P.V. The compound maintains however9some activity on the NK-A system of the R.P.A. Another compound, [Sar ]SP, described several years ago by Sandberg et al. (131, is almost 2.5 times more potent t$an SP on the D.C.A. and is practically inactive on the [Sar ]SP maintains however fu14 activity on the R.P.V. and is R.P.A.; not selective [Safl]SP shows a pharmacological therefore enough. rather different from spectrum [Met(02) ISP, the compound (Table 1) which is completely inactive on the R.P.V. on these findings, the two modifications in positions 9 and 11 of SP Based combined, in order to e~~~tna~~t~~e,aEtivit~~~r~~~~~~oR~~~~~pwi~~ wer 5 and in the R.P.V. Sar activator of NK-P receptors of thz D.C.A. (3.5 times iore potent a potent and is inactive on the other preparations. than two SP) is therefore to be considered a pot$nt and [Sar',Met(O ) 'l]SP igonist of the NK-P receptor. selective Replacementgof Gly 91 Sar to be more favourable than by Pro, since [Pro ,Met(O ) appears 1-f ctiveon :;; ,N:-; alth [Sar ugh ,MetrO:;)ifISP receptors, is only half as ac $ ive as 8
. . ..
its selectivity, that Despite [Sar',Met(O ) 'l1S.P is an undecapeptide contains the N-terminal Arg and Lys w rch are responsible for the release of histamine (26) and some other effects of SP (28). We therefore decided and test shorter sequences, containing the same modifications. to prepare As shown in Table 2, the octapeptide (compound 10) is potent on the D.C.A. and quite selective, while the shorter hexapeptide (compound 11) regains on the R.P.A. and especially on the R.P.V. Further studies some activity to improve affinity and selectivity of hexapeptide sequences are needed for the NK-P receptor.
2.
Selective NK-B receptor agonists experiments were performed in an attempt to identify chemical Further that will favour occupation and activation of NK-B receptors. The changes results of this study are summarized in Table 3, where the activities of neurokinin B in the three pharmacological preparations are compared with As shown those of analogues of two heptapeptides (NKB (4-10) and DiMeC7). (8), the shortening of the NKB sequence by the before by Regoli et al. of three residues at the N-terminal (as in NKB (4-lo)), give a elimination that is 10 fold less active than NKB on the R.P.V., but is almost compound NKB (4-10) active as NKB on the D.C.A. and more potent on the R.P.A. as the minimum active sequence of NKB and it was therefore is however Such selected for further design of NK-B selective agonists {see below). a design was based on the observation that DiMeC7, the compound reported et al. (13), several years ago, although weak on the R.P.V., by Sandberg shows some selectivity on the NK-B receptor (9, 23) system. shown by the results of Table 3, DiMeC7 maintains less than 5% of the on the D.C.A. and 6% on the R.P.V., but is inactive on the NKB activity These findings lead us to utilize into the receptor of the R.P.A. NK-A heptapeptide NKB (4-10) the chemical modifications which are present in 4, 5 and 6 of Table 3. When We therefore prepared compounds DiMeC7. AS
48
tested on the three preparations, it was apparent that the replacement of GAY Shy Sar favours the occupation and activation of NK-P receptors, since [Sar ]NKB (4-10) is potent in the D.C.A., but rather weak on the R.P.A. and R.P.V.
TABLE 3 BIOLOGICAL
ACTIVITIES
OF NEIJROKININ B PEPTIDES.
R.P.V
R.P.A
D.C.A pD2
R.A
pD2
R.A
pD2
R.A.
8.90 8.70
100 63
7.45 7.95
100 316
7.68 6.67
100 10
7.50 7.61 9.26 7.09 7.21
4.0 5.1 229 1.5 2
inact. inact. 6.72 5.50 6.31
8 [Pro7]-NKB(4-lo) 9 [Phe7]-NKB(4-10) 10 [MeVa17]-NKB(4-10)
6.20 8.63 7.64
0.2 54 5.5
11 [MePhe7]-NKB 12 Senktide
7.15 6.39
1.8 0.3
1
Neurokinin
2
NKB(4-10)
B
3
DiMeC7
4 5 6 7
[MePhe7,Sar8]-NKB(4-10) [Sar8]-NKB(4-10) [MePhe7]-NKB(4-10) [Sar7]-NKB(4-10)
as in table 1 relative activity expressed
19 1.1 7.2
6.35 7.03 6.44 7.58 5.86
4.7 22 6 79 1.5
5.65 6.31 5.65
1.6 7 1.6
5.85 5.86 7.68
1.5 1.5 100
5.24 5.46
0.6 0.7
a.30 7.61
417 85
in percent of that of NKB.
The c mbin tion of the two substitutions in positions 7 and 8, as in FI [MePhe' ,Sar INKB (4-lo), gives a compound slightly better then DiMeC7, since it is more active (by 0.7 log units) on the R.P.V., it is inactive on the R.P.A. and it has practically the same activity as DiMeC7 on the D.C.A. These results suggest that the most favourab e changes for NK-B receptor selectivity might be the substitution of Val + with MePhe. This assump ion was confirmed by the biological activities obtained with 5 [MePhe INKB (4-101, which is almost as active as NKB on the R.P.V., bu 5 maintains only 1% of NKB activities in the other two preparations. Val of NKB (4-10) could also be favourably replaced with MeVal: in fact, compound 10 of Table 3 wa found to be as active as NKB on the R.P.V. and $ to was equivalent [MePhe ]NKB (4-10) in the other two prepa ations. 5 Compound 10 is therefore as selective as compound 6. When Val of NKB
49
(4-10) was replaced with other residues, such as Phe, Pro or Sar, the affinities of the resulting compounds (no 8, 9 and 10 of Table 3) on the system of the R.P.V. NX-B were markedly decreased. Some of these compounds (no 8 and 10) showed fairly good affinities in the D.C.A. and R.P.A. Selectivity for NK-B receptor system appears therefore to be favoured by the presence of a N-methyl residue in the center position of NKB (4-10). The validity of this statement is supported by the sequence the comparison of he biological activities7of NKB (4-10) with those of 5 its analogue [MePhe INKB (4-10) and [MeVal INKB (4-lo), in Table 3 (see also Drapeau et al., 1987) (22). To
improve the agonist affinity for NK-B receptors, a decapeptide further MePhe in position analogue of NKB, containing 7, was prepared and As shown in Table 3, this compound is more active than NKB tested. (more than 4 folds) on the R.P.V., but is 150 and 250 fimes less potent in the D.C.A. and R.P.A. than NKB respectively [MePhe INKB is the most agonist for NK-B receptors that we have tested: active it is soluble in water and appears to be promising for physiological and pharmacological studies. the present work was under way, Wormser et al. (14) published the While of the sequence SP (5-ll), containing a N-methyl senktide, an analogue residue in the center position. This compound was also prepared and tested in our laboratory and the results of the biological activities are in Table 3. Senktide appears to be quite selective for the NK-B presented on the R.P.V. recept r, but its affinity is 5 fol less than that of 4 Senktide is more [MePhe'jNKB. active than [MePhe ]NKB on the NK-A receptor system of the R.P.A. Selective
NK-A receptor agonists
Table 4 summarizes
some preliminary
results obtained with NKA-related
TABLE 4 ACTIVITIES OF NEUROKININ A, ITS FRAGMENTS AND ANALOGUES ON SELECTIVE PHARMACOLOGICAL PREPARATIONS. D.C.A.
1 2 3 4
Neurokinin A NKA(4-10) [NlelO]-NRA [Nlelo]-NKA(4-10)
R.P.A
R.P.V.
pD2
R.A.
pD2
R.A.
pD2
R.A.
9.40 8.62 8.04 7.00
100 16.6 4.4 0.4
8.20 8.50 8.00 7.90
100 191 60 49
6.45 6.80 6.50
100 224 112 cl.0
pD : as in Table 1 R.i : relative activity expressed in percent of that of NKA.
50
peptides, prepared with the purpqse of improving agonists affinity and selectivity for the receptors of neurokinin A. As shown before (see Ttble by 1 and the papef0 by Drapeau et al. (17)), the substitution of Met Nle as in by a marked decrease of affinity [Nle INKA, is accompanied with minor changes in the (by approximately 25 folds) in the D.C.A., R.P.A. observations and R.P.V. Because of previous (8, 9) that the C-terminal NKA (4-10) is more potent than NKA on the R.P.A. heptapeptide and in other preparations (for instance the human isolated bronchus (27)), C-terminal with Nle to obtain Met was replaced the sequence the 10 [Nle INKA (4-10). The results in Table 4 confirm previous presented findings that NKA (4-10) is more selective than NKA, since it is twice as but is 6 folds less potent on the D.C.A. ~~~~"]NK"~ ~~"10~" i~heev~~p.~o~e selective , since it maintains almost 50% of activity on the R.P.A., but is practically inactive on the R.P.V. and is two and a half orders of magnitude less active than NKA on the D.C.A. CONCLUSIONS Selective agonists for NK-P receptors have been obtained by reducing the con ormational freedom of the undecapeptide SP with the replacement of 4 have been further improved by Selectivity and potency Gly by ";;;ll oxydizing Histamine releasing ability and metabolic to Met(02). degradation have eliminated or reduced by using been the same modifications in the much [ B_Ala4,sar9,;;~;~e~!~;;p S~4_~~;ll~;pe~~~ho~~ ;;os;ng affinity. potent and selective NK-P agonist: $he shorter hexapeptide sequence is however not selective enough. The chemical essential to improve change selectivity of NKB-related pep$ides for the NK-B receptor has been found to be the replacement of Val by a N-methyl residue, preferably7MePhe. High selectivity for NK-B recept rs has shown by [MePhe INKB (4-10) and especially by been [MePheq]NKB. This compound is selective for NK-B, it is extremely active, and it is soluble 'n water. If adequately protected from metabolic degradation, [MePhe']NKB could provide an excellent ligand for NK-B receptors studies. An attempt has been made to improve selectivity o NKA-related peptides f0 for the NK-A receptors system. Replacement of Met by Nle confers some selectivity as does the reduction 18 f the decapeptide chain by three residues from the N-terminal. [Nle INKA (4-10) represents a first step towards the identification of a selective NK-A receptor agonist. ACKNOWLEDGEMENTS The authors acknowledge the secretarial work of Mrs. C. Theberge and the technical assistance of Mr. M. Boussougou and Mr. R. Laprise. This work was performed with the financial assistance of the Medical Research Council of Canada (M.R.C.C.) and the Quebec Heart Foundation. D.R. is a Career Investigator of the M.R.C.C.
51
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Received Accepted
5 May 1987 19 May 1987
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