(Hypertension. 1996;28:833-839.)
© 1996 American Heart Association, Inc.
Articles |
Structure-Activity Studies of B1 Receptor–Related Peptides
Antagonists
the Department of Pharmacology, Medical School, Universite de Sherbrooke, and the Department of Obstetrics and Gynecology, Centre hospitalier universitaire de Sherbrooke (D.B.) (Quebec, Canada).
Correspondence to D. Regoli, Department of Pharmacology, Medical School, Universite de Sherbrooke, 3001 12th Ave N, Sherbrooke, Quebec J1H 5N4, Canada.
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Abstract |
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We tested several peptides related to des-Arg9-bradykinin as stimulants or inhibitors of B1 (rabbit aorta, human umbilical vein) and B2 (rabbit jugular vein, guinea pig ileum, human umbilical vein) receptors. We also incubated the compounds with purified angiotensin-converting enzyme from rabbit lung to test their resistance to degradation. We evaluated apparent affinities (in terms of the affinity constant pA2) of compounds and their potential residual agonistic activities (
E). Bradykinin and des-Arg9-bradykinin were used as agonists for the B2 and B1 receptors, respectively. Degradation of peptides by the angiotensin-converting enzyme was prevented in the presence of a D-residue in position 7 of des-Arg9-bradykinin. Replacement of Pro7 with D-Tic combined with Leu, Ile, Ala, or D-Tic in position 8 led to weak B1 receptor antagonists, some of which had strong residual agonistic activities on the B2 receptor preparations. The use of D-ßNal in position 7, combined with Ile in position 8 and AcLys at the N-terminal (eg, AcLys[D-ßNal7,Ile8]des-Arg9-bradykinin) gave the most active B1 receptor antagonist (pA2 of 8.5 on rabbit aorta and human umbilical vein), which is also partially resistant to enzymatic degradation. Extension of the N-terminal end by Sar-Tyr-
Ahx (used for labeling purposes) and even cold-labeling of Tyr with iodine were compatible with high, selective, and specific antagonism of the B1 receptors. We compared some compounds with some already known B1 receptor antagonists to underline the novelty of new peptidic compounds.
Key Words: kinins • B1 receptors • adrenergic antagonists • rabbits • guinea pigs • human
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Introduction |
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Recent reports point to an important role of kinin B1 receptors in physiopathology. Dray and Perkins1 have reviewed the possible implications of B1 receptors in various inflammatory states, tissue reactions, and hyperalgesia, particularly the chronic phases of these experimental diseases. This has been further supported by some recent findings from Davis and coworkers2 in inflammatory hyperalgesia in the rat. Using B1 receptor antagonists, Alvarez et al3 have suggested that B1 receptors may be present in spontaneously hypertensive rats, and Chakir et al4 and Zuccollo et al5 have obtained strong evidence that B1 receptors may play a relevant role in streptozotocin-induced diabetes in rat and mouse models, respectively. It thus appears that B1 receptors are formed de novo and take part in the induction and/or maintenance of pathological states. Also, as pointed out by Marceau,6 "it is conceivable that B1 receptors can amplify the responses of injured tissues to kinins and, in some cases, take the relay of B2 receptors in chronic pathologies." B1 receptor antagonists were discovered in the late 1970s,7 8 but no substantial progress has been made in this area despite the evidence of their usefulness in basic pharmacology and various experimental pathologies.
We undertook the present study to search for new peptidic B1 receptor antagonists, starting with the most active structure, Lys[Leu8]des-Arg9-bradykinin,8 and trying to improve it. We made changes at the N-terminal end, to find the most suitable group that maintains affinity and protects against degradation by aminopeptidases, and in positions 7 and 8, to improve affinity and resistance to kininase II (EC 3.4.15.1), the enzyme that plays a major role in the inactivation of the kinins and their des-Arg9 metabolites.8 9 We prepared and tested new compounds in several isolated organs to provide precise pharmacological profiles and to assess the affinity and selectivity for the B1 receptor.
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Methods |
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Peptide Synthesis
All peptides were synthesized with a peptide synthesizer (Applied Biosystems 430 A) using Merrifield-type resins with the first amino acid attached. Amino acids were activated by dicyclohexylcarbodiimide 1-hydroxybenzotriazole (Peptides International) on 1-methyl-2-pyrrolidinone. Peptides were cleaved from the resins with anhydrous hydrogen fluoride in the presence of appropriate scavengers. The resulting peptides were purified by medium-performance reversed-phase (C18) chromatography and if necessary by HPLC. Peptide purity was assessed by analytical HPLC and identity confirmed by ion-spray mass spectrometry (VG Quattro).
Bioassays
Tissues were taken from New Zealand White rabbits (1.5 to 2.5 kg) and Dunken Hartley guinea pigs (250 to 350 g) of either sex that were killed by stunning and exsanguination. Umbilical cords were taken from healthy women 22 to 40 years old after spontaneous delivery at term. The RbJV and GPI, two preparations that contain B2 receptors; the RbA, which contains B1 receptors; and the HUV, a mixed preparation that contains both B1 and B2 receptors,10 were used. Helical strips of RbJV treated with 1 µmol/L captopril to avoid peptide degradation by the ACE, that is, kininase II, were prepared according to Gaudreau et al.11 Helical strips of RbA devoid of endothelium were prepared according to Furchgott and Bhadrakom.12 Longitudinal segments of GPI were prepared with the procedure described by Rang.13 Helical strips of HUV were prepared according to Gobeil et al.10 The tissues were suspended in 10-mL organ baths containing warm (37°C), oxygenated (95% O2/5% CO2) Krebs' solution of the following composition (mmol/L): NaCl 118.1, KCl 4.7, CaCl2·6H2O 2.5, KH2PO4 1.2, MgSO4·7H2O 1.18, NaHCO3 25.0, and D-glucose 5.5. The RbA and HUV were stretched with an initial tension of 2g, and the RbJV and GPI were loaded with 0.5g. Changes of tension produced by the various agents were measured with isometric transducers (model FT 03C, Grass Instrument Co). Myotropic contractions were displayed on a Grass polygraph (model 7D). Before the drugs were tested, the tissues were allowed to equilibrate for 60 to 120 minutes, during which the tissues were repeatedly washed and the tension readjusted every 15 minutes.
At the beginning of each experiment, a submaximal dose of bradykinin (9 nmol/L) was applied repeatedly on the RbJV, GPI, or HUV to ensure that tissues responded with stable contractions. In the RbA, the B1 preparation whose response has been shown to increase during the incubation in vitro,7 des-Arg9-bradykinin (550 nmol/L), was applied 1, 3, and 6 hours after the equilibration period so that the progressive increase of tissue sensitivity, which generally reaches a maximum after 3 to 6 hours, could be monitored. A similar protocol was used for the HUV.
Repeated applications of single and double concentrations of bradykinin (on RbJV, GPI, and HUV) and of des-Arg9-bradykinin (RbA and HUV) were made in the absence and presence of the various peptides for evaluation of their apparent affinities as antagonists in terms of the affinity constant pA2 (-log10 of the molar concentration of antagonist that reduces the effect of a double concentration of agonist to that of a single concentration).14 The antagonists were applied 10 minutes before the myotropic effects of either bradykinin (the B2 receptor agonist) or des-Arg9-bradykinin (the B1 receptor agonist). Pharmacological assays on the HUV (a mixed B1 and B2 receptor preparation) were done in the presence of either Hoe 140 (400 nmol/L; a potent B2 receptor antagonist) or Lys[Leu8]des-Arg9-bradykinin (1 µmol/L; a potent B1 receptor antagonist, applied 10 minutes before the tested agents) for study of B1 and B2 receptors, respectively.10 All kinin antagonists were initially applied to tissues at a concentration of 10 µg/mL for measurement of their potential agonistic activities (E) compared with bradykinin (in the B2 receptor preparations) or des-Arg9-bradykinin (in the B1 receptor preparations).
Experimental Protocol for Biochemical Assays
The metabolic stability of various kinin-related peptides was evaluated by incubation of the peptide in the presence of purified ACE from rabbit lungs (purchased from Sigma Chemical Co) dissolved in phosphate-buffered saline (50 mmol/L, pH 7.5, containing 300 mmol/L NaCl); the final enzyme concentration was 45 µg/mL. The experimental protocol for measurement of ACE activity has been described elsewhere.15 16 Briefly, 200 µmol/L of peptide (40 µL) was placed in a phosphate-buffered saline and incubated for 5 minutes at 37°C before addition of 8.5 µL ACE (total volume of the reaction medium, 190 µL). Samples were withdrawn at 30-minute intervals, immersed in boiling water for 5 minutes, and then cooled on ice and centrifuged (5 minutes) before HPLC analysis. Results of ACE metabolism are expressed as the percentage of kinin degradation after the incubation (30 minutes) of kinin substrates with the enzyme. In these experimental conditions and at the enzymatic dilution indicated above, naturally occurring kinins (eg, bradykinin and des-Arg9-bradykinin) were found to be fully metabolized after 30 minutes.16
HPLC Analysis of Peptide Digests
Pure peptides and metabolic products were analyzed by reversed-phase HPLC on a C18 µBondapak column (4.6 mmx25 cm) (Waters Chromatography) with 10-µm particle size. The peptides and their fragments were eluted at the rate of 2.0 mL/min with a linear gradient ranging from 5% to 65% acetonitrile/trifluoroacetic acid (0.05%)/water over 20 minutes at room temperature. The elution positions of these peptides were determined by following the absorbance at 214 nm. For each assay, 50 µL of the reaction medium (treated or not treated with ACE) was extracted and injected into the column. Concentrations of the peak products, different from the peaks of the peptide substrate, were estimated by a computer software program (Baseline 810, Waters) that also controlled all chromatographic operations. Isolated metabolites and substrates were collected and identified by electrospray mass spectrometry (positive mode).
Drugs
The Hoe 140 derivatives D-Arg[Hyp3,Thi5,D-Tic7,Oic8]des-Arg9-bradykinin (S 0765) and [Hyp3,Thi5,D-Tic7,Oic8]des-Arg9-bradykinin (S 1629)17 were a gift of Dr B. Scholkens (Hoechst, Frankfurt, Germany); D-Arg[Hyp3,D-Hyp7(trans-propyl),Oic8]des-Arg9-bradykinin (NPC 18565) and D-Arg[Hyp3,D-Hyp7(trans-thiophenyl),Oic8]des-Arg9-bradykinin (NPC 18828) were supplied by Dr D.J. Kyle (Scios Inc, Mountain View, Calif). The peptide AcLys[N-MeAla6,Leu8]des-Arg9-bradykinin15 was donated by Dr G. Drapeau (Centre de Recherche Hotel-Dieu de Quebec [Canada]). A series of new peptide analogues of Lys[Leu8]des-Arg9-bradykinin (see abbreviated structures in Table 1
) was prepared in our laboratory with the procedure described by Drapeau and Regoli,18 and they were analyzed as described above. Abbreviations follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature.19 Other abbreviations are described as follows: Hyp, trans-4-hydroxy-L-proline; Thi, ß-(2-thienyl)-L-alanine; Tic, L-(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid); Oic, L-(3aS,7aS)-octahydro-indol-2-carboxylic acid; ßNal: ß-3-(2-naphthyl)-alanine; Sar, sarcosine (N-methyl-glycine); and Ahx, -aminohexanoic acid. Captopril was purchased from Squibb Canada. All chemical agents were obtained from either Bachem or Novabiochem. Concentrated solutions (1 to 5 mg/mL) of peptides and other agents were made in double-distilled and deionized water and kept at -20°C until used.
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Results |
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Tables 1 through 3
present the results obtained with three series of compounds designed to (1) improve antagonistic affinity, (2) eliminate residual agonistic activities, and (3) prevent the degradation of B1 receptor antagonists by ACE. The first series (Table 1) includes compounds (containing a Hyp residue in position 3) that may derive from the conversion by carboxypeptidases of classic B2 receptor antagonists into C-terminal des-Arg9 fragments. The results, summarized in Table 1, present the pharmacological profile of each compound in terms of affinities (pA2) for the B1 receptor (RbA) and affinities (if any) as well as residual agonistic activities (E) on two B2 receptor subtypes (RbJV, GPI). The percentage of degradation (after 30 minutes of incubation) by ACE is also indicated.
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Pharmacological profiles of compounds 1 and 2 are in agreement with those reported by other researchers7 8 and indicate that the presence of a Lys residue at the N-terminal is favorable for antagonism on the B1 receptor, since Lys[Leu8]des-Arg9-bradykinin is 10-fold more active than [Leu8]des-Arg9-bradykinin. These peptides are rapidly inactivated by ACE. The presence of Hyp residue in position 3 and a D-Arg at the N-terminal (two modifications that favor antagonistic affinity on B2 receptors)20 21 22 brings a loss of the antagonistic affinity on B1 receptors (see compound 4) and eventually evokes some residual agonistic activities, especially on the guinea pig B2 receptor subtype. Changes at the N-terminal neither protect from degradation by ACE nor improve affinity for the rabbit B1 receptor. The des-Arg9 metabolites of B2 receptor antagonists of the first generation (compounds 6 and 7)20 21 also show low affinity on the B1 receptor and eventually exert weak antagonism on the B2 receptor of the RbJV. These compounds are expected to be resistant to ACE degradation because of the presence of a D-residue in position 7, which has been shown to protect bradykinin analogues from ACE.23
Results obtained with compounds in which Pro in position 7 was replaced by D-Tic are summarized in Table 2. Thus, D-Tic in position 7 prevents the proteolytic activity of ACE and also markedly reduces affinity for the B1 receptor. Furthermore, two compounds (No. 17 of Table 2 and No. 26 of Table 3) were tested as inhibitors of ACE and were found (at concentrations up to 10-4 mol/L) not to interact with the degradation of des-Arg9-bradykinin by the enzyme (C.F. and F.G., unpublished observations, 1996). To restore antagonistic affinity, we added various residues (Sar, Lys, AcLys, or D-Arg) at the N-terminal of [D-Tic7,Leu8]des-Arg9-bradykinin (compound 8). An increase of antagonistic affinity by 0.8 log unit was obtained with the inclusion of D-Arg (see compound 12). Moreover, the two compounds containing Lys (compounds 10 and 11) act as partial agonists in the B2 receptor preparations (especially on the GPI); these myogenic effects cannot be abolished by Hoe 140 (10 µg/mL), a potent B2 receptor antagonist (not shown). For this series of compounds, the most suitable chemical modifications appear to be D-Arg at the N-terminal and Ile at position 8 (compound 13). In fact, D-Arg[D-Tic7,Ile8]des-Arg9-bradykinin is an antagonist on the B1 receptor, showing a pA2 value of 6.97 with no agonistic activity on the B2 receptor and negligible degradation by ACE. Replacement of the D-Tic stereoisomer (compound 14) in position 7 causes a diminution of the antagonistic potency (pA2 <5.04) on the B1 receptor and a regaining of its susceptibility to being metabolized by ACE. The same results were obtained when the D-Tic residue in position 7 was replaced by a smaller residue, such as Ala (see compound 15). The presence of a Hyp residue in position 3 does not seem to play any particular role (see compounds 16 and 17).
In a third series of compounds (Table 3), Pro in position 7 was substituted by D-ßNal, and the N-terminal was prolonged with either a single residue or a short peptidic chain containing a Tyr residue for iodine labeling. In this series, the residue of choice for the N-terminal appears to be AcLys (see compounds 18 and 19), since D-Arg, D-Lys, and Sar, although suitable for protection from aminopeptidases, reduce antagonistic affinity for the B1 receptor to an extent of 0.5 to 1 log unit. Moreover, compounds 19, 22, 23, 25, and 26 (containing Ile in position 8) are partial agonists on the GPI. Again, these contractile effects are not prevented by Hoe 140 (10 µg/mL) and thus should be considered as nonspecific. As to position 8, Leu has the advantage over Ile of eliminating partial agonistic activities on the GPI (and RbJV) as well as conferring better protection against ACE hydrolysis (see Table 3). However, this latter substitution causes a slight reduction of antagonism potency compared with those compounds containing Ile (see Table 3). AcLys[D-ßNal7,Ile8]des-Arg9-bradykinin (compound 19) represents the most potent B1 receptor antagonist (pA2, 8.40±0.12), with little residual agonistic effect on the GPI (E, 0.38), and is partially metabolized by ACE in our experimental conditions. Furthermore, electrospray mass spectrometric analysis of the composition of the degradation mixture of AcLys[D-ßNal7,Ile8]des-Arg9-bradykinin is in agreement with the proteolysis pattern of des-Arg9 peptides by ACE and led to the identification of C-terminal tetrapeptidyl fragments (not shown). Complete protection from ACE is obtained by replacing Ser in position 6 with N-MeAla, but this is accompanied by a marked loss of B1 receptor antagonism (compounds 21 and 22). These data are consistent with those reported by Drapeau et al.15 A second series of B1 receptor antagonists, designed for labeling, was prepared following the report by Levesque et al24 (see compounds 27, 28, 29, and 30 in Table 3). The four compounds maintain high affinity for the B1 receptors of the RbA (pA2 values of 8.10 to 8.5) and little, if any, residual agonistic activity on the B2 receptors of the RbJV and GPI. They are, however, broken down by ACE, especially those that do not possess a D-amino acid in position 7 (compounds 27 and 28).
B1 and B2 receptor antagonists containing Oic in position 8 are presented in Table 4. These compounds, prepared by investigators at Hoechst and Scios, were tested in the same assays as those of Tables 1 through 3. All compounds exhibit complete resistance to ACE metabolism. All compounds are devoid of partial agonistic activity on both B1 and B2 receptor preparations. The presence of a D-residue in position 7 and of Oic in position 8 appears to be essential for metabolic protection as well as for inactivity as agonists. All compounds presented in Table 4 are nonselective and represent a new class of antagonists that are able to block both B1 and B2 receptors. The spectrum of biological activities shows some differences in that compounds 33 and 34 are more active on the B1 receptor, and compound 31 is more potent on the B2 receptor (see Table 4). Worthy of mention are the interesting pharmacological characteristics of compounds 31, 32, 33, and 34, which are active on the bradykinin B2 receptor of the RbJV as well as on the RbA but are almost inactive on the B2 receptor of the GPI.
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Activities of Some B1 Receptor Antagonists on Human B1 and B2 Receptors
Some peptidic compounds mentioned earlier were also tested on the HUV, a preparation that contains B1 and B2 receptors for the kinins.10 The results, summarized in Table 5, indicate that all compounds selected are pure B1 receptor antagonists, showing no agonistic or antagonistic activities on the human B2 functional site, with the exception of NPC 18828. The presence of Lys at the N-terminal is important for the B1 receptor antagonist because the affinity of Lys[Leu8]des-Arg9-bradykinin is higher than that of [Leu8]des-Arg9-bradykinin by at least 1.5 log units, in agreement with the results of Menke et al,25 who have cloned and characterized the human B1 receptor. The presence of D-Tic in position 7 is unfavorable for B1 receptor antagonism, but the affinity of the antagonist can be markedly increased by the addition of D-Arg at the N-terminal. Other substitutions in this position (with Sar or AcLys) are equally favorable in a series of compounds containing D-ßNal in position 7. A significant gain of affinity for the B1 receptor is observed when position 8 is occupied by Ile. Indeed, AcLys[D-ßNal7,Ile8]des-Arg9-bradykinin is the most active antagonist, showing a pA2 value of 8.49±0.10, which is almost 1.5 log units higher than those of all previous compounds. The stereospecificity of the residue ßNal in position 7 is again crucial, likewise in the rabbit, because the isomeric form L-ßNal shows three orders of magnitude less antagonistic activity than the D-ßNal form. Two of the compounds designed for labeling through an elongation of the N-terminal show very high affinities for the human B1 receptor and are inactive on the human B2 receptor. These results are similar to what has been observed on the rabbit B1 receptor. The Hoechst compound S 0765 is a pure B1 receptor antagonist (pA2, 7.29), in contrast with its mixed B1 and B2 antagonistic activities on the rabbit tissues. NPC 18828 behaves as an antagonist on both human B1 and B2 receptors, with a marked predominance for the former. Worthy of notice is the overall resemblance in the pharmacological profiles of the human and rabbit B1 receptors described in the present study.
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Discussion |
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The present analysis concerns four series of peptides designed to improve B1 receptor antagonism and obtain compounds with high affinity, full selectivity for the B1 receptor, and resistance to degradation by ACE and possibly aminopeptidases. To these goals, substitutions were made in positions 3, 7, and 8 of [Leu8]des-Arg9-bradykinin, and one residue or more were added to the N-terminal end, since Lys[Leu8]des-Arg9-bradykinin has been shown to be at least 10 times more active than [Leu8]des-Arg9-bradykinin in rabbits7 8 and humans.10 25
Recent studies have shown that human kinins have a Hyp residue in position 3.26 27 The reasons for this naturally occurring hydroxylation of the proline at position 3 in the kinin analogue sequences are unknown. It has been shown, by means of functional assays, that Hyp3 contained in bradykinin and its analogues may increase the affinity for the B2 receptor of certain species, such as rabbits and dogs.22 28 The majority of B2 receptor antagonists of the first and second generation also contain a Hyp residue in position 3 (eg, D-Arg[Hyp3,D-Phe7]-bradykinin20 and D-Arg[Hyp3,D-Phe7,Leu8]-bradykinin21 ) and may be converted to des-Arg9 derivatives (antagonists of the B1 receptors) even in vitro by the intramural carboxypeptidases of some isolated organs.29 We therefore designed the first series of compounds to evaluate the role of Hyp3, and the results presented in Table 1 indicate that Hyp in position 3 does not influence the antagonist affinities in the rabbit B1 receptor.
Human and animal kallidins have a Lys residue at the N-terminal end, which is probably the most abundant naturally occurring kinin analogue present in human plasma and urine.30 Furthermore, kallidin and des-Arg10-kallidin are sensitive to aminopeptidases (eg, aminopeptidase M [EC 3.4.11.2]) and can be protected by N-acetylation.15 However, first and second generations of antagonists have D-Arg at the N-terminal, a substitution that has also been shown to protect against degradation by aminopeptidases (see Stewart and Vavrek31 ). In the first three series (Tables 1 through 3), the role of the N-terminal position and the C-terminal portion, especially positions 7 and 8, were investigated together. Because of the predominant role played by ACE, the endothelial enzyme of the pulmonary circulation,32 in the inactivation of both the kinins and their des-Arg9 metabolites, B1 receptor antagonists must be protected if one wishes to prolong their in vivo activities. To this goal, D-Tic, a rigid phenylalanine surrogate that has been shown to be protective in Hoe 140,33 and D-ßNal, a nonnatural aromatic amino acid, were used in the peptide sequence. Results summarized in Tables 2 and 3 clearly indicate that the presence of D-Tic in position 7 not only completely prevents the ACE from acting but is also essential for antagonism on the B1 receptor (compare the two diastereoisomeric compounds 13 and 14 of Table 2). The major limitation of the D-Tic series is their modest antagonistic affinity, which remains 1.5 log units below that of Lys[Leu8]des-Arg9-bradykinin. The fundamental role of a D-residue in position 7 for B1 receptor antagonism is supported by the D-ßNal series (Table 3) and especially by the comparison between compounds 19 (which contains D-ßNal) and 20 (which contains L-ßNal). The presence of D-ßNal in position 7 not only protects (quite efficiently) against degradation by ACE but confers higher affinity (by 3 log units) and selectivity for the rabbit and human B1 receptors. The only limitation of compound 19 is its residual agonistic activity on the GPI, which appears to be due to a contractile effect of unknown nature, since it is not antagonized by either Hoe 140 or losartan, indomethacin, and atropine (data not shown). As for Phe at position 8, several substitutions were made with Leu, Ile, or Ala (in the second and third series), with the result that Ile was found to be the most favorable residue for B1 receptor antagonism. The presence of Ala in position 8 eliminates antagonistic activity, suggesting that a hydrophobic bulky residue is needed in position 8 for B1 receptor antagonism.
Lacking any knowledge of the geometric topography of the B1 receptor interacting sites, one can only speculate on the reasons for differential antagonistic potency observed among the [D-Tic7]des-Arg9-bradykinin analogues (second series) and the [D-ßNal7]des-Arg9-bradykinin derivatives (third series). Indeed, the D-Tic residue, because of its rigid and cyclic structure, may exert a greater influence on peptide conformation than the D-ßNal residue, which has a more flexible side chain. Additionally, both residues, because of their hydrophobic nature and their opposite stereochemical configuration, may enable the accessibility on the receptor via a hydrophobic pouch.
Few peptidic compounds, designed to obtain potential radioligands (to be labeled with 125I), were developed (see Table 3) following the recent report of Levesque et al.24 Two analogues (compounds 27 and 28) based on Lys[Leu8]des-Arg9-bradykinin, the prototypic B1 receptor antagonist, with an additionally extended N-terminal side, were developed. The results demonstrated that they maintained high affinities on the human and rabbit B1 receptors and that they are subject to catabolism by ACE. The insertion at the C-terminal end of enzymatically protecting residues such as D-ßNal in position 7 (compounds 29 and 30) gave rise to more resistant and still selective B1 receptor antagonists. The nonradioactive iodinated peptide (compound 30) was synthesized for assessment of possible changes in affinity and enzymatic resistance of this latter peptide. No significant changes were seen on the pharmacological and biochemical properties of compound 29; thus, SarTyrAhxLys[D-ßNal7,Ile8]des-Arg9-bradykinin (compound 29) may have its usefulness in binding studies on the kinin B1 receptors.
The use of Oic (a nonaromatic, tryptophan-like derivative) in position 8 conjointly with a D–stereo-orientation amino acid in position 7 has been shown to have important effects, such as preservation of B2 receptor antagonism, at least on the rabbit B2 receptor subtype, and thus provides a new category of antagonists acting on both B1 and B2 receptors. These findings are consistent with previous studies34 35 and can be extended with the results obtained with the antagonists NPC 18565 and NPC 18828 (compounds 33 and 34, respectively; Scios). Given the important role played by kinins in the acute (via the B2 receptor) and chronic (via the B1 receptor) phases of inflammation and pain,1 2 this type of compound may become very useful in the assessment of the role of kinins in experimental pathology. In fact, blockade of only one kinin receptor type, when both B1 and B2 receptors are involved, will generally lead to partial protection and underestimation of antagonist affinities. D-Arg[Hyp3,Thi5,D-Tic7,Oic8]des-Arg9-bradykinin (S 0765) and D-Arg[Hyp3,D-Tic7(trans-thiophenyl),Oic8]des-Arg9-bradykinin (NPC 18828) have shown dissimilarities between human and rabbit B2 receptor antagonism (see Tables 4 and 5). This can be explained by the existence of heterogeneous bradykinin B2 receptors among species and/or tissues. Furthermore, this is supported by previous studies that have demonstrated that S 0765 shows differential antagonistic potency among species.34 35 In fact, S 0765 is a poor B2 receptor antagonist in humans (present study), rats,35 and guinea pigs (present study and References 35 and 36), whereas it shows high potency in rabbit tissues35 (present study and Reference 35). As shown in Table 5, NPC 18828 is a very active and quite selective antagonist of the human B1 receptor and interacts with the rabbit but not the guinea pig B2 receptor subtype (Table 4). The present structure-activity study therefore has provided indications on the stereochemical requirements that might contribute to high potency and selectivity for B1 receptor antagonism. From the results presented above, it appears that the chemical features favoring affinity of antagonists on the human B1 receptor are similar to those for the rabbit B1 receptor, as already emphasized by Regoli et al36 and Gobeil et al.10
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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F.G. holds a scholarship from Bio-Mega/Boehringer-Ingelheim, S.N.A. is a student of the Gabon Government, and L.H.P. is a grant holder of the Canadian Stroke and Heart Foundation. D.R. is a career investigator of the Medical Research Council of Canada (MRCC). This work has been supported by the MRCC and by the Canadian Stroke and Heart Foundation.
Received May 31, 1996; first decision June 19, 1996; accepted June 19, 1996.
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References |
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