- Email: [email protected]
Interaction of linear and cyclic peptide antagonists at the human B2 kinin receptor
Peptides 23 (2002) 1457–1463
Interaction of linear and cyclic peptide antagonists at the human B2 kinin receptor Paola Cucchi a,∗ , Stefania Meini a , Laura Quartara b , Alessandro Giolitti c , Sabrina Zappitelli d , Luigi Rotondaro d , Carlo Alberto Maggi a a
Pharmacology Department, Menarini Ricerche S.p.A., 50135 Florence, Italy b Chemistry Department, Menarini Ricerche S.p.A., 50135 Florence, Italy c Drug Design Department, Menarini Ricerche S.p.A., 50135 Florence, Italy d Department of Biotechnology, Menarini Biotech, Rome, Italy Received 18 December 2001; accepted 4 March 2002
Abstract The ligand receptor interactions involving the C-terminal moiety of kinin B2 receptor antagonists Icatibant (H-DArg-Arg-Pro-HypGly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7␥-10␣)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser111 Ala and Ser111 Lys mutant human kinin B2 receptors. Icatibant and [Lys10 ]-Icatibant maintained the same high affinity towards the three receptors. For IcatibantNH2 , [Ala10 ]-Icatibant, MEN 11270 and [Glu10 ]-MEN 11270, the changes in affinity at the WT and Ser111 Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser111 residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg10 ]-Icatibant and [desArg10 ]-Icatibant-NH2 , moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg10 side chain and the C-terminal free carboxylic function favours a high affinity interaction. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Bradykinin; Bradykinin B2 receptor; Bradykinin antagonists; Icatibant; MEN 11270
1. Introduction Kinins are small peptides derived from the enzymatic action of plasma and tissue kallikrein on kininogens [3]. Kinins, such as bradykinin (H-Arg-Pro-Pro-Gly-Phe-SerPro-Phe-Arg-OH), and kallidin (Lys-bradykinin) and their corresponding C-terminal desArg metabolites, exert a number of proinflammatory effects by acting through two types of receptors termed B2 and B1 , respectively [20]. Both receptors belong to the superfamily of seven transmembrane G-protein-coupled receptors, and share a low (36%) degree of homology [10,19]. In recent years a number of mutagenesis studies have been performed to clarify the mode of interaction of peptide ligands with the B2 receptor. It has been reported that the receptor binding epitope of the N-terminal amino acidic portion of peptide agonists and antagonists do not overlap [2,7,9]. However, nuclear magnetic resonance (NMR) and molecu∗ Corresponding author. Tel.: +39-055-5680-732; fax: +39-055-5680-419. E-mail address: [email protected] (P. Cucchi).
lar modelling data suggest that both bradykinin and peptide B2 receptor antagonists [8,12,13,15] present a similar -turn conformation involving the four C-terminal amino acids. Fathy et al. [6] reported that the amino acid residue at position 111 in the third transmembrane domain of the two kinin receptors is crucial for discriminating between the C-terminal residue of B1 and B2 selective peptide ligands. According to these authors, the C-terminal Arg residue of receptor-bound B2 peptide agonists and antagonists would be located in proximity of the Ser111 residue in the kinin B2 receptor sequence. The replacement of Ser111 with Lys, the residue present at the equivalent position in the B1 receptor sequence, may cause a repulsion of the side chain of C-terminal Arg of B2 receptor ligands, while providing a counter-ion for the C-terminal carboxylic function of the desArg-B1 selective ligands [6]. In other words the Ser111 Lys variation would largely account for the remarkably low degree of cross talk between kinins and their des-Arg metabolites at B2 /B1 receptors. The decapeptide Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-OicArg-OH) [11] is a potent and selective B2 receptor antagonist: the importance of its C-terminal -turn conformation
0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 0 8 1 - 5
1458
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
for high affinity interaction with kinin B2 receptor is supported by the findings obtained with MEN 11270 (H-DArgArg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7␥-10␣)) in which the Ser7 residue of Icatibant was replaced by a diaminobutyric residue: this allowed performing a cyclization between the side chain of diaminobutyric and the carboxyl group of the C-terminal Arg residue [18], fixing the Cterminal sequence of the peptide into a -turn conformation [8]. Owing to the structural similarity between MEN 11270 and Icatibant, it could be assumed that the mode of interaction of these two peptide antagonists with the B2 receptor may be largely overlapping [16,18]. However, although MEN 11270 possesses high affinity and selectivity for the kinin B2 receptor, its affinity is somewhat lower than that of Icatibant (two- to six-fold depending on experimental conditions [17,18]), suggesting a mode of interaction not exactly overlapping. To get a deeper insight into the structure–affinity relationships of ligand–receptor interactions involving the C-terminal portion of Icatibant versus MEN 11270, we investigated a series of analogs modified in position 10 at the WT, Ser111 Ala and Ser111 Lys mutant human kinin B2 receptor. 2. Materials and methods 2.1. Site-directed mutagenesis of human B2 receptor cDNA and receptor expression in CHO cells Plasmid pBS/human B2 receptor (kindly provided by Prof. W. Müller-Esterl, Institute for Biochemistry, University Hospital of Frankfurt, Germany) contains a 1.8-kb cDNA for the human kinin B2 receptor cloned in the EcoRI site of the pBlueScript II KS(+) phagemid. The cDNA contains 158 nucleotides of 5 -noncoding sequence in front of the initiating AUG codon [1] in the human B2 receptor cDNA and 391 nucleotides of 3 -noncoding sequence. Site-directed mutagenesis of the cDNA was performed [14] using an in vitro mutagenesis kit (Muta-Gene Phagemid In Vitro Mutagenesis-Version 2-Biorad) according to the manufacturer’s instructions. WT and mutated cDNAs were isolated from NotI + SalI-digested pBS/human B2 receptor and cloned into the polylinker region of a similarly digested pMRS182 vector. pMRS182 was constructed by insertion of NotI and SalI sites by means of a synthetic oligonucleotide between the EcoRV and XbaI sites of pmCMVSV1dhfr. pmCMVSV1dhfr was constructed by removing granulocyte colony-stimulating factor cDNA sequences from XbaI-digested pmCMVG-CSFSV1dhfr (plasmid murine cytomegalovirus beta granulocyte colony-stimulating factor SV1 dhfr) [22]. The complete coding sequence of cDNAs for wild type and mutated human B2 receptors was confirmed by DNA sequencing. Human wild type and mutated B2 receptor cDNAs in pmCMVSV1dhfr were introduced by lipofection into dihydrofolate reductase (DHFR) deficient Chinese hamster ovary
cells (CHO) DUKX-B11 cells and stable DHFR+ transformants were selected as described [21]. 2.2. Binding experiments Cells at confluence were washed out of the medium by Phosphate Buffered Saline Dulbecco’s without calcium and magnesium and harvested by incubating at 37 ◦ C with Hanks Balanced Salt Solution (pH 7.4) with added N-[2-hydroxyethyl]piperazine-N -[2-ethanesulphonic acid] (10 mM), EDTA (1 mM), and a cocktail of peptidase inhibitors: 1,10 phenanthroline (1 mM), ethylene glycol bis (-aminoethyl ether)-N,N,N ,N -tetraacetic acid (1 mM), captopril, leupeptin, soybean trypsin inhibitor, dl-2-mercaptomethyl-3guanidoethylthiopropanoic acid (1 M each), chymostatin (3.3 M), phenylmethyl-sulphonyl fluoride (0.1 mM), and bacitracin (140 g ml−1 ). Cells were then washed in Ntris[hydroxymethyl]methyl-2-aminoethanesulphonic acid (TES, 10 mM, pH 7.4, at 4 ◦ C), containing the above described peptidase inhibitors cocktail, and homogenized with a Polytron (PT 3000, Kinematica), set at 15 000 rpm for 30 s. The homogenate was centrifuged at 45 000 × g for 45 min (4 ◦ C). The pellet was resuspended to obtain 7.5 mg ml−1 membrane protein concentration and frozen immediately in 1 ml aliquots by immersion in liquid nitrogen, and then stored at −80 ◦ C until use. The protein concentration was determined by the method of Bradford [4] with a Bio-Rad kit, using bovine serum albumin as reference standard. The buffer used for binding experiments was TES (10 mM, pH 7.4) containing 1,10 phenanthroline (1 mM), bacitracin (140 g ml−1 ), and bovine serum albumin (1 g l−1 ). Binding assay was performed in a final volume of 0.5 ml. An incubation time of 60 min at 25 ◦ C was used. All incubations were terminated by rapid filtration through UniFilter-96 plates (Packard) that had been pre-soaked for at least 2 h in polyethylenimine (0.6%), using a MicroMate 96 Cell Harvester (Packard Instrument Company). The tubes and filters were then washed five times with 0.5 ml aliquots of Tris buffer (50 mM, pH 7.4, 4 ◦ C). Filters were dried and soaked in Microscint 40 (Packard Instrument Company) and bound radioactivity was counted by a TopCount Microplate Scintillation Counter (Packard Instrument Company). Saturation binding studies with increasing concentrations of [3 H]bradykinin (0.02–20 nM) were performed at the wild type, Ser111 Ala and Ser111 Lys human B2 receptors in order to determine the equilibrium dissociation constants KD . The resulting saturation isotherms approximated to a one site binding model with the following KD and Bmax values: 0.16 ± 0.04 nM and 454 ± 20 fmol mg−1 for the WT, 0.26 ± 0.03 nM and 380 ± 10 fmol mg−1 for the Ser111 Ala and 0.47 ± 0.06 nM and 147 ± 4 fmol mg−1 for the Se111 Lys human kinin B2 receptor, respectively. Competition binding studies were carried out with a radioligand concentration close to the calculated KD for the
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
studied receptor. Competing ligands were tested in a wide range of concentrations (0.3 pM–10 M). 2.3. Data analysis Saturation and competition binding data were analysed using GraphPad Prism (San Diego, CA) in order to determine the maximum binding site density (Bmax ), the equilibrium dissociation constants (KD ) and the concentration of compound producing a 50% inhibition of maximum specific radioligand binding (IC50 ). The equilibrium inhibition constants (Ki ) were calculated using the equation of Cheng and Prusoff [5]: Ki = IC50 /1 + (L/KD ) where IC50 and KD are defined as above and L is equal to the concentration of free radioligand. All values are given as mean ±S.E. of the mean from three experiments, each one performed in duplicate. 2.4. Materials [3 H]bradykinin (specific activity 114 CI mmol−1 ) was provided by Du Pont NEN (Hertfordshire, UK). Bradykinin was obtained from Peninsula (St. Helens, UK). Leupeptin was obtained from Boehringer Mannheim (Germany),
1459
dl-2-mercaptomethyl-3-guanidoethylthiopropanoic acid from Calbiochem (La Jolla, CA, USA). All salts used were purchased from Merck (Darmstadt, Germany). All other materials were obtained from Sigma (St. Louis, MO, USA). All kinin B2 receptor antagonists used were synthesized in Menarini Ricerche (Florence, Italy). All compounds were dissolved in distilled water and stored at −25 ◦ C. 3. Results 3.1. Structure-affinity relationship of Icatibant and analogs modified in position 10 at the WT, Ser111 Ala, and Ser111 Lys human B2 receptors The affinity values (Ki ) of bradykinin, obtained from competition curves at its radioligand binding, were 0.17 nM (95% CI 0.13–0.22), 0.39 nM (95% CI 0.32–0.49) and 0.97 nM (95% CI 0.72–1.3) for the WT, Ser111 Ala and Ser111 Lys receptor, respectively. Icatibant (Fig. 1), displaced [3 H]bradykinin binding with high affinity at the WT receptor (Ki 80 pM) and its affinity was not affected by the Ser111 Ala or Ser111 Lys mutations (Table 1). The C-terminal amide derivative of
Fig. 1. Chemical structure of Icatibant and MEN 11270.
1460
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
Table 1 Affinities of Icatibanta MEN 11270b and their analogs at the WT and mutated human bradykinin B2 receptors Ligand
C-terminal residue (Xaa)
Ki c nM (95% Cl) WT
Ser111 Ala
Ser111 Lys
Icatibant
0.08 (0.06–0.11)
0.05 (0.03–0.07)
0.06 (0.04–0.08)
Icatibant-NH2
0.7 (0.4–1.3)
1.7 (1–2.9)
2.8∗ (1.8–4.3)
[Ala10 ]-Icatibant
2.7 (1.8–4.2)
4.3 (3.1–5.9)
0.25∗ (0.2–0.4)
[desArg10 ]-Icatibant
60.6 (34.3–107)
95.9 (59.7–154)
0.75∗ (0.5–1.1)
[desArg10 ]-Icatibant-NH2
0.2 (0.1–0.3)
0.3 (0.2–0.4)
0.1 (0.1–0.3)
[Lys10 ]-Icatibant
0.12 (0.1-0.14)
0.1 (0.09-0.16)
0.12 (0.1–0.15)
MEN 11270
0.25 (0.2–0.3)
0.1 (0.1–0.2)
1∗ (0.6–1.5)
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
1461
Table 1 (Continued ) Ligand
C-terminal residue (Xaa)
Ki c nM (95% Cl) WT
Ser111 Ala
Ser111 Lys
[Ala10 ]-MEN 11270
1.9 (1.6–2.2)
3.1 (2.1–4)
6∗ (4.5–8)
[Glu10 ]-MEN 11270
4.3 (2.8–6.4)
6.1 (4.5–8.2)
0.4∗ (0.3–0.6)
a
Amino acid sequence of Icatibant and analogs: H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Xaa. Amino acid sequence of MEN 11270 and analogs: H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Xaa)c(7␥-10␣). c The K (equilibrium inhibition constant) values were calculated from the equation of Cheng and Prusoff (see Section 2). Each value was derived i from three independent experiments performed in duplicate. ∗ Statistically different from WT. b
Icatibant, Icatibant-NH2 , showed a nine-fold decrease in affinity at the WT receptor (Table 1). Unlike Icatibant, the affinity of its C-terminal amide derivative was significantly impaired at the Ser111 Lys mutant (four-fold decrease as compared to the WT, Table 1). [Ala10 ]-Icatibant was 34-fold less potent than Icatibant itself competing for [3 H]bradykinin binding at the WT receptor (Table 1). At variance with the behaviour of Icatibant and its amide derivative, the affinity of [Ala10 ]-Icatibant, although overlapping at the Ser111 Ala receptor, was 11-fold higher at the Ser111 Lys mutant (Table 1). [desArg10 ]-Icatibant competed for [3 H]bradykinin binding at the WT receptor with a Ki value of 60.6 nM, being 700-fold less potent than Icatibant (Table 1), whereas the C-terminal amide derivative of [desArg10 ]-Icatibant was only 2.5-fold less potent than Icatibant (Table 1). In other terms [desArg10 ]-Icatibant-NH2 gained 300-fold affinity as compared to [desArg10 ]-Icatibant, which bears a C-terminal free carboxylic function. When tested at the mutated receptors [desArg10 ]-Icatibant-NH2 maintained the same affinity at the Ser111 Ala and Ser111 Lys (Table 1) receptors, whereas [desArg10 ]-Icatibant affinity was 80-fold higher at the Ser111 Lys receptor than at the WT receptor (Table 1). [Lys10 ]-Icatibant showed a comparable affinity to Icatibant at the WT, Ser111 Ala and Ser111 Lys mutant receptors (Table 1). 3.2. Structure–affinity relationship of MEN 11270 and analogs modified in position 10 at the WT, Ser111 Ala, and Ser111 Lys human B2 receptors MEN 11270 (Fig. 1), competed with high affinity for [3 H]bradykinin binding at the WT B2 receptor. Nevertheless, in this cell system, MEN 11270 was three-fold less potent than its linear analog Icatibant (Ki = 0.25 nM, Table 1). Similar to Icatibant-NH2 (see above) its affinity was reduced by four-fold at the Ser111 Lys mutant receptor, whereas no differences were observed at the Ser111 Ala mutant (Table 1).
At the WT receptor [Ala10 ]-MEN 11270 was eight-fold less potent than MEN 11270 itself (Table 1). Interestingly, the same chemical modification in the Icatibant sequence ([Ala10 ]-Icatibant, see above) reduced the antagonist affinity to a much greater extent (34-fold; Table 1). [Ala10 ]-MEN 11270 maintained the same affinity, as compared to the WT receptor, at the Ser111 Ala mutant, whereas it showed a three-fold decrease at the Ser111 Lys mutant (Table 1). On the contrary, [Ala10 ]-Icatibant affinity increased at the Ser111 Lys mutant as compared to the WT receptor (see above). [Glu10 ]-MEN 11270 competed for the [3 H]bradykinin binding at the WT receptor with an affinity 17-fold lower than MEN 11270 (Table 1). The affinity of [Glu10 ]-MEN 11270 was unaffected by the Ser111 Ala mutation, whereas it increased by about 10-fold at the Ser111 Lys mutant (Table 1), contrary to observations in the structure–affinity relationship of MEN 11270 and [Ala10 ]-MEN 11270 at the WT versus the Ser111 Lys receptor.
4. Discussion The relevance of Ser111 in the human kinin B2 receptor, and of the corresponding Lys residue in the human bradykinin B1 receptor, has already been reported in the literature [6]. We started from this observation to perform a two-sided structure–affinity relationship study involving the C-terminal residues of the peptide antagonists Icatibant and MEN 11270 on the one hand and the Ser111 residue of the human kinin B2 receptor on the other. It has been reported that the C-terminal moiety of Icatibant shows the tendency to form a type II -turn conformation [8,12,15]. MEN 11270, on its side, is constrained in the same -turn type [18]. Both peptides bear a charged basic residue in the C-terminal moiety (Arg10 ) but, compared to Icatibant, MEN 11270 lacks the free carboxylic group, involved in cyclization. The affinity of Icatibant did not significantly change at the three receptors under study (WT, Ser111 Ala, Ser111 Lys)
1462
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
whereas MEN 11270 showed a significant decrease in affinity at the Ser111 Lys. These results suggested that in the Icatibant sequence the positive charge of Arg10 side chain could be compensated by its free terminal carboxylic function, and therefore could not interfere with the Lys111 residue in the Ser111 Lys mutant. On the contrary, the side chain of Arg10 in MEN 11270, which lacks in its sequence a negative charged counterpart, could interact unfavourably with Lys111 . This interaction could be possible if the 111 residue of the human kinin B2 receptor is located in proximity to the Arg10 side chain. In order to confirm this hypothesis, the activity of the C-terminal amide derivative of Icatibant, Icatibant-NH2 , was investigated. The affinity for the WT receptor drops by about 10-fold as compared to Icatibant and similarly to MEN 11270, the introduction of a Lys in place of Ser111 yielded a decrease in affinity. In [Ala10 ]-Icatibant the positive charge of the Arg10 was removed, maintaining the negatively charged C-terminal carboxylic function. A significant drop in affinity was observed at WT and Ser111 Ala receptors. With this ligand, in fact, an intramolecular compensation between the side chain of Arg10 and its negative carboxylic group is no longer possible. In principle, the observed decrease in affinity could depend either on the modified charge situation or to an indirect effect on the overall peptide conformation. The higher affinity observed with the Ser111 Lys mutant suggests that charge compensation is the main cause. This is also in agreement with the results obtained with [desArg10 ]-Icatibant, whose affinity was dramatically decreased at the WT receptor but significantly increased at the Ser111 Lys mutant, where the carboxylic function could find a charged counterpart in the receptor. On the contrary, the corresponding C-terminal amide, [desArg10 ]-Icatibant-NH2 , which does not bear the negative charge, showed affinity values at WT receptor higher than [desArg10 ]-Icatibant and unvaried at Ser111 Lys mutant (as observed for Icatibant itself). The positive charge of Arg is delocalized on the guanidinium group, while the Lys has a point charge. Data obtained with [Lys10 ]-Icatibant, which maintains the same affinity of the precursor at the WT and mutated receptors, are in agreement with a completely ionic charge–charge interaction. As previously described, and in agreement with the results obtained with Icatibant-NH2 , the affinity of MEN 11270 was significantly decreased by the unfavourable proximity to the Lys111 positive charge in the Ser111 Lys mutant. Moreover the decrease in affinity at the three receptors of the Ala10 derivative of MEN 11270 suggests the loss of some favourable interaction between the Arg10 side chain of MEN 11270 and the receptor. The charge interaction relevance is confirmed by the derivative [Glu10 ]-MEN 11270; similarly to [Ala10 ]-Icatibant, the affinity of [Glu10 ]-MEN 11270 at the WT receptor is lower than that of MEN 11270 for the possible loss of some favourable interaction, with the additional unfavourable presence of a negative charge. The affinity towards Ser111 Lys was restored to the value of MEN
11270 at the WT receptor, suggesting an equivalent point charge interaction. It is worth noting that none of the modifications of the C-terminal amino acid induced an impairment of ligands affinity to the Ser111 Ala mutant receptor, thus ruling out the possibility that the side chain of Ser111 might participate in a direct interaction with the investigated peptide structures, and confirming the proximity of the C-terminal part of both Icatibant and MEN 11270 to the Ser111 . The comparison of Icatibant and its derivatives with MEN 11270 and its derivatives suggests that the C-terminal -turn structural feature is needed to achieve the active conformation, but is not sufficient for high affinity of the ligand towards the kinin B2 receptor. All these data demonstrated that also the charge compensation at the C-terminal portion of these peptides and the resulting charge–charge interactions with the receptor could affect the affinity. In particular, our results demonstrate that the high affinity of Icatibant is maintained only when the positive charge of the Arg10 side chain could be compensated by the negatively charged carboxylic group. Moreover, although the alternative hypothesis of a ligand conformational change due to sequence modifications cannot be excluded, the data obtained at the Ser111 Lys mutant suggest that most of the changes in affinity could be explained mainly by the presence of a charge–charge interaction.
References [1] AbdAlla S, Godovac-Zimmermann J, Braun A, Roscher AA, Müller-Esterl W, Quitterer U. Structure of bradykinin B2 receptor’s amino terminus. Biochemistry 1996a;35:7514–9. [2] AbdAlla S, Quitterer U, Grigoriev S, Maidhof A, Haasemann M, Jarnagin K, et al. Extracellular domains of the bradykinin B2 receptor involved in ligand binding and agonist sensing defined by anti-peptide antibodies. J Biol Chem 1996b;271:1725–48. [3] Bhoola KD, Figueroa CD, Worhty K. Bioregulation of kinins: kallicreins, kininogens and kininases. Pharmacol Rev 1992;44:1–80. [4] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 1976;75:248–54. [5] Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1 ) and the concentration of inhibitor which causes 50 per cent inhibition (I50 ) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099–108. [6] Fathy DB, Mathis A, Leeb T, Leeb-Lundberg LMF. A single position in the third transmembrane domains of the human B1 and B2 bradykinin receptors is adjacent to and discriminates between the C-terminal residues of subtype-selective ligands. J Biol Chem 1998;273:12210–8. [7] Jarnagin K, Bhakta S, Zuppan P, Yee C, Ho T, Phan T, et al. Mutations in the B2 bradykinin receptor reveal a different pattern of contacts for peptidic agonists and peptidic antagonists. J Biol Chem 1996;271:28277–86. [8] Guba W, Haessner R, Breiphol G, Henke S, Knolle J, Santagada V, et al. Combined approach of NMR and molecular dynamics within a biphasic membrane mimetic: conformation and orientation of the bradykinin antagonist Hoe 140. J Am Chem Soc 1994;116:7532–40. [9] Herzig MCS, Nash NR, Connolly M, Kyle DJ, Leeb-Lundberg LMF. The N terminus of bradykinin when bound to the human bradykinin
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
[10]
[11]
[12]
[13]
[14] [15]
[16]
B2 receptor is adjacent to extracellular Cys20 and Cys277 in the receptor. J Biol Chem 1996;271:29746–51. Hess JF, Borkowski JA, Young GS, Strader CD, Ransom RW. Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor. Biochem Biophys Res Commun 1992;184:260–8. Hock FJ, Wirth K, Albus U, Linz W, Gerhards HJ, Wiemer G, et al. Hoe 140 a new potent and long acting brakykinin antagonist: in vitro studies. Br J Pharmacol 1991;102:769–73. Jarnagin K, Bhakta S, Zuppan P, Yee C, Ho T, Phan T, et al. Mutations in the B2 bradykinin receptor reveal a different pattern of contacts for peptidic agonist and peptidic antagonists. J Biol Chem 1996;271:28277–86. Kotovych G, Cann JR, Stewart JM, Yamamoto H. NMR an CD conformational studies of bradykinin and its agonists and antagonists: application to receptor binding. Biochem Cell Biol 1998;76:257–66. Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 1985;82:488–92. Kyle DJ, Chakravarty S, Sinsko JA, Stormann TM. A proposed model of bradykinin bound to the rat B2 receptor and its utility for drug design. J Med Chem 1994;37:1347–54. Meini S, Patacchini R, Giuliani S, Lazzeri M, Turini D, Maggi CA, et al. Characterization of bradykinin B2 receptor antagonists in human and rat urinary bladder. Eur J Pharmacol 2000a;388:177–82.
1463
[17] Meini S, Patacchini R, Lecci A, Quartara L, Maggi CA. Peptide and non-peptide bradykinin B2 receptor agonists and antagonists: a reappraisal of their pharmacology in guinea-pig ileum. Eur J Pharmacol 2000b;409:185–94. [18] Meini S, Quartara L, Rizzi A, Patacchini R, Cucchi P, Giolitti A, et al. MEN 11270 a novel selective constrained peptide antagonist with high affinity at the human B2 kinin receptor. J Pharmacol Exp Ther 1999;289:1250–6. [19] Menke JG, Borkowski JA, Bierilo KK, MacNeil T, Derrick AW, Schneck KA, et al. Expression cloning of a human B1 bradykinin receptors. J Biochem Chem 1994;269:21583–6. [20] Regoli D, Barabé J. Pharmacology of bradykinin and related kinins. Pharmacol Rev 1980;32:1–46. [21] Renzetti AR, Catalioto RM, Carloni C, Criscuoli M, Cucchi P, Giolitti A, et al. Effects of Tyrosyne289Phenylalanine mutation on binding and functional properties of human tachykinin NK2 receptor stably expressed in Chinese Hamster Ovary Cells. Biochem Pharmacol 1999;57:899–906. [22] Rotondaro L, Mazzanti L, Mele A, Rovera G. High-level expression of cDNA for granulocyte colony-stimulating factor in Chinese hamster ovary cells. Effects of 3 -non-coding sequences. Mol Biotechnol 1997;7:231–40.
Interaction of linear and cyclic peptide antagonists at the human B2 kinin receptor Paola Cucchi a,∗ , Stefania Meini a , Laura Quartara b , Alessandro Giolitti c , Sabrina Zappitelli d , Luigi Rotondaro d , Carlo Alberto Maggi a a
Pharmacology Department, Menarini Ricerche S.p.A., 50135 Florence, Italy b Chemistry Department, Menarini Ricerche S.p.A., 50135 Florence, Italy c Drug Design Department, Menarini Ricerche S.p.A., 50135 Florence, Italy d Department of Biotechnology, Menarini Biotech, Rome, Italy Received 18 December 2001; accepted 4 March 2002
Abstract The ligand receptor interactions involving the C-terminal moiety of kinin B2 receptor antagonists Icatibant (H-DArg-Arg-Pro-HypGly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7␥-10␣)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser111 Ala and Ser111 Lys mutant human kinin B2 receptors. Icatibant and [Lys10 ]-Icatibant maintained the same high affinity towards the three receptors. For IcatibantNH2 , [Ala10 ]-Icatibant, MEN 11270 and [Glu10 ]-MEN 11270, the changes in affinity at the WT and Ser111 Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser111 residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg10 ]-Icatibant and [desArg10 ]-Icatibant-NH2 , moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg10 side chain and the C-terminal free carboxylic function favours a high affinity interaction. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Bradykinin; Bradykinin B2 receptor; Bradykinin antagonists; Icatibant; MEN 11270
1. Introduction Kinins are small peptides derived from the enzymatic action of plasma and tissue kallikrein on kininogens [3]. Kinins, such as bradykinin (H-Arg-Pro-Pro-Gly-Phe-SerPro-Phe-Arg-OH), and kallidin (Lys-bradykinin) and their corresponding C-terminal desArg metabolites, exert a number of proinflammatory effects by acting through two types of receptors termed B2 and B1 , respectively [20]. Both receptors belong to the superfamily of seven transmembrane G-protein-coupled receptors, and share a low (36%) degree of homology [10,19]. In recent years a number of mutagenesis studies have been performed to clarify the mode of interaction of peptide ligands with the B2 receptor. It has been reported that the receptor binding epitope of the N-terminal amino acidic portion of peptide agonists and antagonists do not overlap [2,7,9]. However, nuclear magnetic resonance (NMR) and molecu∗ Corresponding author. Tel.: +39-055-5680-732; fax: +39-055-5680-419. E-mail address: [email protected] (P. Cucchi).
lar modelling data suggest that both bradykinin and peptide B2 receptor antagonists [8,12,13,15] present a similar -turn conformation involving the four C-terminal amino acids. Fathy et al. [6] reported that the amino acid residue at position 111 in the third transmembrane domain of the two kinin receptors is crucial for discriminating between the C-terminal residue of B1 and B2 selective peptide ligands. According to these authors, the C-terminal Arg residue of receptor-bound B2 peptide agonists and antagonists would be located in proximity of the Ser111 residue in the kinin B2 receptor sequence. The replacement of Ser111 with Lys, the residue present at the equivalent position in the B1 receptor sequence, may cause a repulsion of the side chain of C-terminal Arg of B2 receptor ligands, while providing a counter-ion for the C-terminal carboxylic function of the desArg-B1 selective ligands [6]. In other words the Ser111 Lys variation would largely account for the remarkably low degree of cross talk between kinins and their des-Arg metabolites at B2 /B1 receptors. The decapeptide Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-OicArg-OH) [11] is a potent and selective B2 receptor antagonist: the importance of its C-terminal -turn conformation
0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 0 8 1 - 5
1458
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
for high affinity interaction with kinin B2 receptor is supported by the findings obtained with MEN 11270 (H-DArgArg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7␥-10␣)) in which the Ser7 residue of Icatibant was replaced by a diaminobutyric residue: this allowed performing a cyclization between the side chain of diaminobutyric and the carboxyl group of the C-terminal Arg residue [18], fixing the Cterminal sequence of the peptide into a -turn conformation [8]. Owing to the structural similarity between MEN 11270 and Icatibant, it could be assumed that the mode of interaction of these two peptide antagonists with the B2 receptor may be largely overlapping [16,18]. However, although MEN 11270 possesses high affinity and selectivity for the kinin B2 receptor, its affinity is somewhat lower than that of Icatibant (two- to six-fold depending on experimental conditions [17,18]), suggesting a mode of interaction not exactly overlapping. To get a deeper insight into the structure–affinity relationships of ligand–receptor interactions involving the C-terminal portion of Icatibant versus MEN 11270, we investigated a series of analogs modified in position 10 at the WT, Ser111 Ala and Ser111 Lys mutant human kinin B2 receptor. 2. Materials and methods 2.1. Site-directed mutagenesis of human B2 receptor cDNA and receptor expression in CHO cells Plasmid pBS/human B2 receptor (kindly provided by Prof. W. Müller-Esterl, Institute for Biochemistry, University Hospital of Frankfurt, Germany) contains a 1.8-kb cDNA for the human kinin B2 receptor cloned in the EcoRI site of the pBlueScript II KS(+) phagemid. The cDNA contains 158 nucleotides of 5 -noncoding sequence in front of the initiating AUG codon [1] in the human B2 receptor cDNA and 391 nucleotides of 3 -noncoding sequence. Site-directed mutagenesis of the cDNA was performed [14] using an in vitro mutagenesis kit (Muta-Gene Phagemid In Vitro Mutagenesis-Version 2-Biorad) according to the manufacturer’s instructions. WT and mutated cDNAs were isolated from NotI + SalI-digested pBS/human B2 receptor and cloned into the polylinker region of a similarly digested pMRS182 vector. pMRS182 was constructed by insertion of NotI and SalI sites by means of a synthetic oligonucleotide between the EcoRV and XbaI sites of pmCMVSV1dhfr. pmCMVSV1dhfr was constructed by removing granulocyte colony-stimulating factor cDNA sequences from XbaI-digested pmCMVG-CSFSV1dhfr (plasmid murine cytomegalovirus beta granulocyte colony-stimulating factor SV1 dhfr) [22]. The complete coding sequence of cDNAs for wild type and mutated human B2 receptors was confirmed by DNA sequencing. Human wild type and mutated B2 receptor cDNAs in pmCMVSV1dhfr were introduced by lipofection into dihydrofolate reductase (DHFR) deficient Chinese hamster ovary
cells (CHO) DUKX-B11 cells and stable DHFR+ transformants were selected as described [21]. 2.2. Binding experiments Cells at confluence were washed out of the medium by Phosphate Buffered Saline Dulbecco’s without calcium and magnesium and harvested by incubating at 37 ◦ C with Hanks Balanced Salt Solution (pH 7.4) with added N-[2-hydroxyethyl]piperazine-N -[2-ethanesulphonic acid] (10 mM), EDTA (1 mM), and a cocktail of peptidase inhibitors: 1,10 phenanthroline (1 mM), ethylene glycol bis (-aminoethyl ether)-N,N,N ,N -tetraacetic acid (1 mM), captopril, leupeptin, soybean trypsin inhibitor, dl-2-mercaptomethyl-3guanidoethylthiopropanoic acid (1 M each), chymostatin (3.3 M), phenylmethyl-sulphonyl fluoride (0.1 mM), and bacitracin (140 g ml−1 ). Cells were then washed in Ntris[hydroxymethyl]methyl-2-aminoethanesulphonic acid (TES, 10 mM, pH 7.4, at 4 ◦ C), containing the above described peptidase inhibitors cocktail, and homogenized with a Polytron (PT 3000, Kinematica), set at 15 000 rpm for 30 s. The homogenate was centrifuged at 45 000 × g for 45 min (4 ◦ C). The pellet was resuspended to obtain 7.5 mg ml−1 membrane protein concentration and frozen immediately in 1 ml aliquots by immersion in liquid nitrogen, and then stored at −80 ◦ C until use. The protein concentration was determined by the method of Bradford [4] with a Bio-Rad kit, using bovine serum albumin as reference standard. The buffer used for binding experiments was TES (10 mM, pH 7.4) containing 1,10 phenanthroline (1 mM), bacitracin (140 g ml−1 ), and bovine serum albumin (1 g l−1 ). Binding assay was performed in a final volume of 0.5 ml. An incubation time of 60 min at 25 ◦ C was used. All incubations were terminated by rapid filtration through UniFilter-96 plates (Packard) that had been pre-soaked for at least 2 h in polyethylenimine (0.6%), using a MicroMate 96 Cell Harvester (Packard Instrument Company). The tubes and filters were then washed five times with 0.5 ml aliquots of Tris buffer (50 mM, pH 7.4, 4 ◦ C). Filters were dried and soaked in Microscint 40 (Packard Instrument Company) and bound radioactivity was counted by a TopCount Microplate Scintillation Counter (Packard Instrument Company). Saturation binding studies with increasing concentrations of [3 H]bradykinin (0.02–20 nM) were performed at the wild type, Ser111 Ala and Ser111 Lys human B2 receptors in order to determine the equilibrium dissociation constants KD . The resulting saturation isotherms approximated to a one site binding model with the following KD and Bmax values: 0.16 ± 0.04 nM and 454 ± 20 fmol mg−1 for the WT, 0.26 ± 0.03 nM and 380 ± 10 fmol mg−1 for the Ser111 Ala and 0.47 ± 0.06 nM and 147 ± 4 fmol mg−1 for the Se111 Lys human kinin B2 receptor, respectively. Competition binding studies were carried out with a radioligand concentration close to the calculated KD for the
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
studied receptor. Competing ligands were tested in a wide range of concentrations (0.3 pM–10 M). 2.3. Data analysis Saturation and competition binding data were analysed using GraphPad Prism (San Diego, CA) in order to determine the maximum binding site density (Bmax ), the equilibrium dissociation constants (KD ) and the concentration of compound producing a 50% inhibition of maximum specific radioligand binding (IC50 ). The equilibrium inhibition constants (Ki ) were calculated using the equation of Cheng and Prusoff [5]: Ki = IC50 /1 + (L/KD ) where IC50 and KD are defined as above and L is equal to the concentration of free radioligand. All values are given as mean ±S.E. of the mean from three experiments, each one performed in duplicate. 2.4. Materials [3 H]bradykinin (specific activity 114 CI mmol−1 ) was provided by Du Pont NEN (Hertfordshire, UK). Bradykinin was obtained from Peninsula (St. Helens, UK). Leupeptin was obtained from Boehringer Mannheim (Germany),
1459
dl-2-mercaptomethyl-3-guanidoethylthiopropanoic acid from Calbiochem (La Jolla, CA, USA). All salts used were purchased from Merck (Darmstadt, Germany). All other materials were obtained from Sigma (St. Louis, MO, USA). All kinin B2 receptor antagonists used were synthesized in Menarini Ricerche (Florence, Italy). All compounds were dissolved in distilled water and stored at −25 ◦ C. 3. Results 3.1. Structure-affinity relationship of Icatibant and analogs modified in position 10 at the WT, Ser111 Ala, and Ser111 Lys human B2 receptors The affinity values (Ki ) of bradykinin, obtained from competition curves at its radioligand binding, were 0.17 nM (95% CI 0.13–0.22), 0.39 nM (95% CI 0.32–0.49) and 0.97 nM (95% CI 0.72–1.3) for the WT, Ser111 Ala and Ser111 Lys receptor, respectively. Icatibant (Fig. 1), displaced [3 H]bradykinin binding with high affinity at the WT receptor (Ki 80 pM) and its affinity was not affected by the Ser111 Ala or Ser111 Lys mutations (Table 1). The C-terminal amide derivative of
Fig. 1. Chemical structure of Icatibant and MEN 11270.
1460
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
Table 1 Affinities of Icatibanta MEN 11270b and their analogs at the WT and mutated human bradykinin B2 receptors Ligand
C-terminal residue (Xaa)
Ki c nM (95% Cl) WT
Ser111 Ala
Ser111 Lys
Icatibant
0.08 (0.06–0.11)
0.05 (0.03–0.07)
0.06 (0.04–0.08)
Icatibant-NH2
0.7 (0.4–1.3)
1.7 (1–2.9)
2.8∗ (1.8–4.3)
[Ala10 ]-Icatibant
2.7 (1.8–4.2)
4.3 (3.1–5.9)
0.25∗ (0.2–0.4)
[desArg10 ]-Icatibant
60.6 (34.3–107)
95.9 (59.7–154)
0.75∗ (0.5–1.1)
[desArg10 ]-Icatibant-NH2
0.2 (0.1–0.3)
0.3 (0.2–0.4)
0.1 (0.1–0.3)
[Lys10 ]-Icatibant
0.12 (0.1-0.14)
0.1 (0.09-0.16)
0.12 (0.1–0.15)
MEN 11270
0.25 (0.2–0.3)
0.1 (0.1–0.2)
1∗ (0.6–1.5)
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
1461
Table 1 (Continued ) Ligand
C-terminal residue (Xaa)
Ki c nM (95% Cl) WT
Ser111 Ala
Ser111 Lys
[Ala10 ]-MEN 11270
1.9 (1.6–2.2)
3.1 (2.1–4)
6∗ (4.5–8)
[Glu10 ]-MEN 11270
4.3 (2.8–6.4)
6.1 (4.5–8.2)
0.4∗ (0.3–0.6)
a
Amino acid sequence of Icatibant and analogs: H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Xaa. Amino acid sequence of MEN 11270 and analogs: H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Xaa)c(7␥-10␣). c The K (equilibrium inhibition constant) values were calculated from the equation of Cheng and Prusoff (see Section 2). Each value was derived i from three independent experiments performed in duplicate. ∗ Statistically different from WT. b
Icatibant, Icatibant-NH2 , showed a nine-fold decrease in affinity at the WT receptor (Table 1). Unlike Icatibant, the affinity of its C-terminal amide derivative was significantly impaired at the Ser111 Lys mutant (four-fold decrease as compared to the WT, Table 1). [Ala10 ]-Icatibant was 34-fold less potent than Icatibant itself competing for [3 H]bradykinin binding at the WT receptor (Table 1). At variance with the behaviour of Icatibant and its amide derivative, the affinity of [Ala10 ]-Icatibant, although overlapping at the Ser111 Ala receptor, was 11-fold higher at the Ser111 Lys mutant (Table 1). [desArg10 ]-Icatibant competed for [3 H]bradykinin binding at the WT receptor with a Ki value of 60.6 nM, being 700-fold less potent than Icatibant (Table 1), whereas the C-terminal amide derivative of [desArg10 ]-Icatibant was only 2.5-fold less potent than Icatibant (Table 1). In other terms [desArg10 ]-Icatibant-NH2 gained 300-fold affinity as compared to [desArg10 ]-Icatibant, which bears a C-terminal free carboxylic function. When tested at the mutated receptors [desArg10 ]-Icatibant-NH2 maintained the same affinity at the Ser111 Ala and Ser111 Lys (Table 1) receptors, whereas [desArg10 ]-Icatibant affinity was 80-fold higher at the Ser111 Lys receptor than at the WT receptor (Table 1). [Lys10 ]-Icatibant showed a comparable affinity to Icatibant at the WT, Ser111 Ala and Ser111 Lys mutant receptors (Table 1). 3.2. Structure–affinity relationship of MEN 11270 and analogs modified in position 10 at the WT, Ser111 Ala, and Ser111 Lys human B2 receptors MEN 11270 (Fig. 1), competed with high affinity for [3 H]bradykinin binding at the WT B2 receptor. Nevertheless, in this cell system, MEN 11270 was three-fold less potent than its linear analog Icatibant (Ki = 0.25 nM, Table 1). Similar to Icatibant-NH2 (see above) its affinity was reduced by four-fold at the Ser111 Lys mutant receptor, whereas no differences were observed at the Ser111 Ala mutant (Table 1).
At the WT receptor [Ala10 ]-MEN 11270 was eight-fold less potent than MEN 11270 itself (Table 1). Interestingly, the same chemical modification in the Icatibant sequence ([Ala10 ]-Icatibant, see above) reduced the antagonist affinity to a much greater extent (34-fold; Table 1). [Ala10 ]-MEN 11270 maintained the same affinity, as compared to the WT receptor, at the Ser111 Ala mutant, whereas it showed a three-fold decrease at the Ser111 Lys mutant (Table 1). On the contrary, [Ala10 ]-Icatibant affinity increased at the Ser111 Lys mutant as compared to the WT receptor (see above). [Glu10 ]-MEN 11270 competed for the [3 H]bradykinin binding at the WT receptor with an affinity 17-fold lower than MEN 11270 (Table 1). The affinity of [Glu10 ]-MEN 11270 was unaffected by the Ser111 Ala mutation, whereas it increased by about 10-fold at the Ser111 Lys mutant (Table 1), contrary to observations in the structure–affinity relationship of MEN 11270 and [Ala10 ]-MEN 11270 at the WT versus the Ser111 Lys receptor.
4. Discussion The relevance of Ser111 in the human kinin B2 receptor, and of the corresponding Lys residue in the human bradykinin B1 receptor, has already been reported in the literature [6]. We started from this observation to perform a two-sided structure–affinity relationship study involving the C-terminal residues of the peptide antagonists Icatibant and MEN 11270 on the one hand and the Ser111 residue of the human kinin B2 receptor on the other. It has been reported that the C-terminal moiety of Icatibant shows the tendency to form a type II -turn conformation [8,12,15]. MEN 11270, on its side, is constrained in the same -turn type [18]. Both peptides bear a charged basic residue in the C-terminal moiety (Arg10 ) but, compared to Icatibant, MEN 11270 lacks the free carboxylic group, involved in cyclization. The affinity of Icatibant did not significantly change at the three receptors under study (WT, Ser111 Ala, Ser111 Lys)
1462
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
whereas MEN 11270 showed a significant decrease in affinity at the Ser111 Lys. These results suggested that in the Icatibant sequence the positive charge of Arg10 side chain could be compensated by its free terminal carboxylic function, and therefore could not interfere with the Lys111 residue in the Ser111 Lys mutant. On the contrary, the side chain of Arg10 in MEN 11270, which lacks in its sequence a negative charged counterpart, could interact unfavourably with Lys111 . This interaction could be possible if the 111 residue of the human kinin B2 receptor is located in proximity to the Arg10 side chain. In order to confirm this hypothesis, the activity of the C-terminal amide derivative of Icatibant, Icatibant-NH2 , was investigated. The affinity for the WT receptor drops by about 10-fold as compared to Icatibant and similarly to MEN 11270, the introduction of a Lys in place of Ser111 yielded a decrease in affinity. In [Ala10 ]-Icatibant the positive charge of the Arg10 was removed, maintaining the negatively charged C-terminal carboxylic function. A significant drop in affinity was observed at WT and Ser111 Ala receptors. With this ligand, in fact, an intramolecular compensation between the side chain of Arg10 and its negative carboxylic group is no longer possible. In principle, the observed decrease in affinity could depend either on the modified charge situation or to an indirect effect on the overall peptide conformation. The higher affinity observed with the Ser111 Lys mutant suggests that charge compensation is the main cause. This is also in agreement with the results obtained with [desArg10 ]-Icatibant, whose affinity was dramatically decreased at the WT receptor but significantly increased at the Ser111 Lys mutant, where the carboxylic function could find a charged counterpart in the receptor. On the contrary, the corresponding C-terminal amide, [desArg10 ]-Icatibant-NH2 , which does not bear the negative charge, showed affinity values at WT receptor higher than [desArg10 ]-Icatibant and unvaried at Ser111 Lys mutant (as observed for Icatibant itself). The positive charge of Arg is delocalized on the guanidinium group, while the Lys has a point charge. Data obtained with [Lys10 ]-Icatibant, which maintains the same affinity of the precursor at the WT and mutated receptors, are in agreement with a completely ionic charge–charge interaction. As previously described, and in agreement with the results obtained with Icatibant-NH2 , the affinity of MEN 11270 was significantly decreased by the unfavourable proximity to the Lys111 positive charge in the Ser111 Lys mutant. Moreover the decrease in affinity at the three receptors of the Ala10 derivative of MEN 11270 suggests the loss of some favourable interaction between the Arg10 side chain of MEN 11270 and the receptor. The charge interaction relevance is confirmed by the derivative [Glu10 ]-MEN 11270; similarly to [Ala10 ]-Icatibant, the affinity of [Glu10 ]-MEN 11270 at the WT receptor is lower than that of MEN 11270 for the possible loss of some favourable interaction, with the additional unfavourable presence of a negative charge. The affinity towards Ser111 Lys was restored to the value of MEN
11270 at the WT receptor, suggesting an equivalent point charge interaction. It is worth noting that none of the modifications of the C-terminal amino acid induced an impairment of ligands affinity to the Ser111 Ala mutant receptor, thus ruling out the possibility that the side chain of Ser111 might participate in a direct interaction with the investigated peptide structures, and confirming the proximity of the C-terminal part of both Icatibant and MEN 11270 to the Ser111 . The comparison of Icatibant and its derivatives with MEN 11270 and its derivatives suggests that the C-terminal -turn structural feature is needed to achieve the active conformation, but is not sufficient for high affinity of the ligand towards the kinin B2 receptor. All these data demonstrated that also the charge compensation at the C-terminal portion of these peptides and the resulting charge–charge interactions with the receptor could affect the affinity. In particular, our results demonstrate that the high affinity of Icatibant is maintained only when the positive charge of the Arg10 side chain could be compensated by the negatively charged carboxylic group. Moreover, although the alternative hypothesis of a ligand conformational change due to sequence modifications cannot be excluded, the data obtained at the Ser111 Lys mutant suggest that most of the changes in affinity could be explained mainly by the presence of a charge–charge interaction.
References [1] AbdAlla S, Godovac-Zimmermann J, Braun A, Roscher AA, Müller-Esterl W, Quitterer U. Structure of bradykinin B2 receptor’s amino terminus. Biochemistry 1996a;35:7514–9. [2] AbdAlla S, Quitterer U, Grigoriev S, Maidhof A, Haasemann M, Jarnagin K, et al. Extracellular domains of the bradykinin B2 receptor involved in ligand binding and agonist sensing defined by anti-peptide antibodies. J Biol Chem 1996b;271:1725–48. [3] Bhoola KD, Figueroa CD, Worhty K. Bioregulation of kinins: kallicreins, kininogens and kininases. Pharmacol Rev 1992;44:1–80. [4] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 1976;75:248–54. [5] Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1 ) and the concentration of inhibitor which causes 50 per cent inhibition (I50 ) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099–108. [6] Fathy DB, Mathis A, Leeb T, Leeb-Lundberg LMF. A single position in the third transmembrane domains of the human B1 and B2 bradykinin receptors is adjacent to and discriminates between the C-terminal residues of subtype-selective ligands. J Biol Chem 1998;273:12210–8. [7] Jarnagin K, Bhakta S, Zuppan P, Yee C, Ho T, Phan T, et al. Mutations in the B2 bradykinin receptor reveal a different pattern of contacts for peptidic agonists and peptidic antagonists. J Biol Chem 1996;271:28277–86. [8] Guba W, Haessner R, Breiphol G, Henke S, Knolle J, Santagada V, et al. Combined approach of NMR and molecular dynamics within a biphasic membrane mimetic: conformation and orientation of the bradykinin antagonist Hoe 140. J Am Chem Soc 1994;116:7532–40. [9] Herzig MCS, Nash NR, Connolly M, Kyle DJ, Leeb-Lundberg LMF. The N terminus of bradykinin when bound to the human bradykinin
P. Cucchi et al. / Peptides 23 (2002) 1457–1463
[10]
[11]
[12]
[13]
[14] [15]
[16]
B2 receptor is adjacent to extracellular Cys20 and Cys277 in the receptor. J Biol Chem 1996;271:29746–51. Hess JF, Borkowski JA, Young GS, Strader CD, Ransom RW. Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor. Biochem Biophys Res Commun 1992;184:260–8. Hock FJ, Wirth K, Albus U, Linz W, Gerhards HJ, Wiemer G, et al. Hoe 140 a new potent and long acting brakykinin antagonist: in vitro studies. Br J Pharmacol 1991;102:769–73. Jarnagin K, Bhakta S, Zuppan P, Yee C, Ho T, Phan T, et al. Mutations in the B2 bradykinin receptor reveal a different pattern of contacts for peptidic agonist and peptidic antagonists. J Biol Chem 1996;271:28277–86. Kotovych G, Cann JR, Stewart JM, Yamamoto H. NMR an CD conformational studies of bradykinin and its agonists and antagonists: application to receptor binding. Biochem Cell Biol 1998;76:257–66. Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 1985;82:488–92. Kyle DJ, Chakravarty S, Sinsko JA, Stormann TM. A proposed model of bradykinin bound to the rat B2 receptor and its utility for drug design. J Med Chem 1994;37:1347–54. Meini S, Patacchini R, Giuliani S, Lazzeri M, Turini D, Maggi CA, et al. Characterization of bradykinin B2 receptor antagonists in human and rat urinary bladder. Eur J Pharmacol 2000a;388:177–82.
1463
[17] Meini S, Patacchini R, Lecci A, Quartara L, Maggi CA. Peptide and non-peptide bradykinin B2 receptor agonists and antagonists: a reappraisal of their pharmacology in guinea-pig ileum. Eur J Pharmacol 2000b;409:185–94. [18] Meini S, Quartara L, Rizzi A, Patacchini R, Cucchi P, Giolitti A, et al. MEN 11270 a novel selective constrained peptide antagonist with high affinity at the human B2 kinin receptor. J Pharmacol Exp Ther 1999;289:1250–6. [19] Menke JG, Borkowski JA, Bierilo KK, MacNeil T, Derrick AW, Schneck KA, et al. Expression cloning of a human B1 bradykinin receptors. J Biochem Chem 1994;269:21583–6. [20] Regoli D, Barabé J. Pharmacology of bradykinin and related kinins. Pharmacol Rev 1980;32:1–46. [21] Renzetti AR, Catalioto RM, Carloni C, Criscuoli M, Cucchi P, Giolitti A, et al. Effects of Tyrosyne289Phenylalanine mutation on binding and functional properties of human tachykinin NK2 receptor stably expressed in Chinese Hamster Ovary Cells. Biochem Pharmacol 1999;57:899–906. [22] Rotondaro L, Mazzanti L, Mele A, Rovera G. High-level expression of cDNA for granulocyte colony-stimulating factor in Chinese hamster ovary cells. Effects of 3 -non-coding sequences. Mol Biotechnol 1997;7:231–40.