Conformational properties of a cyclic peptide bradykinin B2 receptor antagonist using experimental and theoretical methods (original) (raw)
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Control of Conformational Equilibria in the Human B2 Bradykinin Receptor
Journal of Biological Chemistry, 2001
A prototypic study of the molecular mechanisms of activation or inactivation of peptide hormone G proteincoupled receptors was carried out on the human B 2 bradykinin receptor. A detailed pharmacological analysis of receptor mutants possessing either increased constitutive activity or impaired activation or ligand recognition allowed us to propose key residues participating in intramolecular interaction networks stabilizing receptor inactive or active conformations: Asn 113 and Tyr 115 (TM III), Trp 256 and Phe 259 (TM VI), Tyr 295 (TM VII) which are homologous of the rhodopsin residues Gly 120 , Glu 122 , Trp 265 , Tyr 268 , and Lys 296 , respectively. An essential experimental finding was the spatial proximity between Asn 113 , which is the cornerstone of inactive conformations, and Trp 256 which plays a subtle role in controlling the balance between active and inactive conformations. Molecular modeling and mutagenesis data showed that Trp 256 and Tyr 295 constitute, together with Gln 288 , receptor contact points with original nonpeptidic ligands. It provided an explanation for the ligand inverse agonist behavior on the WT receptor, with underlying restricted motions of TMs III, VI, and VII, and its agonist behavior on the Ala 113 and Phe 256 constitutively activated mutants. These data on the B 2 receptor emphasize that conformational equilibria are controlled in a coordinated fashion by key residues which are located at strategic positions for several G protein-coupled receptors. They are discussed in comparison with the recently determined rhodopsin crystallographic structure.
Journal of Medicinal Chemistry, 1999
The conformational features of H-Lys-Arg-Ado-Ser-Pro-Phe-OH (Ado) 12-aminododecanoic acid), a des-Arg 9 analogue of Lys-bradykinin, have been determined by high-resolution NMR in the presence of a zwitterionic lipid environment. The analogue is the most active member of a series of analogues designed to probe the topological arrangement of the N-and C-termini required for agonistic activity at the B1 kinin receptor. A novel computational procedure for the utilization of NOE constraints from cis and trans configurational isomers is illustrated. Only with this computational methodology could the structural features of the N-terminus of the peptide be determined. Using radical-induced relaxation of the 1 H NMR signals, we measured the topological orientation of the peptide with respect to the zwitterionic lipid interface. The results indicate that the long, alkyl chain of the Ado amino acid imbeds into the lipid surface. The structural features of the C-terminus of the B1-selective analogue consist of a well-defined turn. Although removed from a standard-turn, required for activity at the B2 kinin receptor, the topological orientation of the side chains of the des-Arg 9 compound are surprisingly similar to those previously observed for-turn-containing bradykinin analogues. Therefore, we attribute the high B1 receptor selectivity, observed upon removal of Arg 9 from bradykinin, solely to the loss of a charged amino acid and not to altered structural features.
Structure-based design of six novel classes of nonpeptide antagonists of the bradykinin B2 receptor
Bioorganic & Medicinal Chemistry Letters, 2000
ÐSix classes of nonpeptide bradykinin antagonists were designed using a template derived from structural studies of peptide antagonists. Several compounds from each class were synthesized and assayed for binding to the human bradykinin B 2 receptor. Each family showed compounds active at the level of the smallest template peptide; three classes contained compounds with K d <8 mM. These results provide diverse leads for a medicinal chemistry-based optimization program.
A Proposed Model of Bradykinin Bound to the Rat B2 Receptor and Its Utility for Drug Design
Journal of Medicinal Chemistry, 1994
A putative model of bradykinin bound to the rat B2 receptor was generated using a combination of homology modeling (from the known transmembrane structure of bacteriorhodopsin), energy minimization, molecular dynamics, and a two-stage conformational search as a docking simulation. Overall, the proposed bound ligand adopts a twisted "S" shape, wherein a C-terminal @turn is buried in the receptor just below the extracellular boundary of the cell membrane and the N-terminus is interacting with negatively charged residues in extracellular loop 3 of the receptor (most notably Asp268 and Asp2"). Mutagenesis experiments describing mutations which result in both a loss of bradykinin affinity as well as those which have no effect on bradykinin affinity are in good agreement with the proposed structure. In short, the mutagenesis results and the computational simulations each point to the same region of the receptor as likely to bind bradykinin. A double mutation, predicted as being likely to have a dramatic effect on bradykinin binding affinity, was confirmed experimentally, adding some validation to the proposed complex. Moreover, a new pseudopeptide bradykinin receptor antagonist (D-Argo-Argl-[ 12-aminododecanoyll 2-Ser3-~-Ti~4-0i~5-Arp6) was designed on the basis of the model, and found to have good receptor affinity. Speculation regarding other possible sites for mutagenesis are also described.
Design and Synthesis of New Linear and Cyclic Bradykinin Antagonists
Journal of Medicinal Chemistry, 1996
We report here on the synthesis and pharmacological properties of a new series of small linear and cyclic peptides derived from the five C-terminal amino acid residues of second-generation bradykinin receptor antagonists. Variations of the two first residues of the pentapeptide (Thi-Ser-D-Tic-Oic-Arg) were shown to modulate the biological activities of the analogs on bradykinininduced smooth muscle contractions in rabbit jugular vein (RJV), a tissue preparation specific of the B2 bradykinin receptor. Several analogs showed pA 2 values around 7 on this tissue preparation, and one cyclic compound, c[-Gly-Thi-D-Tic-Oic-Arg-], 24, in which Thi-Ser was replaced by Gly-Thi, displayed a pA 2 of 7.4 on RJV. On the basis of these results, three cyclic molecules and their linear counterparts (compounds 22-24 and 4-6, respectively) were tested on human umbilical vein, a tissue specific of the human B2 receptor. The pK B values obtained for these compounds on these tissue preparations were equivalent to those obtained for the decapeptide NPC 567 (4.8 < pA 2 < 5.1). NMR and molecular modeling studies performed on compound 24 clearly demonstrated a type II′-turn structure. This analog may serve as a new lead for the design of nonpeptide ligands of the bradykinin B2 receptor subtype.
New conformationally homogeneous ?-turn antagonists of the human B2 kinin receptor
Journal of Peptide Science, 2001
We have designed and synthesized a conformationally homogeneous series of cyclic pentapeptides of the general structure c[Pro-aa i -D-Tic-Oic-aa i + 3 ] which adopt a type-II% i-turn conformation believed important for high affinity antagonism of the bradykinin (BK) B 2 receptor. We incorporated D-Tic and octahydroindole-2-carboxylic acid (Oic) residues (present in known active antagonists) in a cyclic pentapeptide that would place the D-aa in the i +l position of the i-turn and a proline as a bridge between the C-and N-termini sides of the turn. In positions i and i +3 alkyl, aromatic, polar or charged amino acids could be introduced without dramatically changing the overall structure. Ten analogues were studied using 1 H nuclear magnetic resonance (NMR) and evaluated for their binding affinity for the human B 2 receptor. The NMR data in dimethylsulfoxide (DMSO) confirmed the structural homogeneity within the class and, on the basis of this, one representative member of the series was chosen for a detailed structure determination using NMR data in sodium dodecylsulphate (SDS) micelles and molecular dynamics calculations. Despite the structural similarity, the binding affinity of the ten analogues was strongly influenced by the nature of the side-chains in positions i and i+ 3, with the doubly charged analogue 49 (pK i =6.2) proving best. This compound may serve as the starting point for the discovery of new non-peptide bradykinin B 2 receptor antagonists.
Characterization of the conformational domains of bradykinin by computational methods
Journal of Peptide Science, 1995
The AMBER 4.0 force field was used to perform a characterization of the conformational profile of the nonapeptide bradykinin. A thorough conformational search was carried out using molecular dynamics as sampling technique, by computing cycles of high (900 K) and low (300 a temperature trajectories. A total of 2400 mintma were generated and subsequently clustered using the root-mean-square of the backbone dihedral angles as criterium. After the use of a tolerance value of 20", the conformations were clustered in 233 unique conformations with energies up to 40 kcal/mol above the lowest minimum. The analysis of the lowenergy conformations indicate that the peptide exhibits a high tendency to adopt a p-turn at the C-terminus and a propensity to adopt a bent structure at the N-terminus. These results are in agreement with the experimental evidence reported in the literature and provide detailed information necessary to understand the conformational preferences of the peptide.