A CD and an nmr study of multiple bradykinin conformations in aqueous trifluoroethanol solutions

CD and nmr studies have been carried out on aqueous trifluoroethanol (TFE) solutions of bradykinin (BK) and a bradykinin antagonist. The CD results exhibit a striking effect of TFE on the spectra of BK, with sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, with sequence D -Arg-Arg-Pro-Hyp-Gly-Thi-D -Ser-D -Cpg-Cpg-Arg [where Hyp is 4-hydroxy-L -proline; Thi refers to β-(2-thienyl)-L -alanine and Cpg refers to α-cyclopentylglycine]. The effect of increasing concentration of TFE in water on the difference ellipticity at 222 nm was examined and showed that BK may be a mixture of at least two different conformers, one of which largely forms when the TFE concentration is increased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreement with that shown by the BK antagonist, indicating that the conformation of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both one-dimensional and two-dimensional ¹H-nmr techniques. The total correlation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H₂O solution at 10°C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H_α(i) – NH(i + 1) or the H_α(i) – H_δδ′(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY for BK in the 95/5% (v/v) TFE/H₂O solution shows that there are two major conformations in the solution with about equal population. The NOESY experiment shows two new important cross peaks for one conformation, namely Pro²(α)-Pro³ (α) and the Pro²(α)-Gly⁴(NH), indicating a cis Pro²-Pro³ bond and a type VI β-turn between residues Arg¹ and Gly⁴ involving cis proline at position 3, respectively. The low temperature coefficient of Gly⁴ for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa β-turn is present. The other conformation is all trans and extended. The BK antafonist shows difference CD spectra in TFE solutions referred to H₂O that are superficially indicative of a β-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-trans extended confirmation over the range of TFE concentrations. The BK-antagonist CD data suggest that solvent perturbation of the CD of an extended confirmation perturbation of the optical activity of the thienyl moiety of the peptide since the CD spectrum of N-acetyl-β-thienyl-L -alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relationship between CD and nmr. © 1994 John Wiley & Sons, Inc.     

Conformational studies of TOAC-labeled bradykinin analogues in model membranes

Summary Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg¹ (TOAC⁰-BK) or replacing Pro³ (TOAC³-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism (CD) approach was used to examine the conformational properties of the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC³-BK's EPR spectral lineshapes are broader than those of TOAC⁰-BK, indicating a more restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC⁰-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidly interconverting forms), while TOAC³-BK's spectra correspond to a more ordered structure. SDS binding induces drastic changes in BK and TOAC⁰-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. In contrast. TOAC³-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC³-BK to acquire the correct receptor-bound conformation leading to loss of biological activity, On the other hand, the greater flexibility of TOAC⁰-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.     

Conformational studies of TOAC-labeled bradykinin analogues in model membranes

Spin-labeled analogues of bradykinin (BK) were synthesized containing the amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) either before Arg¹ (TOAC⁰-BK) or replacing Pro³ (TOAC³-BK). Whereas the latter is inactive, the former retains about 70% of BK's activity in isolated rat uterus. A combined electron paramagnetic resonance (EPR)-circular dichroism(CD) approach was used to examine the conformational propertiesof the peptides in the presence of membrane-mimetic systems (negatively charged sodium dodecyl sulfate, SDS, and zwitterionicN-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, HPS). While the peptides bind to both monomeric and micellar SDS, no interaction occurs with HPS, evincing the contribution of electrostatic interactions. TOAC³-BK's EPR spectral lineshapes are broader than those of TOAC⁰-BK, indicating amore restricted degree of motion at position 3. Moreover, the motional freedom of both peptides decreased upon binding to SDS. BK and TOAC⁰-BK solution CD spectra indicate highly flexible conformations (possibly an equilibrium between rapidlyinterconverting forms), while TOAC³-BK's spectra correspondto a more ordered structure. SDS binding induces drastic changesin BK and TOAC⁰-BK spectra, indicating stabilization of similar folds. The micelle interface promotes a higher degree of secondary structure by favoring intramolecular hydrogen bonds. Incontrast, TOAC³-BK spectra remain essentially unchanged. These results are interpreted as due to TOAC's ring imposing a more constrained conformation. This rigidity is very likely responsible for the inability of TOAC³-BK to acquire the correct receptor-bound conformation, leading to loss of biological activity. On the other hand, the greater flexibility of TOAC⁰-BK and the similarity between its conformational behavior and that of the native hormone are probably related to their similar biological activity.     

Bradykinin analogs containing α-aminoisobutyric acid (Aib)

All seven possible bradykinin (BK) analogs containing Aib in place of proline have been synthesized by the solid phase method and assayed for in vitro myotropic activity on the guinea pig ileum and rat uterus, and in vivo on the rat blood pressure, both by intravenous and intra-aortic administration. [Aib^2,3]-BK, [Aib^2,7]-BK, and [Aib^2,3,7]-BK had no in vivo or in vitro activities; [Aib²]-BK, [Aib³]-BK and [Aib^3,7]-BK had moderate BK-like activities and a significantly increased resistance to pulmonary inactivation in the rat ([Aib^3,7]-BK was totally resistant). [Aib⁷]-BK was found to be the most active position seven BK analog yet assayed on the rat blood pressure, and shows remarkably high ileum (4 times BK) and intravenous rat blood pressure (6 times BK) activity.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing alpha-aminoisobutyric acid

The conformation in aqueous solution of several alpha-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and 1H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro2 and/or Pro3 in BK stabilizes a degree of beta-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB7]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a beta-turn involving their common Arg1-Pro2-Pro3-Gly4 moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis' Pro and the propensity for a beta-turn at the N-terminus of the peptide.     

Bradykinin-induced biphasic response in the rat isolated stomach fundus: Functional evidence for a novel bradykinin receptor

The responses of the rat isolated stomach fundus to bradykinin (BK) and des-Arg⁹-BK (DA-BK) have been examined. In rat isolated stomach fundus pre-contracted with BaCl₂ (0.5-1 mM), BK caused concentration-dependent biphasic responses characterized by relaxation followed by contraction. DA-BK also caused marked relaxations, but, unlike BK, induced only small contractions. Removal of the mucosal layer initially abolished the relaxant responses to BK and both responses to DA-BK without affecting BK-induced contractions, but repeated challenges with BK or DA-BK revealed a time-depeendent reappearance of the relaxant responses, suggesting “de novo” synthesis of BK receptors. Pretreatment of rat stomach fundus with tetrodotoxin (1 μM), atropine (1 μM), captopril (3 μM), prazosin (1 μM) or glibenclamide (1 μM) did not significantly modify the biphasic responses to BK (300 nM). The biphasic responses to DA-BK were antagonized selectivley by the B₁ receptor antagonist des-Arg⁹-[Leu ⁸]-BK (DAL- BK) (1 μM). In contrast, the biphasic responses to BK were unaffected by DAL-BK or by several selective peptide antagonists of B₂ receptors including NPC 431 (Thi^5,8, D-Phe⁷)-BK, NPC 349 (D-Arg Hyp³, Thi^5,8, D-Phe⁷)-BK, NPC 567 (D-Arg -Hyp³, D-Phe⁷)-BK and NPC 361 (D-Phe⁷)-BK (3 to 10 μM). These results are consistent with the view that the biphasic responses of the rat isolated stomach fundus to BK appear to be mediated by a novel BK receptor which is insensitive to blockade by B₁ and B₂ selective BK receptor antagonists.     

Circular dichroism spectra of bradykinin analogs containing β-homoamino acids

The CD spectra of β-homoprolyl⁷-bradykinin (βHProB) and β-homophenylalanyl⁸-bradykinin (βHPheB) were compared to those of bradykinin. The spectra were analyzed in terms of models that have been proposed for the solution conformation of bradykinin. Cann $\text{et al}$. (1) proposed 3 → 1 hydrogen-bonding across Pro³, Pro⁷ and Phe⁸ in bradykinin, as shown by a 234 nm trough in the CD. The extra CH₂ groups in the chains of the two bradykinin analogs would be expected to facilitate the proposed hydrogen-bonding, but in the case of βHProB the 234 nm trough is eliminated, and is reduced in magnitude for βHPheB. Ivanov $\text{et al}$. (2) proposed a cyclic conformation for bradykinin, stabilized by ionic attraction between the side-chain of Arg¹ and the carboxylate terminal. The extra CH₂ groups of these two analogs would be expected to increase the stability of such a conformation, and there was some evidence that the ionic effects on the CD spectra of the two analogs were different from those on the bradykinin spectra. Alternatively, the effects could be attributed to cis-trans isomerizations around the prolyl peptide bonds.     

The neuropeptide bradykinin stimulates phosphoinositide turnover in HSDM1C1 cells: B2-antagonist-sensitive responses and receptor binding studies

Bradykinin (BK) and its analogs (1 nM-100 M) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC₅₀) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg⁹-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 M) of the peptides. In contrast, the analogs [D-Arg⁰-HYP³-Thienyl^5,8-D-Phe⁷]-BK (HYP³-antagonist), [D-Arg⁰-HYP³-Thienyl,^5,8-D-Phe⁷]-BK (Thienyl antagonist) and Des-Arg⁹-Leu⁸-BK were inactive, as agonists, at 0.1 nM-1 M in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B₂-type of BK receptors. This was confirmed further by the fact that both the B₂-selective Hyp³- and Thienyl-antagonists inhibited BK-induced PI turnover with K_BS of 369±51 nM and 368±118 nM respectively while the B₁-selective antagonist, Des-Arg⁹-Leu⁸-BK, was inactive at 1 M. [³H]BK receptor binding studies revealed two binding sites, one with high affinity (K_d=0.24±0.06 nM; B_max=1.4±0.4 pmol/g tissue) and the other with low affinity (K_d=18.5±0.95 nM; B_max=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [³H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B₂-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [³H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A₂.     

Dehydrophenylalanyl analogs of bradykinin: Synthesis and biological activities

We have synthesized three analogs of the potent vasodilator peptide bradykinin, ArgProProGlyPhe SerProPheArg (BK), containing dehydrophenylalanine (Δ^zPhe) in place of the phenylalanyl residues at positions 5 and/or 8. The analogs, [Δ^zPhe⁵]BK, [Δ^zPhe⁸]BK, and [Δ^zPhe^5,8]BK, were assayed for their effects on isolated smooth muscle tissues and on the systemic arterial blood pressure of rats. In these assays [Δ^zPhe⁵]BK showed considerably high biological activities, particularly in terms of its blood pressure-lowering effects, being over 23 times more potent than BK when given intravenously. [Δ^zPhe⁸]BK was less potent than BK and [Δ^zPhe^5,8]BK had effects comparable to those of BK. All three synthetic analogs appear to be more resistant than BK to enzymic degradation during passage through the pulmonary vascular bed.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

Paramagnetic bradykinin analogues as substrates for angiotensin I-converting enzyme: Pharmacological and conformation studies

This study uses EPR, CD, and fluorescence spectroscopy to examine the structure of bradykinin (BK) analogues attaching the paramagnetic amino acid-type Toac (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 3, 7, and 9. The data were correlated with the potencies in muscle contractile experiments and the substrate properties towards the angiotensin I-converting enzyme (ACE). A study of the biological activities in guinea pig ileum and rat uterus indicated that only Toac⁰-BK partially maintained its native biological potency among the tested peptides. This and its counterpart, Toac³-BK, maintained the ability to act as ACE substrates. These results indicate that peptides bearing Toac probe far from the ACE cleavage sites were more susceptible to hydrolysis by ACE. The results also emphasize the existence of a finer control for BK-receptor interaction than for BK binding at the catalytic site of this metallodipetidase. The kinetic kcat/Km values decreased from 202.7 to 38.9 μM⁻¹ min⁻¹ for BK and Toac³-BK, respectively. EPR, CD, and fluorescence experiments reveal a direct relationship between the structure and activity of these paramagnetic peptides. In contrast to the turn-folded structures of the Toac-internally labeled peptides, more extended conformations were displayed by N- or C-terminally Toac-labeled analogues. Lastly, this work supports the feasibility of monitoring the progress of the ACE-hydrolytic process of Toac-attached peptides by examining time-dependent EPR spectral variations.     

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.     

Gamma Ray Irradiation of the Vasoactive Peptide Bradykinin Reveals a Residue- and Position-Dependent Structural Modification

In this work the effect of radical species generated by gamma ray irradiation of aqueous solution upon structure of vasoactive peptide bradykinin (BK, RPPGFSPFR) was investigated. Increasing doses of 1–15 kGy Co⁶⁰ gamma radiation were applied to BK solutions and a progressive degradation of its structure in a non-linear mode was observed. Two main peptide derivatives generated by these treatments were isolated and characterized through a combined amino acid analysis and daughter ion scanning mass spectrometry approach. Notably, it was observed that only the Phe residue located at position 8 and not 5 of BK was oxidized by reactive hydroxyl radical species given rise to Tyr⁸-BK and m-Tyr⁸-BK analogues. Comparative circular dichroism (CD) experiments of these peptides revealed that BK presents greater conformational similarity to Tyr⁸-BK than to m-Tyr⁸-BK. These results are in agreement with the biological potencies of these compounds measured in rat uterus and guinea pig ileum muscle contractile experiments. In summary, gamma irradiation of BK solutions revealed a residue- and surprisingly, position-structural modification effect of reactive radicals even in small peptides. Also of value for peptide chemistry field, the approach of applying controlled strong electromagnetic radiation in solution seems to be an alternative and unique strategy for generating, in some cases, peptides derivatives with uncommon structures and valuable for their further therapeutic potential evaluations.     

Short peptide constructs mimic agonist sites of AT1R and BK receptors

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT₁R and BKRB₁ or BKRB₂ G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S–S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac¹-AngII and Toac⁰-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac³-AngII and Toac³-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT₁R and BKRB cyclic constructs based on the structure of the CXCR₄, an α-chemokine GPCR-type receptor.     

Type 2 Diabetes Elicits Lower Nitric Oxide,Bradykinin Concentration and Kallikrein Activity Together with Higher DesArg9-BK and Reduced Post-Exercise Hypotension Compared to Non-Diabetic Condition

This study compared the plasma kallikrein activity (PKA), bradykinin concentration (BK), DesArg⁹-BK production, nitric oxide release (NO) and blood pressure (BP) response after moderate-intensity aerobic exercise performed by individuals with and without type 2 diabetes. Ten subjects with type 2 diabetes (T2D) and 10 without type 2 diabetes (ND) underwent three sessions: 1) maximal incremental test on cycle ergometer to determine lactate threshold (LT); 2) 20-min of constant-load exercise on cycle ergometer, at 90% LT and; 3) control session. BP and oxygen uptake were measured at rest and at 15, 30 and 45 min post-exercise. Venous blood samples were collected at 15 and 45 minutes of the recovery period for further analysis of PKA, BK and DesArg⁹-BK. Nitrite plus nitrate (NOx) was analyzed at 15 minutes post exercise. The ND group presented post-exercise hypotension (PEH) of systolic blood pressure and mean arterial pressure on the 90% LT session but T2D group did not. Plasma NOx increased ~24.4% for ND and ~13.8% for T2D group 15min after the exercise session. Additionally, only ND individuals showed increases in PKA and BK in response to exercise and only T2D group showed increased DesArg⁹-BK production. It was concluded that T2D individuals presented lower PKA, BK and NOx release as well as higher DesArg⁹-BK production and reduced PEH in relation to ND participants after a single exercise session.     

Total synthesis,structural, and biological evaluation of stylissatin A and related analogs

The natural product cyclic peptide stylissatin A ( 1a ) was reported to inhibit nitric oxide production in LPS‐stimulated murine macrophage RAW 264.7 cells. In the current study, solid‐phase total synthesis of stylissatin A was performed by using a safety‐catch linker and yielded the peptide with a trans‐Phe⁷‐Pro⁶ linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d ‐allo‐Ile in place of l ‐Ile and a different site for macrocyclization. The resulting product (d ‐allo‐Ile²)‐stylissatin A ( 1c ) was also found to have the trans‐Phe⁷‐Pro⁶ peptide conformations like rotamer 1b . Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a , 1b , and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2–7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product ( 1a ), the rotamer ( 1b ), and (d ‐allo‐Ile²)‐stylissatin A ( 1c ) exhibited significant inhibition of NO^.. Further investigations were focused on 1b , which also inhibited proliferation of T‐cells and inflammatory cytokine IL‐2 production. The analogs 2–7 weakly inhibited NO^. production, but strongly inhibited IL‐2 cytokine production compared with synthetic peptide 1b . All analogs inhibited the proliferation of T‐cells, with analog 7 having the strongest effect. In the analogs, the Pro⁶ residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor

The bradykinin (BK) B₁ receptor (B₁R) is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B₁R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP) (enhanced green FP [EGFP] or mCherry) prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg⁹-BK, [Leu⁸]des-Arg⁹-BK, respectively). The design of the spacer-ligand joint peptide was validated by a competition assay for [³H]Lys-des-Arg⁹-BK binding to the human B₁R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B₁R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy). Both assays indicated that the best design was FP-(Asn-Gly)_n-Lys-des-Arg⁹-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B₁R antagonist. EGFP-(Asn-Gly)₁₅-Lys-des-Arg⁹-BK competed for the binding of [³H]Lys-des-Arg⁹-BK to human recombinant B₁R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg⁹-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B₂ receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B₁R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology).     

Structural and positional studies of the antimicrobial peptide brevinin‐1BYa in membrane‐mimetic environments

Brevinin‐1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow‐legged frog Rana boylii, shows broad‐spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin‐1BYa was investigated in various solution and membrane‐mimicking environments by proton nuclear magnetic resonance (¹H‐NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2‐trifluoroethanol (TFE‐d₃)‐H₂O solvent mixture, as well as in membrane‐mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix‐hinge‐helix motif, with the hinge located at the Gly¹³/Pro¹⁴ residues, and the two α‐helices extending from Pro³ to Phe¹² and from Pro¹⁴ to Thr²¹. Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5‐doxyl‐labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non–pore‐forming mechanism.     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹ and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β-branched D -aliphatic residues at position 7 combined with bulky L -aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D -Arg⁰, Hyp³, Thi⁵, D -Cpg⁷, Cpg⁸]-BK [Cpg: α-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L -proline; Thi: β-(2-thienyl)-L -alanine] ( I ), [D -Arg⁰, Hyp³, D -Cpg⁷, Cpg⁸] -BK ( II ), as well as its variant with D -Cpg⁷ replaced by Cpg⁷, namely [D -Arg⁰, Hyp³, Cpg⁷, Cpg⁸]-BK ( III ). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II , in direct correlation with the peptide activities. No turn-like structure was found for residues 6–9. In peptide III , a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists ( I , II ) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D -Cpg⁷]-BK ( IV ) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.     

Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.