Influence of polyfluorination of the phenylalanine ring of angiotensin II on conformation and biological activity

[Phe(F5)8]angiotensin II was synthesized by the solid phase method and purified by reverse-phase HPLC. In rat uterus and rabbit aorta bioassays the analogue had 10 and 50%, respectively, of the contractile activity of angiotensin II and demonstrated antagonist properties. These findings illustrate that inversion of the Phe8 ring quadrupole moment in angiotensin II decreases agonist activity and invokes antagonist properties. 1H-NMR studies at 400 MHz in DMSO-d6 demonstrated the presence of cis and trans isomers in the ratio 1:3 due to restricted rotation of the His-Pro bond. Downfield shifts of the His C2 and C4 protons in [Phe(F5)]ANG II compared to ANG II suggest that the Phe(F5) residue may be involved in a parallel-plate ring pairing interaction with the imidazole group. However heteronuclear NOE studies, carried out by measuring the proton difference spectrum before and after saturation of the fluorine resonances, showed the absence of any NOE enhancement illustrating that electrostatic influences of the Phe(F5) ring occur at relatively long range.     

Proton NMR studies of angiotensin II and its analogs in aqueous solution

The¹H nuclear magnetic resonance (NMR) spectra of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and five of its octapeptide analogs as well as angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) and angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) in aqueous solutions (90% H₂O/10% D₂O) were completely assigned by two-dimensional COSY and ROESY experiments. All of the peptides give rise to two distinct sets of signals. The minor set accounts for about 5% of the total population belowpH 5.5 and increases to 12–20% aroundpH 7.0. The two sets of signals result from acis-trans isomerization of the His-Pro peptide bond with the major resonances arising from thetrans isomer. One analog in which the Pro is replaced with a D-Pro displays a very different isomerization behavior. The measured coupling constants J_NH-CH, the temperature dependence of the amide proton shifts and the relative intensities of the intraresidue and sequential NH-CH ROEs, are all indicative of an extended backbone conformation for ANGII. However, some evidence for the existence of conformers with local structure involving preferred sidechain positions for the Tyr, His, Phe, and the carboxyl group of the Phe was found, particularly in the ROESY andpH-titration experiments. Moreover,pH effects and the unusual amide exchange behavior of the Arg NH suggests the presence of interactions between the Asp and Arg sidechains of ANGII. At low temperatures the Arg guanidinium NH₂ protons were detected as two broad peaks which are related by sizeable exchange peaks in ROESY experiments. This behavior could be useful as a general probe for the study of Arg sidechain mobility and accessibility in other peptides and proteinsPreliminary results of this work have been presented at the XIIth International Conference on Magnetic Resonance in Biological Systems in abstract form (1988).     

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.     

Synthesis and biological activities of novel nonpeptide angiotensin II receptor antagonists based on benzimidazole derivatives bearing a heterocyclic ring

A series of benzimidazole derivatives bearing a heterocyclic ring imidazole (1), 5-chloroimidazole (2), 1,2,4-triazol (3), and imidazoline (4) were synthesized and evaluated for angiotensin II antagonistic activities. The synthetic compounds 1-4 were biologically evaluated in vitro using an AT(1) receptor binding assay, where compounds 1 and 3 provided weak binding affinity, compound 2 showed moderate binding affinity, and compound 4 showed good binding affinity. Moreover, compound 4 was found to be almost equipotent with telmisartan in vivo biological evaluation study.     

Binding of a spin-labeled phenylalanine analog to sickle hemoglobin: EPR and NMR studies

We have synthesized a spin-label analog of phenylalanine as a competitive inhibitor probe of the sickle hemoglobin aggregation process. Sickle hemoglobin gelation measurements indicate that the spin-label phenylalanine analog is a potent inhibitor of deoxy sickle hemoglobin aggregation. We have also used spin label EPR and high-resolution proton NMR to study the interaction of the phenylalanine analog with hemoglobin, and find that the kinetic off-rate is comparable to, or slower than the hemoglobin rotational rate (i.e., greater than or equal to 10(8) s-1), and that at least one, and perhaps two significant localized interaction region(s) exist within a few angstroms of the beta chain N- and C-termini. Correlation with other known structural information suggests that the observed interaction sites may be relevant to the mechanism for inhibition of sickle hemoglobin aggregation.     

Oxytocin and its analogs, methyl-substituted in ortho-, meta- or para-position of aromatic ring of phenylalanine in position 2: NMR study and biological activities

Complete analyses of NMR data of oxytocin (OT) and 4 analogues, ([o-MePhe(2)]OT, [mMe-Phe(2)]OT, [m-OMePhe(2)]OT and [p-MePhe(2)]OT), are given. The same conformational behavior in solution on one hand and large differences in biological activities on the other hand indicate that the compounds adopt a "biologically active conformation" at the stage of interaction with the receptor when the character of the substituent and its position on the aromatic ring may play a role in hindering attaining the ideal complementarity of both interacting components.     

Conformation and activity in angiotensin II peptides

Angiotensin II and its competitive inhibitor [Sar¹, Ile⁸]-angiotensin II, as well as several analogs of these two compounds specifically chosen for their well-defined pharmacological properties, were studied by circular dichroism and nuclear magnetic resonance methods at various pH values in aqueous solution and in d₆-dimethylsulfoxide. The results were compared with their biological activities. This allowed us to establish relationships between conformation and pressor activity, explaining most of the properties of angiotensin II, its inhibitor, and the analogs successively substituted in positions 3 and 5.     

Synthesis and biological activity of cyclic analogs of angiotensin

Biological properties of five novel angiotensin analogues synthesized, using the conventional methods of peptide chemistry, have been studied. Cyclization was attained by means of amide linkage with the aid of diphenylphosphorylazide or pentafluorophenyl esters. Unlike the natural hormone, the cyclic analogues of angiotensin show no pressor activity, but elicit a depressor effect untypical of angiotensin. A slight pressor activity was exhibited by the compound containing aspartic acid in position 1. The cyclic analogues in question release histamine from peritoneal mast cells in rats.     

Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.     

Calcitroic acid: biological activity and tissue distribution studies

Calcitroic acid was recently identified as a major metabolite of 1,25-dihydroxyvitamin D₃ (Esvelt, Schnoes, and DeLuca, Biochemistry 18, 3977, 1979). The metabolite was found to have little, although significant, activity in healing rickets, and causing bone mineral mobilization but elicited no significant elevation in intestinal calcium transport. The compound showed little affinity for either the serum 25-hydroxyvitamin D binding protein or the intestinal cytosol receptor for 1,25-dihydroxyvitamin D₃. Various tissues of the rat were examined for the presence of calcitroic acid following a 120-ng dose of 1,25-dihydroxy-[3α-³H]vitamin D₃. The metabolite was detected in liver, intestinal mucosa, kidneys, and blood with livers and mucosa containing the highest concentrations. In each of these tissues the calcitroic acid content increased during the period between 4 and 12 h after the dose. The presence of calcitroic acid in femurs was indicated but could not be confirmed. Bile duct cannulation reduced but did not abolish the intestinal calcitroic acid content. In addition to calcitroic acid, other polar metabolites of 1,25-dihydroxyvitamin D₃ were detected in these experiments.     

Cyclic melanotropins. Part VII: Modified ring structures--synthesis and biological activity

The highly potent cyclic analogue of alpha-MSH, Ac-[Cys4,Cys10]-alpha-MSH4-13-NH2, was structurally modified in position 4. Four analogues were prepared and their biological activities in the in vitro frog and lizard skin bioassays were determined. It was shown that removing the terminal acetylamino group to give [Mpa4,Cys10]-alpha-MSH4-13-NH2 resulted in little change in the biological activity, but a change in the stereochemistry of cysteine in position 4 to give Ac-[D-Cys4,Cys10[-alpha-MSH4-a3-NH2 led to a small decrease of activity in both bioassays. Decreasing the size of the intramolecular ring by removing one methylene group to give [Maa2,Cys10]-alpha-MSH4-13-NH2, resulted in an analogue with lower activities in both assays (about 3 times in the lizard and 500 times in the frog), and increasing the size of the righ by methylene group to give Ac-[Hcy4,Cys10]-alpha-MSH4-13-NH2 led to much lower activities in the lizard system and similar effects were seen upon decreasing the ring size in the frog skin assay.     

β-endorphin release by angiotensin II: studies on the mechanism of action

Blood-borne angiotensin II induces release of β-endorphin-like immunoreactivity (β-EI) from rat anterior pituitary gland. To study the mechanism of action we investigated in rats the effect of transection of subfornical organ efferent projections on angiotensin-induced β-EI release in vivo and also the direct action of angiotensin II on β-EI release from isolated adenohypophyses in vitro. (i) No effect of transection of subfornical organ efferents on the increase in plasma β-EI following intravenous infusions of angiotensin II was found. (ii) When anterior pituitary quarters were continuously superfused in vitro, angiotensin II (1 – 10 nM) caused release of β-EI into the superfusion medium in a dose-dependent manner. The stimulatory effect of angiotensin II (3 nM) was blocked by the receptor antagonist saralasin (300 nM). We conclude that β-endorphin release by blood-borne angiotensin II, in contrast to other central effects of angiotensin, is not mediated by the subfornical organ; instead a direct action of angiotensin II on the adenohypophysis could be a mechanism of action responsible.     

Anti-plasmodium activity of angiotensin II and related synthetic peptides

Plasmodium species are the causative agents of malaria, the most devastating insect-borne parasite of human populations. Finding and developing new drugs for malaria treatment and prevention is the goal of much research. Angiotensins I and II (ang I and ang II) and six synthetic related peptides designated Vaniceres 1-6 (VC1-VC6) were assayed in vivo and in vitro for their effects on the development of the avian parasite, Plasmodium gallinaceum. Ang II and VC5 injected into the thoraces of the insects reduced mean intensities of infection in the mosquito salivary glands by 88% and 76%, respectively. Although the mechanism(s) of action is not completely understood, we have demonstrated that these peptides disrupt selectively the P.gallinaceum cell membrane. Additionally, incubation in vitro of sporozoites with VC5 reduced the infectivity of the parasites to their vertebrate host. VC5 has no observable agonist effects on vertebrates, and this makes it a promising drug for malaria prevention and chemotherapy.