De novo identification of lipid II binding lipopeptides with antibacterial activity against vancomycin-resistant bacteria.

Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor lipid II. In doing so we identified unique lipid II binding peptides that upon lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides.

Novel Bradykinin Analogues Modified in the N-Terminal Part of the Molecule with a Variety of Acyl Substituents.

In the current work we present some pharmacological characteristics of ten new analogues of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) modified in the N-terminal part of the molecule with a variety of acyl substituents. Of the many acylating agents used previously with B(2) receptor antagonists, the following residues were chosen: 1-adamantaneacetic acid (Aaa), 1-adamantanecarboxylic acid (Aca), 4-tert-butylbenzoic acid (t-Bba), 4-aminobenzoic acid (Aba), 12-aminododecanoic acid (Adc), succinic acid (Sua), 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 3-(4-hydroxyphenyl)propionic acid and 6-hydroxy-2-naphthoic acid. Biological activity of the compounds was assessed in the in vivo rat blood pressure test and the in vitro rat uterus test. Surprisingly, N-terminal substitution of the bradykinin peptide chain itself with aforementioned groups resulted in antagonists of bradykinin in the pressor test and suppressed agonistic potency in the uterotonic test. These interesting findings need further studies as they can be helpful for designing more potent B(2) receptor blockers.

Novel analogues of bradykinin conformationally restricted in the C-terminal part of the molecule.

In the present work, achiral non-coded amino acids, N-(Bzl)-Gly, X(1) or X(2) , were substituted at position 7 of the model B(2) receptor antagonist [D-Arg(0) , Hyp(3) , Thi(5, 8) , D-Phe(7) ]-BK. The N-terminal amino group of the analogues was either free or acylated with 1-Aca or Aaa. Biological activity of the compounds was assessed in the in vitro rat uterus test and the in vivo rat blood pressure test. The X(1) (7) substitution resulted in a decrease in antagonistic potency of the new peptide in both assays. The X(2) (7) and N-(Bzl)-Gly(7) substituted analogues showed weak agonistic properties in the rat uterus test. Interestingly, the latter compound exhibited dual activity in the pressor test, i.e. intrinsic vasodepressor action and at the same time a weak antagonistic effect. Acylation of the N-terminus enhanced antagonistic properties of the resulting peptides in the rat blood pressure test in the case of compounds containing X(1) or X(2) modification. Our studies provide new information about structure-activity relationship of the BK antagonists which may be helpful for designing more potent B(2) receptor blockers.

New bradykinin B(2) receptor antagonists - influence of C-terminal segment modifications on their pharmacological properties.

In the present study we describe the synthesis and some pharmacological properties of eight new analogues of bradykinin (BK). Two peptides were designed by substitution of position 7 or 8 of the known [D-Arg(0),Hyp(3),Thi(5,8),D-Phe(7)]BK antagonist (Stewart's antagonist) with L-pipecolic acid (L-Pip). The next two analogues were obtained by replacement of the d-Phe residue in position 7 of the Stewart's peptide with L-beta(2)-isoproline (L-beta(2)-iPro) or L-beta(3)-homoproline (L-beta(3)-hPro). The four analogues mentioned above were also prepared in N-acylated form with 1-adamantaneacetic acid (Aaa). Biological activity of the compounds was assessed by isolated rat uterus and rat blood pressure tests. Our results showed that L-Pip in position 7 slightly increased antagonistic potency in the blood pressure test, but it turned the analogue into an agonist in the rat uterus test. Replacement of Thi by L-Pip in position 8 also enhanced antagonism in the rat pressure test but preserved the antagonism in the rat uterus test. L-beta(2)-iPro or L-beta(3)-hPro in position 7 decreased the potencies in both tests. We also demonstrated that acylation of the N-terminus did not increase, as was claimed previously, the antagonistic potencies of the resulting peptides. The results thus support the hypothesis about the existence of different types of BK receptors in the rat uterus and blood vessels. Our studies provide new information about the structure-activity relationship of BK antagonists which may help in designing more potent BK receptor blockers.

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.

New bradykinin analogues acylated on the N-terminus: effect on rat uterus and blood pressure.

Our previous studies suggested that acylation of the N-terminus of several known B2 antagonists with various kinds of bulky acyl groups consistently improved their antagonistic potency in rat blood pressure assay. On the other hand, our earlier observations also seemed to suggest that the effects of acylation on the contractility of isolated rat uterus depended substantially on the chemical character of the acyl group, as we observed that this modification might either change the range of antagonism or even transform it into agonism. Bearing all this in mind, we decided to synthesize seven new analogues of bradykinin by N-terminal acylation with various acyl groups of a moderately potent B2 antagonist, previously synthesized by Stewart's group, D-Arg-Arg-Pro-Hyp-Gly-Thr-Ser-D-Phe-Thi-Arg. The analogues were tested in vitro for their blood pressure-lowering and uterotonic activities. The modifications either preserved or increased the antagonistic potency in the rat blood pressure test. On the other hand, all seven substituents negatively influenced the interaction with the rat uterine receptors. Our results may be helpful for designing new B2 agonists and antagonists.

New bradykinin analogues substituted in positions 7 and 8 with sterically restricted 1-aminocyclopentane-1-carboxylic acid.

A sterically constrained non-coded amino acid, 1-aminocyclopentane-1-carboxylic acid (Apc), was introduced in position 7 or 8 of the bradykinin (BK) B(2) receptor antagonist, [D-Arg(0), Hyp(3), Thi(5, 8), D-Phe(7)]BK, previously synthesized by Stewart's group. This modification is believed to reduce the flexibility of the peptides, thereby forcing the peptide backbone and side chains to adopt specific orientations. Apc substitution was combined with acylation of the N-terminus with 1-adamantaneacetic acid (Aaa). The activity of four new analogues was assayed in isolated rat uterus and in rat blood pressure tests. The results clearly demonstrated that the Apc residue inserted in position 7 led to a reduction of antagonistic properties in the rat uterus assay or even restored the agonism in the blood pressure test, whereas Apc at position 8 enhanced antagonistic potency in both the tests. In both cases, acylation of the N-terminus led to the enhancement of the antagonistic potency. On the basis of these findings, new potent and selective B(2) blockers might be designed.