Is the Use of Surface-Enhanced Infrared Spectroscopy Justified in the Selection of Peptide Fragments That Play a Role in Substrate-Receptor Interactions? Adsorption of Amino Acids and Neurotransmitters on Colloidal Ag and Au Nanoparticles.

This paper describes an application of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and surface-enhanced infrared spectroscopy (SEIRA) to characterize the selective adsorption of four peptides present in body fluids such as neuromedin B (NMB), bombesin (BN), neurotensin (NT), and bradykinin (BK), which are known as markers for various human carcinomas. To perform a reliable analysis of the SERIA spectra of these peptides, curve fitting of these spectra in the spectral region above 1500 cm-1 and SEIRA measurements of sulfur-containing and aromatic amino acids were performed. On the basis of the analyses of the spectral profiles, specific conclusions were drawn regarding specific molecule-metal interactions and changes in the interaction during the substrate change from the surface of silver nanoparticles (AgNPs) to gold nanoparticles (AuNPs).

Adsorption of (Phe-h5)/(Phe-d5)-substituted peptides from neurotensin family on the nanostructured surfaces of Ag and Cu: SERS studies.

This work describes an application of Raman (RS) and surface-enhanced Raman scattering (SERS) to characterize the selective adsorption of two peptides belonging to the neurotensin family peptides, such as kinetensin (KN) and xenopsin-related peptide 2 (XP-2) that are known to stimulate the growth of human tumors. To perform a reliable analysis of SERS spectra, the L-Phe residue (at position 8 or 1 in the amino acid sequence of these peptides) was replaced with L-Phe-d5 (five protons of L-phenylalanine ring substituted by deuterium). Native and (Phe-d5)-isotopically labeled peptides were deposited on electrochemically nanostructured surfaces of Ag (AgORC) and Cu (CuORC) from an aqueous solution (H2O). To determine the share of amide bonds in the interaction with the metallic substrate, SERS spectra of peptides adsorbed on AgORC from heavy water (D2O) were measured. Also, to determine the effect of the C-end on the SERS spectrum, measurements were made for the KN analog in which the C-terminal L-leucine was removed ([desLeu9]KN). Based on the analyses of the spectral profiles, in the spectral range of 600-1650 cm-1, specific conclusions have been drawn regarding specific aromatic ring···metal interactions and changes in the interaction during substrate change.

Gonadotropin receptor variants are linked to cumulative live birth rate after in vitro fertilization.

PURPOSE: The objective was to investigate if the gonadotropin receptor variants N680S (N: asparagine, S: serine, rs6166) in the follicle-stimulating hormone receptor (FSHR) and N312S (rs2293275) in the luteinizing hormone/human chorionic gonadotropin receptor (LHCGR) predicted cumulative live birth rate after in vitro fertilization (IVF). METHODS: A total of 665 women were consecutively enrolled for IVF during the period 2007-2016. Inclusion criteria were < 40 years of age, body mass index < 30 kg/m2, non-smoking, regular menstruation cycle of 21-35 days, and bilateral ovaries. A blood sample was drawn for endocrine hormonal analysis and for DNA extraction with subsequent genotyping of the FSHR N680S and LHCGR N312S polymorphisms. Statistical analyses were done on all completed IVF cycles. RESULTS: Women homozygous for S in both receptors combined (4S) had significantly higher live birth rate compared to those with other receptor variants when combining the first three IVF cycles (OR = 2.00, 95% CI [1.02, 3.92], p = 0.043). Cumulatively higher chance of live birth rate, during all IVF cycles, was also evident (HR = 1.89, 95% CI [1.00, 3.57], p = 0.049). CONCLUSIONS: Gonadotropin receptor variants are promising candidates for the prediction of the possibility to have a baby to take home after IVF treatment.

Tip-enhanced Raman spectroscopy of bradykinin and its B2 receptor antagonists adsorbed onto colloidal suspended Ag nanowires.

The tip-enhanced Raman scattering (TERS) spectra of bradykinin (BK) and its potent B2 BK receptor antagonists, [d-Arg(0),Hyp(3),Thi(5,8),l-Pip(7)]BK and [d-Arg(0),Hyp(3),Thi(5),d-Phe(7),l-Pip(8)]BK, approximately with a size of about 40 nm, adsorbed onto colloidal suspended Ag nanowires with diameter in the range of 350-500 nm and length of 2-50 µm were recorded. The metal surface plasmon resonance and morphology of the Ag nanowires were studied by ultraviolet-visible (UV-Vis) spectroscopy and scanning electron microscopy (SEM). Briefly, it was shown that two C-terminal amino acids of BK and [d-Arg(0),Hyp(3),Thi(5,8),l-Pip(7)]BK are involved in the interaction with the colloidal suspended Ag nanowire surface, whereas three last amino acids of the [d-Arg(0),Hyp(3),Thi(5),d-Phe(7),l-Pip(8)]BK sequence attached the Ag surface. Thus, BK adsorbs on the colloidal suspended Ag nanowires mainly through the Phe(5/8) ring (tilted orientation) and the one oxygen atom of the carboxylate group and the H2N-C-NH-CH2- fragment of Arg(9). In the case of [d-Arg(0),Hyp(3),Thi(5,8),l-Pip(7)]BK, the Thi(8) ring (through the lone electron pair on the sulfur atom) and the both oxygen atoms of the carboxylate group and the amine group of Arg(9) mainly participated in the interaction with the Ag nanowire surface. For [d-Arg(0),Hyp(3),Thi(5),d-Phe(7),l-Pip(8)]BK, the d-Phe(7) ring, the Pip(8) ring, and the Arg(9) side-chain assisted in the peptide interaction with the Ag surface. The obtained results emphasize the importance of the C-terminal part of these peptides in the adsorption process onto the colloidal suspended Ag nanowires.

Analogues of arginine vasopressin and its agonist and antagonist modified in the N-terminal part of the molecule with l-beta-homophenylalanine.

In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l-beta-homophenylalanine (beta-Hph). Two of them had just this single modification, the next two peptides are analogues of the V2 agonist, namely [3-mercaptopropionic acid (Mpa)1]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V(1a) antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)1]AVP, with beta-Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa(1),beta-Hph2]AVP, [Cpa1,beta-Hph2]AVP, [Cpa1,beta-Hph3]AVP, turned out to be uterotonic antagonists with pA2 = 6.3 +/- 0.2, 6.3 +/- 0.1, 6.0 +/- 0.3 respectively. The last one exhibited antipressor properties also (pA2 = 6.4 +/- 0.1).

Analogues of arginine vasopressin modified in the N-terminal part of the molecule with enantiomers of N-methylphenylalanine.

Four new analogues of arginine vasopressin (AVP) substituted in positions 2 and 3 with all possible combinations of enantiomers of N-methylphenylalanine were synthesized and studied to assess the influence of N-methylation of the peptide bonds between the first three amino acids on the pharmacological properties of the resulting peptides. The next three analogues were designed to learn how the shortening of the peptide chain, by removal of one of the N-methylphenylalanine residues, would affect pharmacological properties of the resulting compounds. The activity of the analogues was tested in the in vitro uterotonic, pressor and antidiuretic tests. None of the prepared analogues displayed significant biological activity with the exception of [Me-d-Phe(2), Me-Phe(3)]AVP and [Me-d-Phe(2,3)]AVP, which showed low antiuterotonic activity (pA(2) = 6.6 and pA(2) = 6.4, respectively). Our results, while not impressive in terms of biological activity, may be helpful for designing potent and selective oxytocin antagonists.

Potent bradykinin antagonists containing N-benzylglycine or N-benzyl-l-alanine in position 8.

Two new analogues of a previously designed bradykinin (BK) antagonist, d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Phe-Thi-Arg, substituted in position 8 by N-benzylglycine and N-benzyl-l-alanine were designed, synthesized and bioassayed. The results show an impressive enhancement of B2 antagonistic potencies of both peptides in comparison with the model. In two further analogues these modifications were combined with acylation of the N-terminus with 1-adamantanacarboxylic acid. Acylated analogues exhibited higher antagonistic potency in comparison with the parent compounds, however, the range of effect was not as high as in previously described cases. The activity of analogues was assessed by their ability to inhibit vasodepressor response to exogenous BK (rat blood pressure test). Our results may be of value in the design of more potent BK antagonists.

From pro defensins to defensins: synthesis and characterization of human neutrophil pro alpha-defensin-1 and its mature domain.

Human neutrophil alpha-defensins (HNPs) are small, cationic, Cys-rich antimicrobial proteins that play important roles in innate immunity against infectious microbes such as bacteria, fungi and enveloped viruses. Synthesized as inactive precursors in vivo (pre-proHNPs), HNPs are activated through proteolytic removal of the inhibitory pro-peptide required for subcellular sorting and correct folding. We seek to understand the molecular basis for the recognition between the 45-residue pro-peptide and the C-terminal functional domain. Here we described, total chemical synthesis of the 75-residue human neutrophil pro alpha-defensin-1 (proHNP1) via native chemical ligation. After oxidative folding, proHNP1 is cleaved by cyanogen bromide at the Met45-Ala46 peptide bond to release the mature form. The native disulfide connectivity in HNP1, i.e. Cys1-Cys6, Cys2-Cys4 and Cys3-Cys5, is verified by mass mapping of peptide fragments generated by proteolytic digestion and Edman degradation. Fluorescence spectroscopy studies and antimicrobial activity assays further support that synthetic proHNP1 and HNP1 are correctly folded. While largely unstructured in aqueous solution, the pro-peptide binds to HNP1 intermolecularly with an apparent Kd value of 6.2 microM at pH 7.4, confirming the mode of intramolecular inactivation of human alpha-defensin precursors.

New analogues of bradykinin containing a conformationally restricted dipeptide fragment in their molecules.

The present paper describes the synthesis and some pharmacological properties of two new bradykinin analogues containing the ethylene-bridged dipeptide Phe-Phe in their molecules. In a further two peptides this modification was combined with acylation of the N-terminus with 1-adamantaneacetic acid. Finally, we synthesized four analogues by removing the Ser6 residue from the four peptides mentioned above. The activity of the new analogues was assayed on isolated rat uterus (RUT) and in rat blood pressure tests (BPT). The results clearly indicate that the proposed modification, alone or in combination with other changes, resulted in either a drop in antiuterotonic activity or even in conversion to an agonism. Although this tendency is not so distinct in blood pressure assays, the antagonistic potency of the new analogues is also diminished. Nevertheless, it was demonstrated that the D-amino acid in position 7 which, until recently, was considered necessary for antagonism, may be replaced, together with the amino acid occupying position 8, by a suitable, sterically restricted L,L-dipeptide unit.

New bradykinin analogs in contraction of rat uterus.

In this study, we evaluated 20 of our previously synthesized peptides on isolated rat uterus by Holton's procedure with minor modifications, and compared their activity with that assessed previously by their ability to inhibit vasodepressor response to exogenous bradykinin (BK) in conscious rats. We used [D-Arg(0), Hyp(3), Thi(5, 8), (D-Phe)(7)]BK, the B(2) antagonist of Vavrek and Stewart as a model when designing our analogs. We observed that, in the case of the rat uterus test, the activity of peptides modified by acylation of the N-terminus with various bulky groups depends substantially on the chemical character of the substituent. We also learned that, contrary to previous examples, acylation of the N-terminus of antagonists, which contain a sterically restricted fragment in the C-terminal part, may not improve their antagonistic potencies. Besides an improved characterization of a series BK analogs, our studies have provided new information on the structure-activity relationship, which in turn may be of value in the design of more potent and selective bradykinin antagonists. The results of our studies appear to support the hypothesis of others about the presence of different subtypes of B(2) receptors in rat uterus and blood vessels.