Dariusz Sobolewski

Novel analogues of arginine vasopressin containing α-2-indanylglycine enantiomers in position 2

Continuing our efforts to obtain potent and selective analogues of AVP we synthesized and pharmacologically evaluated ten new compounds modified at position 2 with α‐2‐indanylglycine or its D‐enantiomer (Igl or D‐Igl, respectively). All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of these compounds to human OT receptor. The Igl2 substitution resulted in a significant change of the pharmacological profile of the peptides. The new analogues were moderate or potent OT antagonists (pA2 values ranging from 7.19 to 7.98) and practically did not interact with V1a and V2 receptors. It is worth emphasizing that these new peptides were exceptionally selective. On the other hand, the D‐Igl2 substituted counterparts turned out to be weak antagonists of the pressor response to AVP and displayed no antidiuretic activity. Some of the results were unexpected, e.g. dual activity in the rat uterotonic test in vitro: the D‐Igl peptides showed a strong antioxytocic potency (pA2 values ranging from 7.70 to 8.20) at low concentrations and full agonism at high concentrations. The results provided useful information about the SAR of AVP analogues. Copyright

Conformational preferences of proline derivatives incorporated into vasopressin analogues: NMR and molecular modelling studies.

In this study, arginine vasopressin analogues modified with proline derivatives – indoline‐2‐carboxylic acid (Ica), (2S,4R)‐4‐(naphthalene‐2‐ylmethyl)pyrrolidine‐2‐carboxylic acid (Nmp), (2S,4S)‐4‐aminopyroglutamic acid (APy) and (2R,4S)‐4‐aminopyroglutamic acid, (Apy) – were examined using NMR spectroscopy and molecular modelling methods. The results have shown that Ica is involved in the formation of the cis peptide bond. Moreover, it reduces to a great extent the conformational flexibility of the peptide. In turn, incorporation of (2S,4R)‐Nmp stabilizes the backbone conformation, which is heavily influenced by the pyrrolidine ring. However, the aromatic part of the Nmp side chain exhibits a high degree of conformational freedom. With analogues IV and V, introduction of the 4‐aminopyroglumatic acid reduces locally conformational space of the peptides, but it also results in weaker interactions with the dodecylphosphocholine/sodium dodecyl sulphate micelle. Admittedly, both analogues are adsorbed on the micelle’s surface but they do not penetrate into its core. With analogue V, the interactions between the peptide and the micelle seem to be so weak that conformational equilibrium is established between different bound states.

Interaction of Bradykinin and B2 Bradykinin Receptor Antagonists with Colloidal Au Surface Explored by Surface-Enhanced Raman Scattering

Bradykinin (BK), an endogenous peptide hormone, which is involved in a number of physiological and pathophysiological processes, and the potent B2 bradykinin receptor antagonists, [D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, Aaa[D-Arg0,Hyp3,Thi5,8,L-Pip7]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, and Aaa[D-Arg0,Hyp3,Thi5,D-Phe7,L-Pip8]BK, were deposited onto colloidal Au particles of 20 nm size. Interaction of these molecules with colloidal Au surface was explored by surface-enhanced Raman scattering (SERS). Briefly, it was shown that BK adsorbs on the Au surface mainly through the Phe5/Phe8 residues. In case of the BK specifically modified analogues mainly the Pip and Thi rings are involved in the interaction process; however, Pip and Thi adopt slightly different orientation with respect to Au for each of the analogues. In addition, the lack of the Aaa vibrations, together with the enhancement of the Thi, Pip, or Phe modes, emphasizes the importance of the C-terminus in the interaction with the Au surface.

Analogues of AVP modified in the N-terminal part of the molecule with Pip isomers: TFA-catalysed peptide bond hydrolysis.

Using SPPS techniques, six new analogues of AVP and some of its agonists were synthesised. The peptides were designed by substitution of Phe at position 3 of AVP, [Mpa1] AVP (dAVP) and [Mpa1,Val4,D‐Arg8]VP (dVDAVP) with L‐ or D‐Pip, a non‐coded α‐imino acid, also called homoproline. Surprisingly enough, both the analogues of AVP and [Mpa1]AVP with identical substituents at position 2 turned out to be highly sensitive to TFA used for deprotection and cleavage of the synthesised peptides from the resin and it was the reason why we were not able to obtain these four peptides. The mechanisms of their fragmentation were proposed in this study. Unfortunately, all the new analogues were inactive in bioassays for the pressor, antidiuretic and uterotonic invitro activities in the rat. Copyright

Potent antidiuretic agonists, deamino-vasopressin and desmopressin, and their inverso analogs: NMR structure and interactions with micellar and liposomic models of cell membrane.

Deamination of vasopressin (AVP) enhances its antidiuretic activity. Moreover, introduction of D‐Arg8 instead of its L enantiomer in deamino‐vasopressin (dAVP) results in an extremely potent and selective antidiuretic agonist ‐ desmopressin (dDAVP). In this study we describe the synthesis, pharmacological properties and structures of these two potent antidiuretic agonists, and their inverso analogs. The structures of the peptides are studied in micellar and liposomic models of cell membrane using CD spectroscopy. Additionally, three‐dimensional structures in mixed anionic‐zwitterionic micelles are obtained using NMR spectroscopy supported by molecular dynamics simulations. Our conformational studies have shown that desmopressin in a membrane mimicking environment adopts one of the characteristic for vasopressin‐like peptides β‐turn ‐ in position 3,4. Furthermore, dDAVP shows the tendency to create a β‐turn in the Cys6‐Gly9 C‐tail, considered to be important for the antidiuretic activity, and also some tendency to adopt a 5,6 β‐turn. In desmopressin, in contrast to the native vasopressin, deamino‐vasopressin and [D‐Arg8]‐vasopressin (DAVP), the Arg8 side chain, crucial for the pressor and antidiuretic activities, is very well exposed for interaction with the receptor, whereas Gly9, crucial for the pressor and uterotonic activities, is situated together with the C‐terminal amide group very close to the tocin ring. The arrangements of the Gln4 and Asn5 side chains, being crucial for OT activity, also differ in desmopressin as compared to those of AVP, dAVP and DAVP. These differences in arrangement of the important for activities side chains are likely to explain extremely potent and selective antidiuretic activities of desmopressin.

Arginine-, d-arginine-vasopressin, and their inverso analogues in micellar and liposomic models of cell membrane: CD, NMR, and molecular dynamics studies

We describe the synthesis, pharmacological properties, and structures of antidiuretic agonists, arginine vasopressin (AVP) and [d-Arg8]-vasopressin (DAVP), and their inverso analogues. The structures of the peptides are studied based on micellar and liposomic models of cell membranes using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic–zwitterionic micelles are obtained using NMR spectroscopy and molecular dynamics simulations. NMR data have shown that AVP and DAVP tend to adopt typical of vasopressin-like peptides β-turns: in the 2–5 and 3–6 fragments. The inverso-analogues also adopt β-turns in the 3–6 fragments. For this reason, their inactivity seems to be due to the difference in side chains orientations of Tyr2, Phe3, and Arg8, important for interactions with the receptors. Again, the potent antidiuretic activity of DAVP can be explained by CD data suggesting differences in mutual arrangement of the aromatic side chains of Tyr2 and Phe3 in this peptide in liposomes rather than of native AVP. In the presence of liposomes, the smallest conformational changes of the peptides are noticed with DPPC and the largest with DPPG liposomes. This suggests that electrostatic interactions are crucial for the peptide–membrane interactions. We obtained similar, probably active, conformations of the antidiuretic agonists in the mixed DPC/SDS micelles (5:1) and in the mixed DPPC/DPPG (7:3) liposomes. Thus it can be speculated that the anionic–zwitterionic liposomes as well as the anionic–zwitterionic micelles, mimicking the eukaryotic cell membrane environment, partially restrict conformational freedom of the peptides and probably induce conformations resembling those of biologically relevant ones.

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, X1 or X2, were substituted at position 7 of the model B2 receptor antagonist [D‐Arg0, Hyp3, Thi5, 8, D‐Phe7]‐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 X17 substitution resulted in a decrease in antagonistic potency of the new peptide in both assays. The X27 and N‐(Bzl)‐Gly7 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 X1 or X2 modification. Our studies provide new information about structure–activity relationship of the BK antagonists which may be helpful for designing more potent B2 receptor blockers. Copyright

Influence of Conformationally Constrained Amino Acids Replacing Positions 2 and 3 of Arginine Vasopressin (AVP) and Its Analogues on Their Pharmacological Properties

Synthesis of thirteen new analogues of arginine vasopressin (AVP) has been described. Amino acid residues at positions 2 and 3 of AVP, [3-mercaptopropionic acid (Mpa)(1)]AVP (dAVP), [Mpa(1),d-Arg(8)]VP (dDAVP) and [Mpa(1),Val(4),d-Arg(8)]VP (dVDAVP) were replaced with one amino acid residue using sterically constrained non-proteinogenic amino acids, 4-aminobenzoic acid (Abz), cis-4-aminocyclohexanecarboxylic acid (ach) or its trans-isomer (Ach). In the case of a potent V(1a) antagonist, [1-mercaptocyclohexaneacetic acid (Cpa)(1)]AVP, only one similar analogue has been prepared by replacing positions 2 and 3 with Abz. Unfortunately, all new peptides were inactive in bioassays for the pressor, antidiuretic and uterotonic in vitro activities in the rat.