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.     

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).     

The influence of 1-aminocyclopentane-1-carboxylic acid at position 2 or 3 of AVP and its analogues on their pharmacological properties.

This study describes the synthesis and some pharmacological properties of eight new analogues of arginine vasopressin (AVP) substituted at position 2 or 3 with cycloleucine (1-aminocyclopentane-1-carboxylic acid, Apc). All new peptides were tested for their pressor, antidiuretic and uterotonic in vitro potency. The Apc3 modification resulted in an almost complete loss of potency in all three tests, which is interpreted as a loss of interaction with all three neurohypophyseal hormone receptors. On the other hand, the Apc2 modification resulted in compounds having differently modified activities (high antidiuretic potency, low and graded pressor activity and either no activity or low oxytocin antagonizing activity in the uterotonic in vitro test) thus selectively altering the interaction with the receptors similar to that of 1-aminocyclohexane-1-carboxylic acid (Acc). The results obtained may be helpful for designing new analogues of arginine vasopressin.     

Raman and surface-enhanced Raman spectroscopy investigation of vasopressin analogues containing 1-aminocyclohexane-1-carboxylic acid residue

In this work, Raman spectroscopy (RS) was employed to characterize molecular structures of [Arg8]vasopressin (AVP) and its [Acc2,D-Arg8]AVP, [Acc3]AVP, and [Cpa1, Acc3]AVP analogues. The RS band assignments have been proposed. To determine the mechanism of adsorption of the above-mentioned compounds adsorbed on a colloidal silver surface, surface-enhanced Raman spectra (SERS) were measured. The SERS spectra were used to determine relative proximity of the adsorbed functional groups of [corrected] investigated peptides and their orientation on the silver surface. The AVP and [Acc3]AVP SERS spectra (Acc: 1-aminocyclohexane-1-carboxylic acid) show that the L-tyrosine (Tyr) lies far from the metal surface, whereas the [Cpa1,Acc3]AVP spectrum (Cpa: 1-mercaptocyclohexaneacetic acid) provides evidence that Tyr interacts with the silver surface. These results suggest that [corrected] the binding of the Tyr-ionized phenolic group might be responsible for the selectivity of the analogues. We show that the aromatic ring of L-phenylalanine (Phe) of AVP and [Acc2,D-Arg8]AVP interacts with the silver surface. The strength of this interaction is considerably weaker for [Acc2,D-Arg8]AVP than for AVP. This might be due either to a longer distance between the Phe ring and the silver surface, or to the almost perpendicular orientation of the Phe ring towards the surface. The carbonyl group of the L-glutamine [corrected] (Gln) or L-asparagine [corrected](Asn) of AVP, [Acc2,D-Arg8]AVP, and [Acc3]AVP is strongly bound to the silver surface. We have also found that all peptides adsorb on the silver surface via sulfur atoms of the disulfide bridge, adopting a "GGG" conformation, except [Cpa1,Acc3]AVP, which accepts a "TGG" geometry.     

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 V_1a 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 © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence that the effects of arginine-8-vasopressin (AVP) on pituitary corticotropin (ACTH) release are mediated by a novel type of receptor

Ovine corticotropin releasing factor (oCRF-41) and AVP act synergistically to stimulate pituitary ACTH secretion. In the present study we have investigated whether the effect of AVP, either in the presence or in the absence of oCRF-41 (0.5 nmol/l), could be blocked by V1 (pressor)-antagonists. Furthermore, oxytocin, and [1-deamino,8-D-arginine] vasopressin (dDAVP) were tested for their ability to release ACTH. All experiments were carried out in vitro, using segments of rat anterior pituitary glands. The V1-antagonist [1-deamino,penicillamine(o-methyl-tyrosine)]AVP inhibited ACTH release induced by AVP or AVP + oCRF-41. However, it also had some agonistic activity which was more pronounced in the presence of oCRF-41. An equally potent V1-antagonist, [1-beta-mercapto-beta, beta-cyclopentamethyleneproprionic acid (o-methyl-tyrosine)]AVP, failed to inhibit AVP-stimulated ACTH secretion, and also had weak agonist potency. The relatively selective V2 (antidiuretic)-agonist dDAVP was 20-30 fold less potent than AVP. Oxytocin, a weak V1- and V2-agonist was only 4-8 fold less potent than AVP. These data are compatible with the suggestion that AVP receptors on pituitary corticotrope cells are neither classical V1- nor V2-receptors.     

Novel linear antagonists of the antidiuretic (V2) and vasopressor (V1) responses to vasopressin

We report the solid phase synthesis of a series of 16 linear analogues of the cyclic antagonist of the antidiuretic (V2) and the vasopressor (V1) responses to arginine vasopressin (AVP), d(CH2)5[D-Tyr(Et)2, Val4]AVP(A). Peptide 1, the linear precursor of (A), (CH2)5(SH)-CH2-CO-D-Tyr(Et)-Phe-Val-Asn-Cys-Pro-Arg-Gly-NH2 was modified at position six with alpha-L-aminobutyric acid (Abu) to give peptide 2. Further modifications of the Abu6 analogue (No. 2) at position one by substituting cyclohexylacetic acid (Caa), cyclohexylpropionic acid (Cpa), 1-adamantaneacetic acid (Aaa), phenylacetic acid (Phaa), tert.-butylacetic acid (t-Baa), isovaleric acid (Iva), propionic acid (Pa), L-penicillamine (P), tert.-butoxycarbonyl (Boc) or omitting any substituent at this position, and/or in combination with Arg-NH2(9), Ala-NH2(9), D-Arg8-Arg-NH2(9), and desGly9 modifications yielded the remaining 14 peptides. All 16 peptides were examined for agonistic and antagonistic potencies in AVP V2 and V1 assays in rats. Apart from the Cpa analogue and the analogue lacking any substituent in the 1-position, all exhibit substantial V2 and V1 antagonism. A number are as potent as (A) as V2 antagonists. With an anti-V2 pA2 = 8.11 +/- 0.07, Aaa-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (No. 6) is as potent as any cyclic AVP V2 antagonist reported to date. The PaI analogue of No. 6 exhibits promising anti-V2/anti-V1 selectivity. These findings prove conclusively that a ring structure is not a requirement for recognition of or for binding to AVP V2 or V1 receptors. This discovery thus offers a promising new approach to the design of peptide and non-peptide antagonists of AVP and perhaps also to other cyclic peptides such as somatostatin, atrial-natriuretic factor, insulin, and the recently discovered endothelin. Some of these linear antagonists may be of value as pharmacological tools and as therapeutic agents.     

Analogues of a potent oxytocin antagonist with truncated C-terminus or shorter amino acid side chain of the basic amino acid at position 8.

Twelve analogues were synthesized, their structure derived from modifications of [(S)Pmp1, D-Trp2, Pen6, Arg8]oxytocin, PA, in which (S)Pmp = beta,beta-(3-thiapentamethylene-beta-mercaptopropionic acid). PA is a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 8.86) and in the baboon. Truncated analogues of PA from the C-terminus were systematically prepared ending in either the free acid or the amide, i.e. PA1-9 acid, PA1-8 acid, PA1-7 acid, PA1-6 acid, PA1-8 amide, PA1-7 amide and PA1-6 amide. PA1-8 amide was roughly as potent as PA in the rat uterotonic assay in vitro, and the shorter amides were only somewhat weaker antagonists. All four acid analogues were weaker antagonists than PA but still maintained rather high antagonistic potency. These findings suggest that, if these truncated acids form as metabolites in vivo, they may contribute to the overall biological effect of PA and their contribution should be taken into account. Furthermore, using these analogues, the radioimmunoassay measurements of PA may be standardized, as they may cross react with PA antibodies and interfere with the determination. In addition, five analogues were made by substituting Arg8 of PA with Lys, Orn8, Dab8, Dap8 and Cit8. All of these analogues maintained high potency as OTAs in the uterotonic assay, although their activity was only about 1.5-3 times lower than PA. The most potent analogue in the uterotonic assay, [Dap8]PA, pA2 = 8.53, had weak pressor activity (pA2 = 6.90) and no antidiuretic effect. The pressor activity was lower for all tested acids, and for PA1-6 acid it was even below the detection limit. Additionally, PA1-9 acid, PA1-7 acid and PA1-6 acid showed no antidiuretic activity. Hence, the PA1-6 acid is a potent OTA with pA2 = 8.27 and no measurable effect in the pressor or antidiuretic tests and thus it is a pure oxytocin antagonist. This fact makes it an attractive candidate for further studies on inhibition of OT biological effects and on preterm labour.     

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.     

Analogs of arginine vasopressin modified in position 3 with (R)-alpha-hydroxymethylphenylalanine.

This study describes the synthesis and some pharmacological properties of three new analogs of arginine vasopressin (AVP) substituted in position 3 with (R)-alpha-hydroxymethylphenylalanine ([R]-HmPhe). All new peptides were tested for vasopressor and antidiuretic as well as uterotonic activity. None of the 3 analogs showed any pressor activity and their uterotonic activity was negligible. Only analog [Mpa1,(R)-HmPhe3]AVP exhibited significant antidiuretic activity.     

Conformational studies of vasopressin analogues modified with N-methylphenylalanine enantiomers in dimethyl sulfoxide solution

The conformations of [Arg8]vasopressin (AVP) analogues substituted at positions 2 and 3 with N-methylphenylalanine (MePhe) enantiomers were earlier investigated by using nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. A comparison of the results obtained in H2O/D2O (9:1) and DMSO-d6 has shown the structures in the first solution to be more flexible than those in DMSO-d6. This is manifested by a higher percentage of minor conformations in H2O/D2O. The largest differences between the NMR spectra in both solvents were noticed for [MePhe2, D-MePhe3]AVP (II) and [D-Cys1,MePhe2,D-MePhe3]AVP (III). Namely, in the ROESY spectra in aqueous solution, the cis/trans isomerization between MePhe2-DMePhe3 and D-Cys1-MePhe2 for II and III, respectively, is observed, while in DMSO-d6, the appropriate cross peaks indicate isomerization across the Cys6-Pro7 peptide bond. In the case of the remaining peptides, the position of cis/trans isomerization is the same in aqueous solution and in dimethyl sulfoxide. [D-MePhe2,MePhe3]AVP (V) displays low antiuterotonic and antipressor activities, while [D-MePhe2,)]AVP (IV) is a weak but selective blocker of oxytocin (OT) receptors in the uterus. The former shows similar conformational preferences as another antagonist of V1a and OT receptors-namely, [Acc2,D-Arg8]VP (Acc: 1-aminocyclohexane-1-carboxylic acid)-investigated by us. In the case of IV, the cis peptide bond between residues at positions 2 and 3 might be the reason for selectivity.     

Antagonists of oxytocin featuring replacement with modified beta-mercaptopropionic acids at position 1.

Twenty analogues were synthesized of [Pmp1, D-Trp2, Arg8]oxytocin, PA, (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid), a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 7.77) and in the baboon. Systematic substitution of Pmp1 was made with beta-mercaptopropionic acids featuring replacement of the 4-methylene group of the cyclohexyl ring of Pmp with isosteric O, S, NH or with C=O. Since the more hydrophilic NH and C=O substitutions showed a sharply decreased antagonistic potency (rat uterotonic test in vitro), additional modifications were made to reduce their hydrophilicity. Acylation of the NH group with various acyl groups, and ketalization or thioketalization of C=O with more or less bulky substituents led to a partial restoration of potency, the N-carbamyl- and the 2-mercapto-2-adamantaneacetyl analogues being equipotent with PA. Internal cyclization by amidation of the NH-group with Gly-9, resulted in a bicyclic analogue, (cyclo 1-9)[(HN)Pmp1, Gly9]PA which was equipotent with PA. When Pen-6 was introduced into the bicyclic derivative instead of Cys-6, to reduce the flexibility of the rings, the resulting (cyclo 1-9)[(HN)Pmp1, Pen6, Gly9]PA had somewhat better potency (pA2 = 8.17) in the uterotonic test and no detectable activity in the antidiuretic assay. In the case of substitution of PA with beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, (S)Pmp, there was also an increase in inhibitory potency in the uterotonic test (pA2 = 8.08): the analogue had extremely weak antidiuretic activity. To establish the importance of the steric effects of the Pen-6 substitution, analogues [Pen6]PA and [(S)Pmp1, Pen6]PA were made and found to be very potent, with a pA2 of 8.72 and 8.86, respectively. The high potency of the latter analogue and its extremely weak action in the diuretic assay makes it an attractive candidate for studies on the inhibition of the biological effects of oxytocin and for the prevention of preterm labour.     

Desensitization of renal vasopressin-stimulated adenylate cyclase by a vasopressor and antidiuretic antagonist, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-ethyl)-D-tyrosine, 4-valine]arginine vasopressin

Desensitization of the renal antidiuretic (V2) receptor has been regarded as agonist-specific. We found that a vasopressor and antidiuretic (V1V2) antagonist d(CH2)5DTyr(Et)VAVP induced desensitization of the receptor and that a vasopressor (V1) antagonist d(CH2)5Tyr(Me)AVP, which is a weak antidiuretic (V2) agonist, counteracted the potent desensitizing action of 1-deamino-8-D-arginine-vasopressin and endogenous arginine vasopressin.     

Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists

In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26).     

Stimulation of inorganic-phosphate incorporation into phosphatidylinositol in rat thoracic aorta mediated through V1-vasopressin receptors.

Vasopressin stimulates the incorporation of [32P]Pi into phosphatidylinositol but not into other phospholipids in rat thoracic aorta strips. The relative abilities of three vasopressin analogues to stimulate phosphatidylinositol labelling in rat aorta are similar to their relative pressor potencies in vivo and to their relative potencies in stimulating the metabolism of rat hepatocytes, but very different from their relative antidiuretic potencies. The vasopressor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin competitively inhibits [Arg8]vasopressin-stimulated phosphatidylinositol labelling in rat aorta with a pA2 of 8.1. It is concluded that the Ca2+-mobilizing vasopressin receptors (V1-receptors) of the rat aorta stimulate phosphatidylinositol metabolism, probably by enhancing phosphatidylinositol breakdown.     

Pancreatic vasopressin V1b receptors: characterization in In-R1-G9 cells and localization in human pancreas

Vasopressin (AVP) receptors present in In-R1-G9 cells, a hamster glucagon-secreting alpha-pancreatic cell line, were characterized using SSR-149415, a selective nonpeptide V1b receptor antagonist, and reference AVP compounds. Binding experiments, using [3H]AVP as a ligand, identified a single population of high-affinity binding sites. SSR-149415 competitively inhibited this binding and exhibited nanomolar and stereospecific affinity for these sites. The affinity of various AVP/oxytocin ligands confirmed a V1b binding profile. In functional studies, AVP was a potent stimulant in inducing intracellular Ca2+ increase, glucagon secretion, and cell proliferation. These effects were fully antagonized by SSR-149415 with a nanomolar potency, whereas its diasteroisomer as well as two selective V1a and V2 receptor antagonists were much less potent. Additionally, the order of potency of AVP agonists and antagonists was in agreement with V1b-mediated effects. By RT-PCR, we confirmed the presence of V1b receptor mRNA in both In-R1-G9 cells and in human pancreas. The distribution pattern of V1b receptors investigated in human pancreas by immunohistochemistry showed strong labeling in islets of Langerhans, and colocalization studies indicated that this receptor was expressed in alpha-glucagon, beta-insulin, and somatostatin pancreatic cells. Thus, in In-R1-G9 cells, AVP mediates intracellular Ca2+ increase, glucagon secretion, and cell proliferation by activating V1b receptors, and these effects are potently antagonized by SSR-149415. Moreover, the presence of V1b receptors also found in human Langerhans islets could suggest hormonal control of AVP in human pancreas.     

Novel approach to the design of synthetic radioiodinated linear V1A receptor antagonists of vasopressin.

We report the solid phase synthesis of six analogs of the potent and selective linear AVP vasopressor (V1a receptor) antagonist: Phaa1-D-Tyr(Et)2-Phe3-Gln4-Asn5-Lys6-Pro7-Arg-NH(8)2(A) (where Phaa = phenylacetyl) in which the Phaa1 residue is replaced by hydroxyphenylacetyl (HO-Phaa), hydroxyphenylpropionyl (HO-Phpa) and phenylpropionyl (Phpa) and the D-Tyr(Et)2 and Lys6 residues by D-Tyr(Me)2 and Arg6 substituents. The phenolic-containing peptides were synthesized to test the feasibility of using this approach for the design of high affinity selective ligands for AVP V1a receptors. The following analogs of A were synthesized: 11 [(HO)Phaa1]; 2. [(HO)Phaa1,D-Tyr(Me)2]; 3. [(HO)Phaa1,D-Tyr(Me)2, Arg6]; 4. [(HO)Phaa1,Arg6]; 5. [Phpa1]; 6. [(HO)Phpa1]. All six peptides were examined for agonistic and antagonistic potencies in vasopressor (V1a-receptor) and antidiuretic (V2-receptor) and in vitro oxytocic assays in rats. The affinities of the phenolic-containing peptides for hepatic V1a and uterine receptors were also determined. The phenolic-containing peptides all exhibit potent V1a antagonism. Their anti-V1a pA2 values range from 8.23 to 8.63 (the anti-V1a pA2 value of A = 8.69). Their inhibition constants (Ki in nM) range 0.4 to 1.0. They are weak antidiuretic agonists with activities ranging from 0.022 U/mg to 0.13 U/mg (A = 0.033 U/mg). They all exhibit OT antagonism in vitro. Their anti-OT pA2 values range from 7.28 to 7.71 (A = 7.62). All five phenolic compounds were iodinated using iodine chloride and tested in the same in vivo and in vitro assay system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: molecular dynamics simulation of the agonist-bound state in the membrane-aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.     

Position 4 analogues of [deamino-Cys(1)] arginine vasopressin exhibit striking species differences for human and rat V(2)/V(1b) receptor selectivity.

Arginine vasopressin (AVP) mediates a wide variety of biological actions by acting on three distinct G-protein coupled receptors, termed V(1a) (vascular), V(1b) (pituitary) and V(2) (renal). It also binds to the oxytocin (OT) receptor. As part of a program aimed at the design of selective agonists for the human V(1b) receptor, we recently reported the human V(1b), V(1a), V(2) and OT receptor affinities of the following position 4 substituted analogues of [deamino-Cys(1)] arginine vasopressin (dAVP)-(1) d[Leu(4)]AVP, (2) d[Orn(4)]AVP, (3) d[Lys(4)]AVP, (4) d[Har(4)]AVP, (5) d[Arg(4)]AVP, (6) d[Val(4)]AVP, (7) d[Ala(4)]AVP, (8) d[Abu(4)]AVP, (9) d[Nva(4)]AVP, (10) d[Nle(4)]AVP, (11) d[Ile(4)]AVP, (12) d[Phe(4)]AVP, (13) d[Asn(4)]AVP, (14) d[Thr(4)]AVP: (15) d[Dap(4)]AVP. With the exception of Nos. 7 and 12, all peptides exhibit very high affinities for the human V(1b) receptor. Furthermore, peptides 1-4 exhibit high selectivities for the human V(1b) receptor with respect to the V(1a), V(2) and OT receptors and, with d[Cha(4)]AVP, in functional tests, are the first high affinity selective agonists for the human V(1b) receptor (Cheng LL et al., J. Med. Chem. 47: 2375-2388, 2004). We report here the pharmacological properties of peptides 1-4, 5 (from a resynthesis), 7, 9-13, 15 in rat bioassays (antidiuretic, vasopressor and oxytocic) (in vitro: no Mg(++)) with those previously reported for peptides 5, 6, 8, 14. We also report the rat V(1b), V(1a), V(2) and OT receptor affinities of peptides 1-5 and the rat V(2) receptor affinities for peptides: 7-15.The antidiuretic activities in units/mg of peptides 1-15, are: 1=378; 2=260; 3=35; 4=505; 5=748; 6=1150; 7=841; 8=1020; 9=877; 10=1141; 11=819, 12=110; 13=996; 14=758; 15=1053. Peptides 1-4 exhibit respectively the following rat and human (in brackets) V(2) receptor affinities: 1=3.1 nm (245 nm); 2=3.4 nm (1125 nm); 3=24.6 nm (11,170 nm); 4=0.6 nm (1386 nm). Their rat V(1b) receptor affinities are 1=0.02 nm; 2=0.45 nm; 3=9.8 nm; 4=0.32 nm. Their rat V(1a) receptor affinities are 1=1252 nm; 2=900 nm; 3=1478 nm; 4=32 nm. Their rat oxytocin (OT) receptor affinities are 1=481 nm; 2=997 nm; 3=5042 nm; 4=2996 nm. All four peptides have high affinities and selectivities for the rat V(1b) receptor with respect to the rat V(1a) and OT receptors. However, in contrast to their high selectivity for the human V(1b) receptor with respect to the human V(2) receptor, they are not selective for the V(1b) receptor with respect to the V(2) receptor in the rat. These findings confirm previous observations of profound species differences between the rat and human V(2) receptors. Peptides 1-4 are promising leads to the design of the first high affinity selective agonists for the rat V(1b) receptor.     

Vasopressin antisense peptide interactions with the V1 receptor

The molecular recognition hypothesis, that peptide ligands and their receptor binding sites are encoded by complementary nucleotide sequences, was tested for arginine vasopressin (AVP) and its V1 receptor. Binding of [125I] [d(CH2)5,Sar7]AVP (a selective V1 vasopressin antagonist radioligand) or [3H]AVP to rat liver plasma membranes was inhibited by peptides known to bind to V1 receptors but not by the AVP complementary peptide (Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala) (PVA). Rabbit anti-PVA antibodies were nonimmunoreactive with any protein in rat liver membranes or in a partially purified preparation from rat liver containing reconstitutable vasopressin binding activity. Furthermore, there was no suppression of the AVP pressor effect by PVA in vivo using a rat blood pressure bioassay. These findings do not support the hypothesis that the V1 receptor binding site is encoded by the antisense DNA strand to AVP.