Design of neurohypophyseal peptides that exhibit selective agonistic and antagonistic properties.

Within the spectrum of the characteristic pharmacological activities (oxytocic (O), milk-ejecting (ME), antidiuretic (A), pressor (P) associated with the known natural and synthetic analogs of oxytocin and vasopressin it is possible to discern patterns of selectivity of these types: 1) interpeptide-like (a) O/A, (b) O/P; 2) intraoxytocin-like (a) O/ME; (b) ME/O; 3) intravasopressinlike (a) A/P, (b) P/A. Consideration of structural modifications of oxytocin or vasopressin, which individually or in combination give rise to peptides possessing enhanced selectivity of a given type, can in some cases provide a rational basis for the design of peptides with even greater selectivity. [1-Deamino-4-valine-8-D-arginine]vasopressin, the most highly specific antidiuretic peptide known to date, was designed in this fashion. By contrast, intraoxytocin-like selectivity, is manifested to only a minor degree in all peptides studied to date. Enhanced interpeptide-like selectivity of the type 1a (O/A; O/P) is readily attainable by specific single substitutions at positions 4 or 7 in oxtocin. Substitution of threonine in the 4 position of the oxytocic antagonist [1-deaminopenicillamine]oxytocin brought about a threefold enhancement in oxytocic inhibitory activity. Thus [1-deaminopenicillamine-4-threonine]oxytocin (dPTOT) is the most potent antagonist of the in vitro oxytocic response to oxytocin known to date. Thus analysis of the pharmacological data from over 300 analogs of oxytocin and vasopressin allows the delineation of those structural modifications that can optimize selectivities. The potential and limitations of this approach for the design of peptides possessing desired agonistic or antagonistic selectivity for potential clinical use and for studies on oxytocin and vasopressin receptors is discussed.     

The influence of modification at position 2 on the side-chain conformation in oxytocin analogs

The nonapeptide oxytocin (OT) is used in medicine to help begin and/or continue childbirth. Its analogs can be also used to control bleeding following fetus delivery. The main function of oxytocin is to stimulate contraction of uterus smooth muscle and the smooth muscle of mammary glands, thus regulating lactation. This paper describes theoretical simulations of the distribution of the torsional angles chi1 in the non-standard methylated phenylalanine residues of three oxytocin analogs: [(Phe)(2)o-Me]OT, [(Phe)(2)m-Me]OT, [(Phe)(2)p-Me]OT. The conformations of the oxytocin analogs were studied both in vacuum and in solution. We found some correlations between the biological activity of the considered peptides and the side-chain conformations of amino-acid residues 2 and 8.     

Oxytocin analogs with oxygen-containing side chains in position 3

We have synthesized three oxytocin analogs containing an oxygen atom in the amino acid side chain in position 3 to determine the influences of increased side chain length and of hydrophilicity on the potencies and specificities of the resulting analogs. These three analogs: [3-O-methylhomoserine] oxytocin, [3-O-ethylserine] oxytocin, and [3-O-methylthreonine] oxytocin - have the following activities in U/mg: 490, 208, 265, milk ejection; 125, 129, 63, uterus in vivo; 0.2, 16, 0.03, antidiuretic; and 0.1, 0.5, 0.1, pressor. The results show that a longer side chain, [3-O-methylhomoserine] and [3-O-ethylserine] vs. [3-O-methylthreonine], tends to increase all activities. Moving the hydrophilic oxygen farther away from the peptide backbone, on the other hand, decreases vasopressin-like activities but increases or has no effect on oxytocin-like activities.     

Fluorescent, photoaffinity, and biotinyl analogs of oxytocin

This study reports the solid phase synthesis and biological activities of two oxytocin analogs, [1-desamino, 4-lysine,7-(L-3,4,-dehydroproline)]oxytocin and [1-desamino, 4-threonine,7-(L-3,4-dehydroproline),8-lysine]oxytocin, and several fluorescent, photoaffinity, or biotinylated derivatives of these analogs and of oxytocin. The activities (in IU/mg) of the lysine-containing parent compounds, respectively, were as follows: uterus (without Mg++) 4.8 and 54; uterus (with Mg++) 19 and 440; milk ejection 65 and 414. The above analogs were coupled through the chemically reactive epsilon-amino group of lysine in position 4 or 8 or, in the case of oxytocin, through the N-terminal amino group of fluoresceine, photoaffinity, or biotinyl ligands. Fluoresceine coupled in position 1 of oxytocin gave an analog of low to moderate uterine (3.8 without Mg+ and 1.9 with Mg++) and milk ejection (7.9) activities. Analogs with biotin or fluoresceine coupled to lysine in position 4 had moderate uterine (11 and 23 without Mg++; 38 and 11 with Mg++) and milk ejection (33 and 13) activities. Analogs with fluoresceine, photoaffinity, or biotinyl labels coupled to lysine in position 8 retained good uterine (106, 62, and 147 without Mg++; 79, 78, and 509 with Mg++) and milk ejection (101, 181, and 247) activities and represent potentially useful experimental tools for studying hormone-receptor interactions and for receptor localization and isolation.     

Analysis of the dual mechanism of ACTH release by arginine vasopressin and its analogs in conscious rats

Plasma ACTH and/or corticosterone levels were measured in conscious rats 30 min after subcutaneous administration of arginine vasopressin (AVP), oxytocin (OT) and various analogs with a large range of activity on the vasopressor (V1), antidiuretic (V2) or oxytocic receptors. The comparison of their dose-response curves indicated that two different mechanisms are involved in the release of ACTH by neurohypophysial peptides and their analogs. AVP itself and a specific vasopressor agonist (Phe2, Orn8, OT) displayed a similar, high slope dose-response curve. Non-vasopressor analogs, such as dDAVP were characterized by a low slope dose-response curve. Furthermore, dDAVP potentiated CRF and neither its own ACTH-releasing action nor its potentiation of CRF were sensitive to previous VI- or V2-receptor blockade. These results, together with other available data, are interpreted as indicative of the existence of two mechanisms of action for ACTH release by AVP and its analogs in vivo: an indirect action via endogenous CRF release, mediated by a VI receptor mechanism, and a direct action on the pituitary, shared by dDAVP and other non-vasopressor analogs, with receptor characteristics different to both the V1 and the V2 classical types.     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.     

Synthesis and biological activities of oxytocin and lysine vasopressin analogs containing glutamic acid gamma-hydrazide in position 4

Solution methods, using N-hydroxysuccinimide esters, were used to synthesize [Glu(NHNH2)4] oxytocin and [Glu(NHNH2)4, Lys8] vasopressin. In these analogs of neurohypophyseal hormones, the side-chain carboxamide function of a glutamine residue is formally replaced by a hydrazide group at position 4. The hormone analogs were assayed for uterototonic activity, milk ejection activity, antidiuretic activity, and rat pressor activity. The specific biological activities of the oxytocin and vasopressin analogs were decreased compared to the respective parent hormones in all assay systems.     

The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine(6)-oxytocin and penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs.

Six [Pen(6)]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa(1)]oxytocin, [dPen(1)]oxytocin, [5-t-BuPro(7)]oxytocin, [Mpa(1), 5-t-BuPro(7)]oxytocin and [dPen(1), 5-t-BuPro(7)]oxytocin. When tested in the uterotonic test in vitro [Pen(6)]oxytocin, [Pen(6), 5-t-BuPro(7)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen(1), Pen(6)]Oxytocin and [dPen(1), Pen(6), 5-t-BuPro(7)]oxytocin were found to be pure antagonists with similarly high pA2 values of approximately 8.2. Replacement of proline by 5-tert-butylproline increased binding affinity by a factor of two in [Pen(6)]oxytocin and had no influence on the binding affinity of [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. Assignment of the proton signals for prolyl amide cis- and trans-isomers by NMR experiments in water indicated that the Pen(6)-5-tert-BuPro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5-tert-butylproline(7) analogs of [Pen(6)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. This augmentation in cis-isomer population was correlated with a 21-fold reduction in the agonistic potency and 2-fold augmentation in antagonistic potency for [Pen(6), 5-t-BuPro(7)]oxytocin relative to [Pen(6)]oxytocin. Augmentation of cis-isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa(1), Pen(6)]oxytocin to [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin. In the potent oxytocin antagonist, [dPen(1), Pen(6)]oxytocin, substitution of 5-tert-butylproline for proline augmented the cis-isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen(6)]oxytocin and [Pen(6), 5-t-BuPro(7)]oxytocin analogs 1-6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis-isomer population caused by 5-tert-butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.     

Conformationally restricted analogs of oxytocin; stabilization of inhibitory conformation

Analogs of oxytocin containing tetrahydroisoquinoline carboxylic acid (Tic) of L or D configuration in position 2 were synthesized and their biological activities were tested. Both analogs showed negligible agonist activity in uterotonic, galactogogic, and pressor assays, but they are in vitro uterotonic inhibitors. In comparison with oxytocin analogs containing L- or D-phenylalanine in position 2, the analog with the D-configuration of the conformationally fixed aromatic residue has significantly increased inhibitory activity which suggests that the proper conformation for the interaction with the receptor, but not for its activation, was stabilized. 1H NMR and CD studies, supported by theoretical calculations, suggest that the conformational properties of the analog containing D-tetrahydroisoquinoline carboxylic acid are similar to those of [2-D-phenylalanine]oxytocin.     

Synthesis and receptor binding of oxytocin analogs containing conformationally restricted amino acids

Summary We report the solid-phase synthesis and receptor-binding properties of eleven oxytocin analogs (Mpa-Xxx-Ile-Gln-Asn-Cys-Sar-Arg-Gly-NH₂) containing non-coded amino acids in position 2: D-α- and L-α-(2-indanyl)glycine, R,S-6-methoxy-2-aminotetralin-2-carboxylic acid, D- and L-pentafluorophenylalanine, D,L-2,4-dimethylphenylalanine, D,L-2,4,6-trimethylphenylalanine, R,R- and S,S-1,2,3,4-tetrahydro-1-methyl-β-carboline-3-carboxylic acid and R- and S-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid. Some of these amino acid analogs (2-indanylglycine and D-pentafluorophenylalanine) were earlier successfully applied for the synthesis of potent bradykinin antagonists [1, 2]. Their receptor bindings were tested on isolated guinea-pig uterus, rat liver and rat kidney inner medulla plasma membranes. The extent of binding of the peptides to the oxytocin receptor was in several cases was even higher than that of the parent hormone (oxytocin). However, the real pharmacological value of these analogs can be evaluated only afterin vivo measurements of their inhibition of uterine motor activity.     

Dual duplication of neurohypophysial hormones in an Australian marsupial: mesotocin, oxytocin, lysine vasopressin and arginine vasopressin in a single gland of the northern bandicoot (Isoodon macrourus)

Neurohypophysial hormones of an Australian marsupial, the Northern bandicoot (Isoodon macrourus), have been identified by their retention times in high-pressure reverse-phase liquid chromatography using two solvent systems and by their molar pressor or uterotonic activities. Two pressor peptides, arginine vasopressin and lysipressin, and two uterotonic peptides, mesotocin and oxytocin, have been characterized. Because mesotocin and arginine vasopressin have been identified in three other Australian marsupial families, it is assumed that a duplication of each ancestral gene occurred in Peramelidae and subsequent mutations in one copy led to the additional oxytocin and lysipressin. A similar dual duplication of neurohypophysial hormones has previously been discovered in the North-American opossum (Didelphis virginiana) so that the duplication propensity seems peculiar to marsupials in contrast to placental mammals.     

The use of a steric parameter (Yγ) in QSAR calculations for peptide hormones

A steric parameter (Y_γ) has been derived for α-amino acids to characterize the steric hindrances near to the α-carbon atom. This easily computable variable is suitable for quantitative structure-activity relationship (QSAR) calculations for peptide hormones. Successful multivariate analyses were performed for oxytocin and angiotensin II analogs with the help of parameters describing the lipophilic and steric properties and the measure of dispersion forces. In the case of position 4 substituted oxytocin analogs, the steric, lipophilic and H-bridge forming properties equally account for the different biological activities of the derivatives. In the case of position 5 substituted angiotensin II analogs, primarily the steric parameters account for the biological activity of the series of derivatives.     

The composition and sequence specificity of Pro-Ala-Lys-OH for the thrombolytic activities of P6A and related oligopeptides

The in vitro and in vivo thrombolytic activities of Ala-Arg-Pro-Ala-Lys-OH, its analogs and the related peptides were assayed. The results indicate that when (5)Lys of Ala-Arg-Pro-Ala-Lys-OH is changed into (5)Arg, and (3)Lys of Pro-Ala-Lys-OH is changed into (3)Arg the thrombolytic activities are collapsed; when Pro-Ala-Lys-OH is changed into Ala-Pro-Lys-OH, and Ala-Arg-Pro-Ala-Lys-OH is changed into Ala-Arg-Ala-Pro-Lys-OH the thrombolytic activities are also collapsed; when (5)Lys of Ala-Arg-Pro-Ala-Lys-OH is changed into (5)nLeu the thrombolytic activities are again collapsed. All of the results indicate that for the thrombolytic activities of Ala-Arg-Pro-Ala-Lys-OH and the related peptides Pro-Ala-Lys-OH exhibits either amino acid composition specificity or sequence specificity. The composition and sequence specificity of Pro-Ala-Lys-OH reflects its rule as the pharmacophore of P6A and the related peptides.     

Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity

The synthetic glycopeptides are interesting model systems to study the effect of O-glycosylation in modulating their function and structure. A series of glycosylated analogs of two antibacterial peptides, formaecin I and drosocin, were synthesized by varying the nature of sugar and its linkage with bioactive peptides to understand the influence of structure variation of glycosylation on their antibacterial activities. Higher antibacterial activities of all glycopeptides compared to their respective non-glycosylated counterparts emphasize in part the importance of sugar moieties in functional implications of these peptides. The consequences of the unique differences among the analogs were apparent on their antibacterial activities but not evident structurally by circular dichroism studies. We have shown that differently glycosylated peptides exhibit differential effect among each other when tested against several Gram-negative bacterial strains. The change of monosaccharide moiety and/or its anomeric configuration in formaecin I and drosocin resulted into decrease in the antibacterial activity in comparison to that of the native glycopeptide, but the extent of decrease in antibacterial activity of glycosylated drosocin analogs was less. Probably, the variation in peptide conformation arising due to topological dissimilarities among different sugars in the same peptide resulting in possible modulation in binding properties appears to be responsible for differences in their antibacterial activities. Indeed, these effects of glycosylation are found to be sequence-specific and depend in the milieu of amino acid residues. Interestingly, none of the carbohydrate variants affected the basic property of these peptides, which is non-hemolytic and non-toxicity to eukaryotic cells.     

Actions of vasopressin, oxytocin, and synthetic analogs on vascular smooth muscle

A variety of physiological, pharmacological, and experimental factors are needed to explain why different authors have recorded often confusing and contradictory results of studying blood pressure and blood flow with vasopressin, oxytocin, and their analogs. Vasopressin and a number of synthetic analogs can produce potent constriction of numerous regional arteries and arterioles (e.g., splanchnic, renal, skeletal muscle, carotid, hepatic) in near physiological (i.e., 10(-12) - 10(-10)M) concentrations. Oxytocin appears to be able to elicit much weaker constriction (threshold = 10(-7) - 10(-5)M) and can produce relaxation (i.e., vasodilatation) in the presence of high degrees of tone in skeletal muscle, hepatic, renal, and splanchnic vasculatures; this may account for its blood pressure-lowering actions. With respect to the cerebral, coronary, and pulmonary vasculatures, there appears to be no good direct evidence indicating that either vasopressin or oxytocin can elicit much in the way of constrictor actions. Oxytocin, in contrast to vasopressin, is a potent constrictor of the umbilical-placental vasculature in concentrations found at term pregnancy and during parturition. Blood vessels from animals genetically lacking vasopressin appear to be supersensitive to the contractile actions of neurohypophyseal hormones. The contractile potencies and structure-activity relationships of neurohypophyseal peptides appears to vary with the type of blood vessel; i.v. rat pressor assays do not reflect the latter and cannot be utilized to determine structure-activity relationships of these peptides on vascular smooth muscle. Either a heterogeneity of the vasopressin receptor exists in peripheral blood vessels or there are vasopressin receptor subtypes on vascular muscles. Oxytocin analogs can be designed that are extremely potent splanchnic vasoconstrictors. Further insight into these areas should provide basic information on the role of these peptides in circulatory homeostasis.     

Structure-function analysis of tritrpticin analogs: potential relationships between antimicrobial activities, model membrane interactions, and their micelle-bound NMR structures

Tritrpticin is a member of the cathelicidin family of antimicrobial peptides. Starting from its native sequence (VRRFPWWWPFLRR), eight synthetic peptide analogs were studied to investigate the roles of specific residues in its biological and structural properties. This included amidation of the C-terminus paired with substitutions of its cationic and Phe residues, as well as the Pro residues that are important for its two-turn micelle-bound structure. These analogs were determined to have a significant antimicrobial potency. In contrast, two other peptide analogs, those with the three Trp residues substituted with either Phe or Tyr residues are not highly membrane perturbing, as determined by leakage and flip-flop assays using fluorescence spectroscopy. Nevertheless the Phe analog has a high activity; this suggests an intracellular mechanism for antimicrobial activity that may be part of the overall mechanism of action of native tritrpticin as a complement to membrane perturbation. NMR experiments of these two Trp-substituted peptides showed the presence of multiple conformers. The structures of the six remaining Trp-containing analogs bound to dodecylphosphocholine micelles showed major, well-defined conformations. These peptides are membrane disruptive and show a wide range in hemolytic activity. Their micelle-bound structures either retain the typical turn-turn structure of native tritrpticin or have an extended alpha-helix. This work demonstrates that closely related antimicrobial peptides can often have remarkably altered properties with complex influences on their biological activities.     

Effect of dehydration on the endogenous opiate content of the art neuro-intermediate lobe

The relationship of endogenous opiate peptides of rat neuro-intermediate lobe to the release of neurohypophysial peptides has been investigated. Both dehydrated rats, with increased oxytocin and vasopressin release, as well as rats homozygous for hypothalamic diabetes insipidus (DI) of the Brattleboro strain, with increased oxytocin release, showed significantly decreased levels of pituitary opiate peptides. We suggest that neuro-intermediate lobe opiate peptides may modulate the release of neurohypophysial antidiuretic peptides.     

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F(6)] and [V(8)]) in the proline hinge region with other hydrophobic residues ([W(6)] and [I(8)]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR](3) and KLLKQWPIG[KLLK](3), respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.     

Structure-function analysis of tritrypticin, an antibacterial peptide of innate immune origin.

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity against Escherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and beta-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a beta-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.     

Investigating the cationic side chains of the antimicrobial peptide tritrpticin: hydrogen bonding properties govern its membrane-disruptive activities

The positively charged side chains of cationic antimicrobial peptides are generally thought to provide the initial long-range electrostatic attractive forces that guide them towards the negatively charged bacterial membranes. Peptide analogs were designed to examine the role of the four Arg side chains in the cathelicidin peptide tritrpticin (VRRFPWWWPFLRR). The analogs include several noncoded Arg and Lys derivatives that offer small variations in side chain length and methylation state. The peptides were tested for bactericidal and hemolytic activities, and their membrane insertion and permeabilization properties were characterized by leakage assays and fluorescence spectroscopy. A net charge of +5 for most of the analogs maintains their high antimicrobial activity and directs them towards preferential insertion into model bacterial membrane systems with a similar extent of burial of the Trp side chains. However the peptides exhibit significant functional differences. Analogs with methylated cationic side chains cause lower levels of membrane leakage and are associated with lower hemolytic activities, making them potentially attractive pharmaceutical candidates. Analogs containing the Arg guanidinium groups cause more membrane disruption than those containing the Lys amino groups. Peptides in the latter group with shorter side chains have increased membrane activity and conversely, elongating the Arg residue causes slightly higher membrane activity. Altogether, the potential for strong hydrogen bonding between the four positive Arg side chains with the phospholipid head groups seems to be a determinant for the membrane disruptive properties of tritrpticin and many related cationic antimicrobial peptides.