Emerging approaches in the molecular design of receptor-selective peptide ligands: conformational, topographical and dynamic considerations.
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Animals↗
Biomedical subjects
V J Hruby
Publications and source records attributed to V J Hruby.
Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes.
Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.
8-Bromo Cyclic Adenosine Monophosphate↗
The conformation of deamino-oxytocin: X-ray analysis of the 'dry' and 'wet' forms.
Two crystal structures of (1 beta-mercaptopropionic acid) deamino-oxytocin are reported. The 'dry form' in space group C2 has cell dimensions a = 27.08 +/- 0.03, b = 9.06 +/- 0.01, c = 22.98 +/- 0.02 A, beta = 102.06 +/- 0.03 with one deamino-oxytocin and six water molecules per asymmetric unit. The 'wet form' in space group P2(1) has cell dimensions a = 27.27 +/- 0.02, b = 9.04 +/- 0.01, c = 23.04 +/- 0.02 A, beta = 102.24 +/- 0.02, with two deamino-oxytocin and 13 water molecules per asymmetric unit. A local twofold parallel to the monoclinic axis gives a pseudo C2 packing. Initial phases of the 'dry form' were calculated by the heavy-atom method from the isomorphous and anomalous difference Pattersons and anomalous difference Fouier synthesis. The structure was refined by using restrained least-squares at 1.2 A resolution to a crystallographic R = 0.10. The molecular replacement method yielded the P2(1) structure that was refined with geometric restraints to R less than 0.09, by using all data to 1.09 A resolution. Deamino-oxytocin consists of a cyclic tocin ring formed by six amino acids, closed by a disulphide bridge, S1-S6, and held by two trans-annular hydrogen bonds N2-O5 and N5-O2 with a type II turn at residues 3 and 4. A flexible tripeptide tail has a loosely hydrogen-bonded type I beta-turn between N9 and O6. The sulphur of cysteine at position 1 is disordered in all the molecules leading to alternative hands of disulphide. The conformational flexibility of Ile 3, Asn 5, Pro 7 side chains and the disulphide bridge is consistent with previous models of oxytocin in which flexibility is necessary for biological activity.
Amino Acid Sequence↗
A uterotonic antagonist blocks the oxytocin-induced facilitation of female sexual receptivity.
The nonapeptide oxytocin (OXT) has been shown to facilitate female sexual receptivity when infused into the cerebral ventricles or the basal forebrain. Various selective antagonists have been used to block other behavioral effects of centrally administered OXT. In this study we compared the effects of equal doses of uterotonic, antidiuretic (V2) or vasopressor (V1) antagonists in blocking the facilitative effects of a simultaneous infusion of OXT into the basal forebrain. Ovariectomized (OVXed) animals were implanted with chronic cannulas in the basal forebrain. All animals were then given 0.5 micrograms estradiol benzoate daily for 3 days before testing. On the fourth day animals were tested to 8-10 mounts with a sexually vigorous male before and 20, 40 and 90 min after infusions of 500 ng OXT alone or in combination with a uterotonic, V2 or a V1 antagonist analogue. OXT significantly increased lordosis responding 20 and 40 min after its infusion into the medial preoptic area and anterior hypothalamus when compared to the receptivity of normal saline vehicle infused animals. The uterotonic antagonist significantly blocked the facilitation seen after OXT. The V1 and V2 antagonists at equal doses had no effect on the OXT-induced facilitation of lordosis postures. The V1 antagonist itself facilitated sexual receptivity 90 min after infusion. The facilitative effect of OXT on receptivity appears to be mediated by central uterotonic receptors, while central vasopressor receptors may serve an inhibitory role.
Animals↗
Differentiation between rat brain and mouse vas deferens delta opioid receptors.
Certain enkephalin analogues, including those which contain the conformationally restricted amino acid E-(2R,3S)-cyclopropylphenylalanine [2R,3S)-delta E Phe), have been shown to have high affinity for brain delta opioid receptors but are much less active in mouse vas deferens bioassays. To investigate whether there are differences between delta opioid receptors in brain and mouse was deferens, the ability of a selective delta opioid compound, [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE), and [D-Ala2,(2R,3S)-delta E Phe4,Leu5]enkephalin methyl ester (CP-OMe), to inhibit [3H]pCl-DPDPE binding in both rat brain and mouse vas deferens were measured. pCl-DPDPE recognized brain and mouse vas deferens binding sites with equal affinity, however, CP-OMe showed 33 fold lower affinity in mouse vas deferens compared to brain. This suggests that mouse vas deferens delta opioid receptors may be distinct from brain delta opioid receptors.
Animals↗
1H-NMR assignments and conformational studies of melanin concentrating hormone in water using two-dimensional NMR.
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Amino Acid Sequence↗
Synthesis and actions of a melanotropin conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, on melanocytes and melanoma cells in vitro.
L-Glutamic acid (gamma-4'-hydroxyanilide) (GHB) is oxidized by tyrosinase to a quinone which inhibits DNA polymerase, RNA polymerase, and mitochondrial energy production within mushrooms. It was previously shown that GHB can kill B16 melanoma cells in culture, but lacks cytotoxicity for nontyrosinase-containing cells. We have conjugated this drug to a superpotent melanotropic peptide and examined the bioactivity of this conjugate to melanoma cells. 4'-Hydroxyaniline was attached to glutamic acid at position 5 in the superpotent melanotropin fragment analogue, Ac-[Nle4, D-Phe7]alpha-MSH4-10-NH2. The melanotropin:anilide conjugate, Ac-[Nle4, Glu(gamma-4'-hydroxyanilide)5, D-Phe7]alpha-MSH4-10-NH2, was not cytotoxic to B16 or Cloudman S91 mouse melanoma cells in culture, as determined by cell counts and protein assays. Interestingly, we also found that GHB stimulated melanoma cell tyrosinase above control levels in both melanoma cell lines. In our study, GHB itself also was found not to be cytotoxic to B16 or S91 melanoma cells in culture. In the frog skin bioassay, the melanotropin conjugate was more potent than alpha-MSH or Ac-[Nle4, D-Phe7]alpha-MSH4-10 in stimulating melanosome dispersion. These results demonstrate that putative chemotherapeutic ligands can be incorporated into active-site fragment analogues of MSH without loss of biological activity.
Animals↗
Effects of conformational constraint in 2- and 8-cycloleucine analogues of oxytocin and [1-penicillamine] oxytocin examined by circular dichroism and bioassay.
The analogues of oxytocin and [1-penicillamine]oxytocin, containing a cycloleucine (Cle) residue in position 2 or 8, were investigated by means of circular dichroism measurements in different solvents, and the results examined in terms of their biological activities. A cycloleucine residue in position 2 substantially reduces the free conformational space of the hormone 20-membered ring moiety (including the disulfide group), and stabilizes a conformation which is close to one of the possible conformations of oxytocin and involves a beta-turn. In position 8, the Cle residue affects the conformation of the Tyr2 side chain, apparently forcing it away from the space above the 20-membered disulfide ring. However, it does not appear that the Cle residue has any significant effect on the overall backbone conformation of the hormone. The steric effect of the penicillamine residue in position 1 on the conformation of the disulfide group and Tyr2 side chain from previous investigations is further confirmed. The synthesis and biological potency of [1-penicillamine, 8-cycloleucine]oxytocin is described. This analogue exhibits a strong inhibitory effect on the uterotonic activity of oxytocin in vitro. It also inhibited the vasopressor response to vasopressin.
Animals↗
Modulation of glucagon actions by phorbol myristate acetate in isolated hepatocytes. Effect of hypothyroidism.
Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumulation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2+] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action.
Animals↗
[D-Pen2,D-Pen5]enkephalin analogues with increased affinity and selectivity for delta opioid receptors.
The conformationally restricted, cyclic disulfide-containing enkephalin analogue [D-Pen2,D-Pen5]enkephalin (DPDPE) was modified by halogenation (F, Cl, Br, I) of the phenylalanine-4 residue in the para position. The potency and selectivity of these analogues for the delta opioid receptor was greater than that of the parent peptide. The analogues possessed greater potency and affinity for the delta receptors than DPDPE in the mouse vas deferens assay and in radioreceptor assays (against [3H]DPDPE), respectively. [p-ClPhe4]DPDPE was the most selective in the radioligand binding assays (IC50(mu)/IC50(delta) = 574), being about 5-fold more delta opioid receptor selective than DPDPE in this assay, whereas [p-IPhe4]DPDPE was the most selective in the classical bioassay systems using the mouse vas deferens and guinea pig ileum assays (IC50(GPI)/IC50(MVD) = 17,374), making it nearly 9-fold more selective than DPDPE in direct comparisons using the same assay conditions.
Amino Acid Sequence↗
Design and synthesis of highly potent and selective cyclic dynorphin A analogues.
We have designed and synthesized several cyclic disulfide-containing peptide analogues of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogues exhibit unexpected selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogues has resulted in the kappa/mu opioid receptor ligands [Cys5,Cys11]Dyn A1-11-NH2 (1) and [Cys5,Cys11,D-Ala8]Dyn A1-11-NH2 (2), which possess high kappa and mu opioid receptor affinities centrally (guinea pig brain, GPB), but only weak activity at peripheral kappa and mu opioid receptors (guinea pig ileum, GPI). On the other hand, [Cys8,Cys13]Dyn A1-13-NH2 and [D-Cys8,D-Cys13]Dyn A1-13-NH2 (5) display high kappa potencies and selectivities at the peripheral (GPI) but not at the central (GPB) kappa opioid receptor. The lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays suggests the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral nervous systems.
Amino Acid Sequence↗
In vitro transdermal delivery of a melanotropic peptide through human skin.
A superpotent analogue of alpha-MSH, (Nle4,D-Phe7)-alpha-MSH, when applied topically to mice induces darkening of follicular melanocytes throughout the skin. In vitro studies have demonstrated delivery of the peptide across mouse but not rat skin. This variation in permeability of skin of animal models prompted us to use human skin in vitro. The melanotropin was applied to the surface of human skin samples through a permeation apparatus and allowed to penetrate for 24 h at 36 degrees C. Passage of the analogue was shown by both bioassay and radioimmunoassay. These assays correlated well and demonstrated both the presence and the biologic integrity of the peptide after transdermal passage. Regional differences were noted in the degree of transdermal penetration. In addition, split thickness skin allowed greater penetration suggesting dermal binding of the hormone. This study is the first to show that a melanotropic peptide can be delivered transdermally through human skin in vitro. This has potential importance in the development of therapies for hypopigmentary disorders and for the stimulation of skin tanning without ultraviolet light.
Administration, Cutaneous↗
Cholecystokinin analogues with high affinity and selectivity for brain membrane receptors.
A series of CCK analogues in which positions 28 and 31 have been replaced by N-methylnorleucine residues have been synthesized. It has been found that most of these N-methylnorleucine containing analogues of CCK are highly potent and some are extraordinarily selective for the central vs. peripheral receptor in two animal models (guinea pig and rat). [N-MeNle28,31]CCK26-33 nonsulfated exhibited both high potency (IC50 = 0.13 nM) and selectivity for central vs. peripheral receptors. The pancrease to brain cortex binding affinity ratio for this analogue is 5100 in the rat model. NMR studies reveal that there is cis/trans isomerism about the N-methylnorleucine residue that may be related to high selectivity.
Amino Acid Sequence↗
Design, synthesis, and biological activities of a potent and selective alpha-melanotropin antagonist.
Based on structure-activity relationships of the potent alpha-MSH agonist, Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10-NH2, several analogs of the general formula Ac-Nle4-Asp5-Waa6-Xaa7-Yaa8-Zaa9-Lys10+ ++-NH2 were synthesized and tested on frog and lizard skin bioassays for their possible inhibitory actions against alpha-MSH on melanocyte stimulation. When Waa6 = Trp, Xaa7 = D-Phe, Yaa8 = Nle and Zaa = Trp, a highly potent alpha-MSH antagonist, Ac-Nle-Asp-Trp-D-Phe-Nle-Trp-Lys-NH2, with selectivity on the frog skin alpha-MSH receptor system (pA2 = 8.4) was obtained. However, several modifications in the amino acid sequence of the peptide resulted in a complete loss of antagonistic activity and a recovery of very weak agonistic action. The following changes in the amino acid sequence of the peptide were examined; His or D-Trp for Waa, L-Phe for Xaa, Arg, Ala or Pro for Yaa, and D-Trp for Zaa. All resulted in full agonists with no antagonistic activity. In addition, lactam cyclization between the Asp5 and Lys10 side chains in the antagonist gave a full agonist and a complete loss of antagonistic activity. Efforts to develop a rational approach for the design of selective alpha-MSH antagonists for the frog skin alpha-MSH receptor will be discussed.
Amino Acid Sequence↗
Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified.
A large number of reports have demonstrated the presence of neurohypophysial hormone-like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone-like substances which demonstrate mouse mammary milk-ejection activity (ME-activity) in vitro. Peptides with ME-activity were extracted from more than 5 kg of bovine pineal glands. ME-activity containing peptides were found in both high (Mr approximately 10,000-15,000) and low (Mr approximately 500-1000) Mr species from Sephadex G-25 chromatography of 0.2 N acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone-like peptides were localized to an ultrafiltration Mr 500-1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8-arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non-mammalian nonapeptide AVT was not identified.
Amino Acid Sequence↗
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.
Animals↗
Conformational and structural considerations in oxytocin-receptor binding and biological activity.
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Animals↗
mechanism of glucagon choleresis in guinea pigs.
The present studies were carried out to clarify the mechanism of glucagon choleresis in guinea pigs. At the infusion rate of 1.4 nmol.min-1.kg-1, glucagon increased bile flow from 206.6 +/- 14.3 to 302.6 +/- 35.0 microliters.min-1.kg-1 and bicarbonate biliary concentration from 63.7 +/- 4.2 to 75.5 +/- 5.9 meq/l. Measurements of bile acid excretion in bile, the biliary tree volume, and of the hormone choleretic effect in guinea pigs with proliferated bile ductules/ducts induced by alpha-naphthylisothiocyanate feeding indicated that glucagon, unlike secretin, stimulated canalicular bile flow. Inhibition of prostaglandin synthesis by indomethacin administration (5 mg.kg-1.h-1) did not modify the choleretic effect of glucagon, and infusion of a glucagon analogue (TH-glucagon, 1.4 nmol.min-1.kg-1), which did not increase hepatic formation of adenosine 3'5'-cyclic monophosphate (cAMP), failed to stimulate bile flow. Like the parent hormone, however, TH-glucagon augmented plasma glucose levels and stimulated formation of inositol phosphates. Colchicine pretreatment (0.5 mg/kg ip) almost entirely prevented the choleretic effect of glucagon but did not modify spontaneous and bile acid-induced bile flow and the stimulatory effect of the hormone on glucose release and on hepatic formation of cAMP and inositol phosphates. Finally, glucagon produced a large increase in the biliary entry of horseradish peroxidase, even though this effect was transient and was not coupled to the increase in bile flow. These results indicate that glucagon choleresis in the guinea pig is not secondary to prostaglandin release, is canalicular in origin, involves bicarbonate secretion, is mediated by cAMP, and requires an intact microtubular system.
Animals↗