Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor

The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.     

Potent agonists of growth hormone-releasing hormone. Part I.

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr1, or N-MeTyr1. Some peptides contained one or more additional L- or D-amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28. Compared to the natural sequence of GH-RH(1-29)NH2, [Dat1,Ala15]GH-RH(1-28)Agm (MZ-3-191) and [D-Ala2,Ala15]GH-RH(1-28)Agm (MZ-3-201) were 8.2 and 7.1 times more potent in vitro, respectively. These two peptides contained Met27. Their Nle27 analogs, [Dat1,Ala15,Nle27]GH-RH(1-28)Agm(MZ-2-51), prepared previously (9), and [D-Ala2,Ala15,Nle28]GH-RH(1-28)Agm(MZ-3-195) showed relative in vitro potencies of 10.5 and 2.4, respectively. These data indicate that replacement of Met27 by Nle27 enhanced the GH-releasing activity of the analog when the molecule contained Dat1-Ala2 residues at the N-terminus, but peptides containing Tyr1-D-Ala2 in addition to Nle27 showed decreased potencies. Replacement of Ser28 with Asp in multi-substituted analogs of GH-RH(1-28)Agm resulted in a decrease in in vitro potencies compared to the parent compound. Thus, the Ser28-containing MZ-2-51, and [Dat1,Ala15,D-Lys21,Nle27]GH-RH(1-28)Agm, its Asp28 homolog (MZ-3-149), possessed relative activities of 10.5 and 5.6, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potent trypsin-resistant hGH-RH analogues.

A series of analogues of hGH-RH-(1-29)-NH2 designed to have metabolic stability has been synthesized. Standard Boc-SPPS was employed, modified to permit the guanidinylation of amino side-chains after chain assembly but before release from the resin. [Dat1, Har(11, 12, 20, 21, 29), Ala15, Nle27, Asp28]-, [Dat1, Har(11, 20, 29), Orn12, Ala15, Nle27, Asp28]-, and [Dat1, Gap(11,12, 21, 29), Ala15, Har20, Nle27, Asp28]-hGH-RH-(1-29)-NH2 were completely resistant to trypsin and about 50 times as potent as hGH-RH-(1-29)-NH2 itself when injected subcutaneously in rats. These peptides are candidates for clinical application in the therapy of GH deficiency.     

Evaluation of in vivo [corrected] biological activity of new agmatine analogs of growth hormone-releasing hormone (GH-RH)

The effects of agmatine analogs of growth hormone releasing hormone (GH-RH) were compared to GH-RH(1-29)-NH2 after intravenous (iv) and subcutaneous (sc) administration to pentobarbital-anesthetized male rats. After the iv injection, the analogs [desNH2-Tyr1,Ala15,Nle27] GH-RH(1-28)Agm (MZ-2-51); [desNH2-Tyr1,D-Lys12,Ala15,Nle27] GH-RH(1-28)Agm (MZ-2-57); [desNH2-Tyr1,Ala15,D-Lys21,Nle27] GH-RH(1-28)Agm (MZ-2-75) and [desNH2-Tyr1, D-Lys12,21, Ala15, Nle27] GH-RH(1-28)Agm (MZ-2-87) showed a potency equivalent to 4.4, 1.9, 1.07 and 1.03 times that of GH-RH (1-29)-NH2, respectively, at 5 min and 5.6, 1.8, 1.9 and 1.8 times higher, respectively, at 15 min. After sc administration, analogs MZ-2-51, MZ-2-57 and MZ-2-75 showed to be 34.3, 14.3 and 10.5 times more potent than the parent hormone at 15 min and 179.1, 88.9 and 45.0 times more active, respectively, at 30 min. In addition, MZ-2-51 had prolonged GH-releasing activity as compared to the standard. We also compared the activity of MZ-2-51 and MZ-2-57 with their homologous L-Arg and D-Arg analogs [desNH2-Tyr1,Ala15,Nle27] GH-RH(1-29)-NH2 (MZ-2-117), [des-NH2Tyr1,D-Lys12, Ala15, Nle27] GH-RH(1-29)NH2 (MZ-2-123) and [desNH2-Tyr1,D-Lys12,Ala15, Nle27,D-Arg29] GH-RH(1-29)NH2 (MZ-2-135) after intramuscular (im) injection. MZ-2-51 induced a somewhat greater GH release than MZ-2-117 at 15 min, both responses being larger than the controls (p less than 0.01) at 15 and 30 min. MZ-2-57, MZ-2-123 and MZ-2-135 given i.m. were able to stimulate GH release only at 15 minutes (p less than 0.05). Animals injected i.m. with MZ-2-51, but not with MZ-2-117, showed GH levels significantly higher than the control group (p less than 0.05) at 60 min. GH-RH(1-29)NH2 had low activity intramuscularly when tested at a dose of 2.5 micrograms. No toxic effects were observed after the iv administration of 1 mg/kg of Agm GH-RH analogs. These results indicate that our Agm analogs are active iv, sc and im and that the substitutions made in these compounds produce increased and prolonged GH releasing activity. These analogs, especially MZ-2-51, should be useful for clinical and veterinary purposes.     

Evidence for a C-terminal structural motif in gastrin and its bioactive fragments in membrane mimetic media

The conformational preferences of human little gastrin, [Nle(15)] gastrin-17, and its short analogues, gastrin-4 and [beta-Ala(1)] gastrin-5, which include the C-terminal tetrapeptide sequence Trp-Met-Asp-Phe-NH(2) crucial for gastrin bioactivity, were determined by NMR spectroscopy in aqueous solutions of zwitterionic dodecylphosphocholine micelles. Backbone HN chemical shift temperature variance, Halpha chemical shift deviations and complex non-sequential NOE patterns pointed to the C-terminal of [Nle(15)] gastrin-17 adopting an ordered conformation. Distance geometry calculations and NOE-restrained molecular dynamics simulations in membrane mimetic solvent boxes of decane and water indicated the C-terminal tetrapeptide sequence of all three peptides adopted a similar, well defined structure, with a general type IV beta-turn observed for all three peptides. The conformation of [Nle(15)] gastrin-17 consisted of two short helices between Leu(5)-Glu(9) and Ala(11)-Trp(14), with the one helix terminating in a type I beta-turn spanning Gly(13)-Asp(16). The experimental evidence and conformational characteristics of the three peptides in micellar media support a membrane-associated mechanism of receptor recognition and activation for the gastrin hormone family and furthermore point to a possible biologically relevant structural motif for gastrin activity.     

Evaluation of the biological potency of new agmatine analogs of growth hormone-releasing hormone in the bovine.

Four new growth hormone-releasing hormone (GHRH) analogs with C-terminal agmatine were compared with the parent human GHRH(1-29)NH2 fragment to assess their abilities to increase serum concentrations of growth hormone (GH) in the bovine. The four analogs were: [D-Ala2, Nle27] GHRH(1-28)Agm (JG-73); [desNH2-Tyr1, Ala15, Nle27] GHRH(1-28)Agm (MZ-2-51); [desNH2-Tyr1, Ala15, D-Lys21, Nle27] GHRH(1-28)Agm (MZ-2-75); and [desNH2-Tyr1, D-Lys12,21, Ala15, Nle27] GHRH(1-28)Agm (MZ-2-87). The special characteristic of all four GHRH analogs is that arginine was replaced by agmatine (Agm) in Position 29. Five pregnant Holstein cows received these peptides subcutaneously at the following doses: 0.0156, 0.0625, 0.25, 1, and 4 micrograms/kg body wt. Each cow received each analog-dose combination according to a 5 x 5 Greco-Latin square design repeated for the 5-week treatment. Each cow also received saline vehicle only at the end of the 5-week treatment. Blood samples were collected from 30 min before until 360 min after treatment injection. Total area under the GH response curves for the 6-hr sampling period for each dose of each GHRH analog was compared. There was a linear dose-dependent GH release in response to hGHRH(1-29)NH2 and its four GHRH(1-28)Agm analogs. At the dose of 0.25 micrograms/kg, two GHRH analogs, JG-73 and MZ-2-75, stimulated greater GH release than hGHRH(1-29)NH2 (P less than 0.05). No differences were seen at the two lowest doses, 0.0625 and 0.156 micrograms/kg. When both total area under the GH response curves and GH peak amplitudes for each treatment were averaged for all doses, JG-73 and MZ-2-75 stimulated greater GH release than hGHRH(1-29)NH2 (P less than 0.05). In summary, three GHRH(1-28)Agm analogs, JG-73, MZ-2-75, and MZ-2-51, were found to be 11.8, 11.3, and 6.5 times more potent, respectively, on a weight basis, than hGHRH(1-29)NH2 in stimulating the release of GH in cows.     

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala15 and Nle27 substitutions and one or more additional L- or D-amino acid modifications. [Dat1, Ala15, Nle27]GH-RH(1-28)Agm (MZ-2-51) was the most active analog. Its in vitro GH-releasing potency was 10.5 times higher than that of GH-RH(1-29)NH2 and in the i.v. in vivo assay, MZ-2-51 was 4-5 times more active than the standard. After s.c. administration to rats. MZ-2-51 showed an activity 34 times higher at 15 min and 179 times greater at 30 min than GH-RH(1-29)NH2 and also displayed a prolonged activity. D-Tyr10, D-Lys12, and D-Lys21 homologs of MZ-2-51 also showed enhanced activities. Thus, [Dat1, D-Tyr10, Ala15, Nle27]GH-RH(1-28)Agm (MZ-2-159), [Dat1, D-Lys12, Ala15, Nle27]GH-RH(1-28)AGM (MZ-2-57), and [Dat1, Ala15, D-Lys21, Nle27]GH-RH(1-28)Agm (MZ-2-75) were 4-6 times more active in vitro than GH-RH(1-29)NH2. In vivo, after i.v. administration, analog MZ-2-75 was equipotent and analogs MZ-2-159 and MZ-2-57 about twice as potent as the standard.(ABSTRACT TRUNCATED AT 250 WORDS)     

The parathyroid hormone-like peptide associated with humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells

[Tyr36]human adenylate cyclase stimulating peptide (1-36)-NH2, an amino-terminal analog of a tumor peptide which is associated with hypercalcemia of malignancy, and [Nle8, Nle18, Tyr34]bovine parathyroid hormone (PTH)-(1-34)-NH2 both bind with similar affinities to receptors on rat osteosarcoma cells, ROS 17/2.8, when either of the peptides is used as the radioligand. Pretreatment of the cells with either peptide down-regulates available binding sites for either radioligand and desensitizes the cAMP accumulation stimulated by either peptide. Prior exposure of the cells to dexamethasone increases these responses to both peptides. Photoderivatized radioiodinated [Tyr36]human adenylate cyclase-stimulating peptide (1-36)-NH2 and [Nle8, Nle18, Tyr34]bovine PTH-(1-34)-NH2 both specifically label a Mr = 80,000 membrane protein on ROS 17/2.8 cells. The intensity of labeling this receptor band by either photoprobe is reduced by co-incubation with either peptide over the same dose range. Equivalent dose-dependent down-regulation of receptors which bind both photoprobes is also found when ROS 17/2.8 cells are preincubated with either peptide. Dexamethasone increases the intensity of receptor labeling. Our findings strongly indicate that both peptides recognize the same plasma membrane receptor on ROS 17/2.8 cells. Although the physiological function(s) of human adenylate cyclase-stimulating peptide is unknown, these results could explain why its biological actions on mineral ion metabolism so closely simulate those of PTH and raise interesting questions about the general biological and evolutionary significance of the use of the same receptor by chemically distinct peptides.     

Dissociation of cAMP accumulation and phosphate uptake in opossum kidney (OK) cells with parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP)

Bovine parathyroid hormone (PTH) 1-34 [bPTH(1-34)] and human PTH related protein [hPTHrP(1-34)] stimulated cAMP accumulation in opossum kidney (OK) cells with Km of 5 x 10(-9) M, but inhibition of phosphate uptake was obtained with 17-fold lower Km of 3 x 10(-10) M. Phosphate uptake was partially inhibited with [Nle8.18Tyr34]bPTH(3-34)NH2 without concomitant cAMP stimulation. With hPTHrP(7-34)NH2, cAMP accumulation was increased in parallel to inhibition of phosphate uptake. [D-Trp12Tyr34]bPTH(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 had no agonist activity on cellular cAMP and inhibition of phosphate uptake. bPTH(1-34)-stimulated cAMP accumulation was antagonized by [Nle8.18Tyr34]bPTH(3-34)NH2, [D-Trp12Tyr34]bPTH(7-34)NH2, hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 with Ki of 1.4 x 10(-7), 2 x 10(-7), 4.7 x 10(-7) and 3.7 x 10(-6) M, respectively. But [Nle8.18Tyr34]bPTH(3-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2 reversed the inhibition of phosphate uptake only marginally, and hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 were inactive. With hPTHrP(1-34) the Ki for cAMP accumulation of [Nle8,18Tyr34]bPTH(3-34)NH2 and hPTHrP(7-34)NH2 were 1.9 x 10(-7) and 7.2 x 10(-7) M, and inhibition of phosphate uptake was partially reversed with [Nle8,18Tyr34]bPTH(3-34)NH2, but not with hPTHrP(7-34)NH2. The present results indicate that truncated hPTHrP(7-34)NH2, unlike [Tyr34]hPTH(7-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2, elevates cellular cAMP and inhibits phosphate uptake. bPTH(1-34)- and hPTHrP(1-34)-evoked cAMP accumulation is suppressed by PTH and PTHrP fragments while inhibition of phosphate uptake remains largely unaltered.     

Characterization of the tachykinin NK2 receptor subtype in the rabbit pulmonary artery.

Contractile responses to neurokinin A (NKA), neuropeptide gamma(NP gamma), and the NK2 receptor-selective analogs [Lys5,MeLeu9,Nle10]NKA(4-10) and MDL 28,564 were determined in the endothelium-denuded rabbit pulmonary artery. Responses to NKA, NP gamma, and [Lys5,MeLeu9,Nle10]NKA(4-10) were antagonized by the NK2 receptor antagonist MDL 29,913, with pA2 values of 6.67, 6.46, and 7.32, respectively. Autoradiographic studies failed to demonstrate any specific binding sites for [125I]-iodohistidyl NKA (INKA) over the pulmonary artery. These data suggest the presence in rabbit pulmonary artery of an unusual "nonclassical" NK2 receptor subtype, which appears to lack affinity for INKA.     

Conformationally restricted cyclic analogues of substance P: insight into the receptor binding process

Three new cyclic substance P analogues were prepared to examine the possible role of a pseudocyclic turn structure for receptor recognition. In the guinea pig isolated ileum [Cys5, Cys11]-SP5-11-NH2 and [Cys6, Cys11]-SP5-11-NH2 were inactive at concentrations up to 100 microM, while [Cys5, Cys6, Nle11]-SP was a weak agonist. The order of relative affinities on the rat brain radioreceptor assay was as follows: [Cys5, Cys6, Nle11]-SP greater than [Cys5, Cys11]-SP5-11-NH2 greater than [Cys6, Cys11]-SP5-11-NH2. We interpret these results to indicate that a pseudocyclic structure of the 5-11 sequence may not be an important factor involved in the receptor recognition of substance P.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Effects of hydrophobic substitutions at position 18 on the potency of parathyroid hormone antagonists

Position 18 in a parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2 (ii), was shown to tolerate substitutions by a range of amino acids with retention of inhibitory activity. The effects of hydrophobic substitutions at this position as a means of enhancing binding interactions with the receptor were evaluated. Substitution of Nle at position 18 with either D-Ala, D-Trp, or L-Trp in analog ii or with Trp (D or L) in the recently reported, highly potent antagonist, [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2 (in vitro activities; Kb = 15 nM and Ki = 125 nM), was performed. In terms of activity on renal receptors, one antagonist, [Nle8,D-Trp12,18,Tyr34]bPTH(7-34)NH2, is the most active in vitro PTH antagonist yet reported (Kb = 4 nM; Ki = 30 nM). The rationale for design of this antagonist and the conclusions regarding PTH-receptor interactions are discussed.     

Behavioral effects of [4-norleucine, 7-D-phenylalanine]-alpha-melanocyte-stimulating hormone

The behavioral effects of alpha-melanocyte stimulating hormone (alpha-MSH) were compared to an alpha-MSH analogue that had a norleucine substituted for methionine in the four position and a D-phenylalanine substituted for L-phenylalanine in the seven position. [Nle4, D-Phe7]-alpha-MSH has previously been shown to be a superpotent agonist on melanocytes [17]. The present experiments indicate that [Nle4, D-Phe7]-alpha-MSH is equipotent to alpha-MSH in inducing grooming when administered intraventricularly. In contrast, the analogue has the opposite effect of alpha-MSH on performance of a visual discrimination task. alpha-MSH improves visual performance whereas [Nle4, D-Phe7]-alpha-MSH attenuates such performance. The contrasting activities of [Nle4, D-Phe7]-alpha-MSH on the physiological processes described suggest that this analogue may interact with three distinct melanotropin receptors in different ways. On melanocyte receptors the melanotropin analogue is a superagonist, on CNS melanotropin receptors involved in grooming it is equipotent to alpha-MSH, and on CNS receptors involved in attention, learning and memory [Nle4, D-Phe7]-alpha-MSH may be an antagonist of endogenous melanotropin.     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Actions of several substituted short analogues of porcine galanin on isolated rat fundus strips: a structure-activity relationship.

The activity of porcine galanin (Gal) fragments and analogues were tested in vitro using rat gastric fundus strips. The peptides contracted longitudinal smooth muscle in a concentration-dependent manner with the following order of potency: [Nle4]Gal(1-15), Gal(-15), [Cle4]Gal(1-15), [Hse6]Gal(1-15), [Va14]Gal(1-15), [Ile4]Gal(1-15), [endoTrip2a, Cle4]Gal(1-15), [desThr3, Cle4]Gal(1-15), [D-Leu4] Gal(1-15), [desLeu4]Gal(1-15). On the contrary [desTrp2, Val4]Gal (1-15) remained inactive up to 10 microM. The values of Hill's coefficients estimated from the appropriate concentration-contraction curves for all analogues except for [Val4]Gal(1-15), [Hse6]Gal(1-15), [endoTrp2a,Cle4]Gal(1-15), [desLeu4]Gal(1-15) and [D-Leu4] Gal(1-15) did not significantly differ from unity. Our results indicate that the integrity of the first four N-terminal amino acids of Gal molecule is essential for the full excitatory myogenic action of the peptide in rat gastric fundus. Similarly, substitution, addition or deletion of amino acid residues in positions two, three, four and six can considerably influence the ability of Gal analogues to interact with Gal receptors. The data acquired in the course of our structure-activity study suggest that both N- and C-terminals of Gal molecule contribute towards the affinity and activity of Gal in rat gastric smooth muscle cell receptors.     

Biological activity of parathyroid hormone antagonists substituted at position 13.

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.     

Replacement of Tyr-SO3H by a p-carboxymethyl-phenylalanine in a CCK8-derivative preserves its high affinity for CCK-B receptor.

The sulfated tyrosine present in the sequence of CCK8 Asp26-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-PheNH2, seems to play a critical role in the recognition of CCK-A binding sites. In this work, we have investigated whether the presence of an anionic charge on the tyrosine moiety is strictly necessary and whether the sulfate moiety interacts with a divalent cation in the receptor subsite. For this purpose, the novel amino acids (L,D)Phe(p-CH2CO2H) and (L,D) Phe(p-CH2CONHOH), as well as their L-resolved forms were introduced into the sequence of Ac[X27, Nle28, Nle31]-CCK27-33 by solid phase method. The biological activities of these new derivatives were compared to two almost equiactive analogues of CCK8, Ac[Phe(p-CH2SO3H)27, Nle28, Nle31]-CCK27-33 and Boc[Nle28, Nle31]-CCK27-33 (BDNL) and to the nonsulfated analogue of the latter peptide (BDNL NS). All these new CCK-related analogues behave as agonists in stimulating pancreatic amylase release and display high affinity for brain binding sites (KI approximately 3-11 nM) but the only peptides which retain affinity for CCK-A receptors (KI approximately 20 nM) are those containing a p-carboxymethyl phenylalanine. Thus, introduction of this amino acid under an esterified form on the side chain, into specific and potent CCK-B agonists could allow compounds endowed with good bioavailabilities to be obtained.     

Synthesis of 153N-6 analogues and structure-function analysis at murine melanocortin-1 (MC1) receptors.

153N-6 (H-[Met5,Pro6,D-Phe7,D-Trp9,Phe10]-MSH(5-13)) has emerged as the most potent antagonist of alpha-MSH activity on Xenopus laevis melanophores, from a library of 32 360 peptides based on alpha-MSH(5-13) [22]. A recent report has confirmed our observation that 153N-6 also binds to mammalian melanocortin receptors. Here we report the receptor-binding affinities and biologic activities of 153N-6 and 17 selected alpha-MSH analogues at the native MCI receptor expressed by murine B16 melanoma cells. Our intention is to determine the structural requirements for agonism and competitive antagonism of melanocortin activity at the MC1-R and to discover more potent antagonists. 153N-6 was able to inhibit the action of native alpha-MSH and the potent synthetic agonist, [Nle4,D-Phe7]alpha-MSH, at the murine MC1-R. However, the Ki of 153N-6 was 439 times higher than that of alpha-MSH and 4475 times higher than that of [Nle4,D-Phe7]alpha-MSH; too high to allow 153N-6 to be considered as a practical antagonist for use in vivo (Ki of 153N-6 = 9.0 X 10(-6) M). Because Met4 is an important component of alpha-MSH binding at the MC1-R, we investigated alpha-MSH(1-13) and alpha-MSH(4-13) analogues to produce compounds with higher MC1-R-binding affinity than 153N-6. The binding affinity of 153N-6 was not significantly different from alpha-MSH(5-13), but it was 232 times lower than alpha-MSH(4-13). Coupling of H-Nle (as an isosteric replacement for Met) or acetyl-Nle to the N-terminus of 153N-6 raised the binding affinity by a factor of 46, but this and all full-length alpha-MSH analogues with Met or Nle in position 4 were full agonists of the MC1-R. A full-length alpha-MSH(1-13) derivative of 153N-6 with Ala4 did not exhibit significantly greater binding affinity than 153N-6 and appeared to be a partial agonist at the MC1-R in the cAMP assay. These data suggest that Met4 is an important determinant of the intrinsic efficacy of melanocortins as well as their binding affinity at the MCI-R. Pro6 and Phe10 (with respect to alpha-MSH) were found to be the most influential substitutions that determined the antagonist activity of 153N-6.