The influence of albumin on vitamin D metabolism in fetal chick osteoblast-like cells

Human pancreatic growth hormone releasing factor (1-29)-amide [hpGRF (1-29)-NH2] and the following analogs: [D-Tyr-1]-hpGRF(1-29)-NH2, [D-Ala-2]-hpGRF(1-29)-NH2, [D-Asp-3]-hpGRF(1-29)-NH2, and [N-Ac-Tyr-1]-hpGRF (1-29)-NH2 were synthesized using solid phase methodology and tested for their ability to stimulate growth hormone (GH) secretion in the rat and the pig in vivo. [D-Ala-2]-hpGRF (1-29)-NH2 was approximately 50 times more potent than the parent molecule in eliciting GH secretion in the rat. The other analogs were less active, but all were more potent than the 1-29 amide in the rat. [D-Tyr-1]-hpGRF(1-29)-NH2 was 10 times more potent, [D-Asp-3]-hpGRF(1-29)-NH2 7 times more potent, and the acetylated molecule approximately 12 times more potent than hpGRF(1-29)-NH2.     

Characterisation and visualisation of [3H]dermorphin binding to mu opioid receptors in the rat brain. Combined high selectivity and affinity in a natural peptide agonist for the morphine (mu) receptor

Dermorphin, Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D-amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. 1. Dermorphin was tested for its relative affinity to mu-, delta- and chi-opioid receptors by determining its potency in displacing the selective mu-receptor ligand [3H]Tyr-DAla-Gly-MePhe-Gly-ol (where Gly-ol = glycinol), the prototypic delta-receptor ligand [3H]Tyr-DPen-Gly-Phe-DPen (where DPen = beta, beta-dimethylcysteine) and the chi ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM, 62 nM and greater than 5000 nM respectively for mu, delta and chi sites, indicating a selectivity ratio Ki(delta)/Ki(mu) = 88. Under similar conditions, Tyr-DAla-Gly-MePhe-Gly-ol, which is regarded as one of the most selective high-affinity mu-agonist available, exhibited a selectivity ratio of 84. 2. Specific binding properties of tritium-labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high-affinity binding sites (Kd = 0.46 nM; Bmax = 92 fmol/mg membrane protein). 3. Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other mu-selective radiolabeled ligands. 4. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other mu probes. Binding parameters and selectivity profile of [3H]dermorphin on slide-mounted sections were similar to those obtained with membrane homogenates. 5. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than beta-endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super-agonist towards the morphine (mu) receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Effect of Structural Changes of Pro6 in Dermorphin Molecule on Its Antinociceptive Activity

We studied effect of dermorphin (H-Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH₂) and its analogs with modified amino acid residue proline in position 6, H-Tyr-DAla-Phe-Gly-Tyr-[DPro]-Ser-NH₂, H-Tyr-DAla-Phe-Gly-Tyr-[dehydro-Pro]-Ser-NH₂, and H-Tyr-DAla-Phe-Gly-Tyr-[D-dehydro-Pro]-Ser-NH₂, on nociception in the tail-flick and hot plate tests after intraperitoneal injection. Replacement of LPro with the stereoisomer DPro as well as Pro dehydration (LdHPro) was shown to increase antinociceptive activity. Replacement of LdHPro with DdHPro cancelled the activity in the tail-flick test. All three dermorphin analogs retained antinociceptive activity in the hot plate test; however, the effect of dermorphin was more pronounced.     

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)     

Potent and prolonged melanotropic activities of the alpha-MSH fragment analog, Ac-[Nle4,D-Phe7]-alpha-MSH4-9-NH2

Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 and Ac-[Nle4]-alpha-MSH4-9-NH2, fragment analogs of the tridecapeptide, alpha-melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to alpha-MSH in several melanotropin bioassays. The D-Phe-containing hexapeptide was 10 times more active than alpha-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than alpha-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 was considerably prolonged compared to alpha-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle4, D-Phe7]-substituted analogs reside within this hexapeptide sequence.     

Importance of hydrophobic residues in alpha-human atrial natriuretic peptide (alpha-hANP) for vasorelaxant activity

To investigate the roles of the hydrophobic residues in the ANP molecule on biological activities, we synthesized a series of analogs containing various phenylalanine-homologs in position 8 or methionine-homologs in position 12. Among the analogs [pCl-Phe8]-alpha-hANP(7-28) was 4.8 times as potent as alpha-hANP(7-28) in cGMP accumulation and 3.5 times as potent in vasorelaxant activity. All the analogs showed nanomolar affinity to the receptor. In contrast, vasorelaxation and cGMP accumulation activity of the analogs ranged widely. These results suggest that these hydrophobic residues in the cyclic core are critical for vasorelaxant activity rather than for the "apparent receptor binding", and that these residues may possibly discriminate the "bioactive receptor" which is coupled to guanylate cyclase from the non-coupled receptor.     

Binding characteristics of dermorphin, and [dermorphin1-7]-beta c-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin (beta c-EP) analog in which residues 1-7 correspond to the dermorphin sequence ([Dermorphin1-7]-beta c-EP) have been investigated with respect to their receptor binding characteristics using human and camel beta-EP as reference peptides. Tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin, ethylketocyclazocine and human beta-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel beta-endorphin was the most potent peptide in all experiments. [Dermorphin1-7]-beta c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala2, D-Leu5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human beta-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine (potency relative to camel beta-EP, 1-4%) but it is more potent (9%) in competition with tritiated human beta-endorphin. Dermorphin and the [Dermorphin-1-7]-beta c-EP appear to interact preferentially with mu opiate receptors.     

Potent GnRH agonists containing L-amino acid derivatives in the six position

New agonists related to gonadotropin-releasing hormone (GnRH) have been synthesized that are comparable in potency to the GnRH and its superagonists for release of LH and estrus suppression without substitutions with D- or unnatural amino acids in position 6. We now report a series of L-beta-aspartyl-6 GnRH analogs containing only naturally occurring L-amino acids in the whole sequence, exhibiting considerable in vivo biological activity. Dose and time dependent LH release capability of the different analogs in adult male mice, estrus suppression comparisons and blockade of ovulation in female rats are given. The incorporation of L-Asp-OMe and L-Asp-OBzl in position 6 of GnRH resulted in the most potent GnRH agonists (to 12-20xGnRH potency) in this series inducing a biphasic biological response similar to the D-amino acid-6 substituted superactive GnRH analogs. A correlation between the LH releasing potencies of the analogs and their HPLC retention times was also investigated. Peptide synthesis were achieved using either solid phase or solution phase methodology.     

Structural requirements for dermorphin opioid receptor binding

Structural features influencing binding activity of dermorphin to opioid receptors have been investigated in the rat brain through the synthesis and evaluation of binding affinity of a series of synthetic dermorphin analogs. Tritiated dermorphin was used as primary ligand. The single population of high affinity dermorphin binding sites present in the rat brain is clearly of an opioid nature since bound radiolabeled dermorphin was fully displaced with high affinity either by morphine or naloxone. Displacement of tritiated dermorphin by all alkaloid opiates or dermorphin related peptides tested was monophasic, consistent with simple competitive inhibition at a single population of binding sites. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) was the most potent competitor in all experiments. The D-configuration of the amino acid residue in position 2 was found to be of crucial importance for binding. Replacement of D-Ala2 with L-Ala led to a deleterious effect, this analog being 1/5000th as potent as dermorphin in displacing bound tritiated dermorphin from its receptor. Shorter dermorphin homologs, dermorphin-(1-4)-NH2 and dermorphin-(1-3)-NH2, were found to be 20 and 40-fold less potent, respectively, than dermorphin. The C-terminal carboxamide function is of significant importance for manifestation of the full intrinsic binding potency of dermorphin. Deamidated dermorphin had 1/5th the potency of the parent peptide. This suggests that while the whole dermorphin sequence is required for the expression of the full intrinsic binding activity of the molecule, the N-terminal tripeptide is a key structure as it contains the features which allow receptor recognition.     

Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine

Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure-activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala² residue with d-Arg² makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg² have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg² may provide a new target molecule for developing novel analgesics that have fewer side effects.     

Synthesis and antidiabetic activity of 2,5-disubstituted-3-imidazol-2-yl-pyrrolo[2,3-b]pyridines and thieno[2,3-b]pyridines

In the present investigation, two series of 2,5-disubstituted-3-imidazol-2-yl-pyrrolo[2,3-b]pyridines (2a-l) and thieno[2,3-b]pyridines (3a-l) were designed as analogs of BL 11282 (1). The in vitro glucose dependent insulinotropic activity of all the test compounds was evaluated using RIN5F cell based assay and all the test compounds showed glucose and concentration dependent insulin secretion. The in vivo antidiabetic activities of most potent compounds from each series (2c and 3c) were assessed in C57BL/6J mice. Compounds 2c and 3c showed dose dependent insulin secretion and significant glucose reduction in vivo. In general, compounds 2c and 3c were found to be equipotent at all the three different doses selected and with respect to BL 11282, both the test compounds were found to be more potent, at all the time points.     

Tumor-targeting peptide-PNA-peptide chimeras for imaging overexpressed oncogene mRNAs

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [99sTc] chelator-PNA-D (CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.     

Position 4 substituted somatostatin analogs: increased binding to somatostatin receptors in pituitary and brain

Somatostatin (S-14) analogs with Phe4 substitutions bound to pituitary and cerebrocortical S-14 receptors more avidly than did S-14. The 2-4 fold greater affinities of the Phe4 S-14 as well as analogs with structural modification of the Phe4 residue for binding to pituitary S-14 receptors showed good correlation with their reported potencies for in vivo Gh inhibition. In the cerebral cortex, [Phe4] S-14, [Phe4, D-Trp8] S-14 and [F5-Phe4] S14 were 2-3 times more potent while [p-NH2-Phe4] S-14 was 6 times more potent compared to S-14 in binding to S-14 receptors. The increased binding affinities of the Phe4 analogs in these two tissues does not appear to be due to differential stability of the analogs compared to S-14 under the experimental conditions used. [Thr4] S-14 exhibited very low binding in both these tissues. Thus structural modification of the position 4 moiety of the S-14 molecule does not result in dissociated affinities for binding to S-14 receptors in the brain and the pituitary. The increased receptor binding affinities of the Phe4 analogs in the cerebral cortex suggest that they may be more potent than S-14 in the CNS.     

Peptide N-alkylamides by solid phase synthesis

Three new resins have been developed that allow for the solid phase synthesis of C-terminal peptide N-alkylamides using Boc amino acids, usual side chain protecting groups and hydrogen fluoride cleavage and deprotection. These resins were prepared by reacting the appropriate alkylamine (NH2CH3, NH2CH2CH3, NH2CH2CF3) to Merrifield's 1% divinylbenzene cross-linked chloromethylated polystyrene resin. The application of these resins to the synthesis of C-terminal GnRH N-alkylamides illustrates the versatility of this approach. GnRH analogs were tested for their ability to release LH from cultured rat anterior pituitary cells. [DGlu6, Pro9-NHCH2CH3]-GnRH was synthesized for the first time using the solid phase approach and found to be three times more potent than [DGlu6]-GnRH. Other analogs including [DTrp6, Pro9-NHCH2CH3]-GnRH, [DAla6, Pro9-NHCH2CF3]-GnRH and related peptides were found to be equipotent and to have the same properties (HPLC retention times, amino acid analysis and specific rotation) as the corresponding peptides synthesized using less amenable strategies; yields were equivalent or better than those reported earlier.     

Synthesis and biological activity of carboxyl terminally extended dermorphins

Dermorphinoyl(DMR)-glycine, DMR-sarcosine and DMR-glycyl-arginine have been prepared in order to examine the effect of C-terminal extension of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) on opioid activity. On GPI preparation the addition of Gly, Sar, or Gly-Arg to the carboxyl terminus of dermorphinoic acid was detrimental to mu activity: dermorphinoyl-derivatives, in fact, retain only 5-20% of dermorphin potency. Following intracerebroventricular administration (tail-flick test), whereas the analgesic activities of compounds showed the trend dermorphin greater than DMR-Sar greater than DMR-Gly-Arg greater than DMR-Gly greater than morphine, the nonapeptide displayed highest activity after subcutaneous injection in mice: DMR-Gly-Arg was 2.5 and 10 times more potent than dermorphin and morphine, respectively.     

Comparison of biologic activities of synthetic lipopentapeptide analogs of bacterial lipoprotein in mice

Mitogenicity, lethal toxicity, induction of tumor necrotizing factor (TNF), and antitumor activity against Meth A fibrosarcoma of four chemically synthesized lipopentapeptide analogs, S-[2,3-bis(palmitoyloxy)-2R (designated as KAB-1), -2S(KAB-3)-propyl]-N-palmitoyl-(R)-cysteinyl-(S)-seryl- (S)-seryl-(S)-asparaginyl-(S)-alanine, S-[2,3-bis(palmitoyloxy)-2R(KAB-2), and -2S(KAB-4)-propyl]-N-[(2,2,2)-trichloroethoxycarbonyl]-(R)- cysteinyl-(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine, of bacterial lipoprotein were investigated. These four analogs, as well as bacterial lipopolysaccharide (LPS) or synthetic Escherichia coli-type lipid A (506), were capable of increasing of [3H]thymidine into splenocytes of C3H/He mice. Although LPS and 506 did not exhibit the mitogenic activity in C3H/HeJ mice, KAB compounds showed remarkable mitogenicity. These analogs did not show the lethal toxicity at a high dose of 50 micrograms/mouse in galactosamine-loaded C57BL/6 mice. Peritoneal macrophages, stimulated with four analogs, caused the production of TNF which induces the L929 cell lysis in vitro. Twice, intravenous injections of 50 micrograms/mouse of these analogs showed weak growth inhibition of Meth A fibrosarcoma in BALB/c mice. The inhibitory effect of KAB-2 compound, which caused the strong TNF-induction among the four analogs, was the most potent. These results indicate that the biological activity of KAB-2 (R-configuration of the C-2 position in glycerol moiety with dipalmitoyl) is stronger than that of the other three analogs.     

TUMOR-TARGETING PEPTIDE-PNA-PEPTIDE CHIMERAS FOR IMAGING OVEREXPRESSED ONCOGENE mRNAS

We have optimized a method involving continuous solid phase synthesis of chelator-peptide-PNA-peptide probes in order to noninvasively image oncogene mRNAs overexpressed in tumors. The PNA (peptide nucleic acid) probes carry cyclized peptide ligand analogs specific for receptors overexpressed on malignant breast or colorectal cancer cells, and chelators to bind radioactive metal ions, or a fluorophore. In vivo scintigraphic imaging of MCF7 xenografts in immunocompromised mice indicated that CCND1 and MYC [^99mTc]chelator-PNA-D(CSKC) probes concentrated in MCF7 cells up to 7 times more than the corresponding mismatch controls.     

Antibiotic activity and structural analysis of the scorpion-derived antimicrobial peptide IsCT and its analogs

IsCT is a non-cell-selective antimicrobial peptide isolated from the scorpion Opisthacanthus madagascariensis that has potent cytolytic activity against both mammalian and bacterial cells. To investigate the structure-activity relationships of IsCT and to design novel peptide antibiotics with bacterial cell selectivity, we synthesized several analogs of IsCT and determined their three-dimensional structures in solution by 2D-NMR spectroscopy. IsCT has a linear alpha-helical structure from Gly3 to Phe13, and [K7]-IsCT has a linear alpha-helical structure from Leu2 to Phe13. [K7, P8, K11]-IsCT, which has a bend in its middle region, exhibited the highest antibacterial activity without hemolytic activity, suggesting that its proline-induced bend is an important determinant of this selectivity. Tryptophan fluorescence showed that the high selectivity of [K7, P8, K11]-IsCT toward bacterial cells is closely correlated with its highly selective interaction with negatively charged phospholipids. Its potent activity against antibiotic-resistant bacteria suggests that [K7, P8, K11]-IsCT may serve as a promising lead candidate in the development of new peptide antibiotics.