Synthesis and biological properties of ovine corticotropin-releasing factor (CRF)

The 41-residue sequence of recently identified ovine corticotropin-releasing factor (CRF) was assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptide was purified by gel filtration and reverse-phase, medium pressure, followed by high-performance liquid chromatography (HPLC). In addition to the usual criteria, the homogeneity of the final material, obtained in 7% yield, was assessed by the isolation and examination of cyanogen bromide cleavage and tryptic digestion fragments by HPLC and amino acid analysis. The synthetic 41 amino acid CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column, the responses being related to the log-dose of CRF in the range of 0.05-125 ng/ml. The synthetic peptide also augmented in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal, as assessed by the measurement of serum corticosterone. The data indicates chemical purity and high biological activity of synthetic material.     

Distribution of corticotropin-releasing factor in ovine brain determined by radioimmunoassay

We have developed and used a sensitive and specific radioimmunoassay to demonstrate the presence of CRF-like immunoreactivity in extra-hypothalamic areas of ovine brain. Synthetic CRF displaced antibody bound tracer at an ED50 value of 200 pg and there was no cross-reactivity with LHRH, TRH, ACTH, beta-endorphin and several other peptides. Displacement of bound 125I-CRF by brain extracts exhibited curves parallel to synthetic CRF standards. Highest concentrations (1 ng/mg tissue) of CRF-like immunoreactivity were found in the median eminence but surprisingly, high concentrations of CRF-like immunoreactivity were found in frontal, parietal, occipital and particularly temporal areas of cerebral cortex. Much lower concentrations were found in other brain areas including the basal ganglia, limbic system and brain stem.     

Development of the hypothalamic-pituitary-axis in the ovine fetus: ontogeny of action of ovine corticotropin-releasing factor

In samples from twenty chronically cannulated ovine fetuses the plasma immunoreactive adrenocorticotrophin (ACTH) concentrations were 12.5 +/- 3.2(8), 15.2 +/- 4.1(9) and 21.2 +/- 5.6(8) pg/ml at periods, prior to parturition, of -30 to -35, -25 to -29 and -20 to -24 days respectively. Values are mean +/- SEM (number of samples). These values were not significantly different from each other but were significantly lower (P less than 0.02) than values in the next two age groups -36.0 +/- 4.9(7) pg/ml at -19 to -15 days, and 39.6 +/- 6.6(11) pg/ml at -14 to -9 days. A further significant increase (P less than 0.05) occurred in the -8 to -3 day period, ACTH being 53.9 +/- 5.4(12) pg/ml. On day of delivery two samples had values of 325 and 360 pg/ml. A single injection, intravenously of 1.0 microgram ovine corticotrophin-releasing factor (O-CRF), caused a significant increase in fetal plasma ACTH concentrations in fetuses of -6 to -23 days prior to delivery but not in fetuses -24 to -35 days prior to parturition. The maximum values of ACTH after O-CRF were significantly greater in fetuses -2 to 0 days prior to parturition than in younger fetuses (P less than 0.01). In 6 experiments in 4 fetuses (parturition -1 to -13 days) the effect of 1.0 microgram O-CRF persisted for at least 2.5 h. The results support the hypothesis that the pituitary release of ACTH changes sensitivity to hypothalamic O-CRF at least twice during the last fifth of gestation; an increasing sensitivity is seen as term approaches.     

The synthesis and biological properties of some 5-substituted-2'-deoxyuridines

The reaction of various nucleophiles with 5-vinyl-2'-deoxyuridine has been studied in an attempt to explain the in vitro toxicity of the compound. Attempts to synthesise pro-drugs of 5-vinyl- and 5-ethynyl-2'-deoxyuridine are described.     

Synthesis, purification and biological evaluation of porcine corticotropin-releasing factor

The 41-residue sequences of recently identified porcine corticotropin-releasing factors [Ile40]pCRF and [Asn40]pCRF were assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptides were purified by HPLC. The homogeneity of the final materials, obtained in 0.2% and 0.4% overall yield for [Ile40]pCRF and [Asn40]pCRF respectively, was assessed after the isolation by HPLC and amino acid analysis. Both sequences of the synthetic 41-residue pCRF stimulated the release of corticotropin (ACTH) from superfused rat pituitary cells on a column, the responses being related to a log-dose of CRF in the range of 1-20 ng/ml. [Ile40]pCRF and [Asn40]pCRF also augmented the in vivo release of ACTH in rats pretreated with chlorpromazine, morphine and Nembutal. [Ile40]pCRF appeared to be equipotent to ovine CRF and about twice as active as [Asn40]pCRF. The data indicate that synthetic porcine [Ile40]pCRF and [Asn40]pCRF have high biological activity.     

Solid-phase synthesis of naphthylamidines as factor VIIa/tissue factor inhibitors

Reductive amination followed by acylation of polymer-linked formyl aryl amidines generate combinatorial libraries of aryl amidines 8-13. Potent small molecule naphthylamidine inhibitors 12 (Ki<100 nM) of FVIIa/TF have been discovered and their activity against other serine proteases in the coagulation cascade is reported.     

Solid-phase synthesis and biological activities of gastrointestinal hormones: Secretin and motilin

Many successful solid-phase syntheses of peptide chains in the region of 20–40 amino acid residues have now been routinely reported. Utilizing standard solid-phase synthetic methodologies but, particularly, new and powerful purification techniques we have been developing rapid and efficient preparative routes for the numerous neuro-gastrointestinal peptides. In the present study, secretin and motilin were obtained in 16% and 10% yields, respectively, after simplified two-step purification of hydrogen fluoride-cleaved peptides by gel filtration followed by preparative high performance liquid chromatography. Peptides were essentially homogeneous by TLC and analytical high performance liquid chromatography. Secretin was found to have full biological activity when tested against a standard sample of natural material for effects on pancreatic secretion in the dog. Motilin exhibited full biological activity on interdigestive motility in the dog. Secretin has been reported to undergo rearrangement with loss of bioactivity during purification and prolonged storage. We observed no obvious problems during our abbreviated purification schedule and have found no loss of purity of peptide which has been kept for 6 months as powder lyophilized from dilute acetic acid.     

The synthesis and some biological properties of N-(6-purinyl)peptides

The chemical synthesis and biological properties of N-(6-purinyl)peptides are described. N-(6-Purinyl)amino-acid derivatives were synthesized and condensed with amino acid esters and peptide esters using the dicyclohexylcarbodiimide/N-hydroxysuccinimide method. The products were isolated via gel filtration on Sephadex G-10 in 0.05M NH4HCO3 followed by either ion exchange chromatography on SP-Sephadex or by preparative HPLC. The methyl esters were saponified and the tert-butyl ester group was removed by treatment with trifluoroacetic acid without damaging the purinyl residue. N-(6-Purinyl)peptides were characterised by chromatographic and spectroscopic methods. Acid hydrolysis of N-(6-purinyl)-L-amino acids caused the racemization of the neighbouring L-amino acid. Model studies were performed with N-(6-purinyl)-L-alanine, N-(6-purinyl)-D-alanine, N-(6-purinyl)-L-alanyl-L-leucine and N-(6-purinyl)-D-alanyl-L-leucine. After acid hydrolysis the N-(6-purinyl)amino acids were totally racemized and the N-(6-purinyl)dipeptides formed 14% of the enantiomer of alanine. The N-(6-purinyl)-omega-amino acids and the N-(6-purinyl)peptides were screened in a limited number of tests as immunomodulators (antibody-secretion, phagocytosis, cytostatic activity of macrophages) and as cytotoxic agents.     

Solid phase synthesis of ovine corticotropin releasing factor

Ovine corticotropin releasing factor was synthesized by the stepwise solid phase method under conditions anticipated to maximize yield. Final yields of fully active hormone were 34% based on peptide which could be removed from the resin and 24% on initial resin substitution.     

In vitro potentiation of the activity of synthetic ovine corticotropin-releasing factor by arginine vasopressin

The ability of arginine vasopressin (AVP) to potentiate the actions of synthetic ovine corticotropin-releasing factor (CRF) was examined using anterior pituitary fragments. Marked potentiation of ACTH release was observed upon incubating the fragments with a combination of 2 nM AVP and 1 nM CRF. Potentiation of CRF-induced ACTH release was also observed when the fragments were incubated with a combination of 1 nM AVP and 0.5 nM CRF. These results suggest that AVP may play a role in the release of ACTH from the adenohypophysis.     

Solid-phase synthesis and biological evaluation of a uridinyl branched peptide urea library

A 1000-member uridinyl branched peptide library was synthesized on PS-DES support using IRORI technology. High-throughput screening of this library for anti-tuberculosis activity identified several members with a MIC₉₀ value of 12.5 μg/mL.     

Effect of synthetic ovine corticotropin-releasing factor on growth hormone secretion in patients with acromegaly

In a significant proportion of patients with acromegaly, a non-specific increase in plasma growth hormone (GH) has been recognized following administration of thyrotropin-releasing hormone (TRH) or luteinizing hormone-releasing hormone (LH-RH), probably due to the lack of the specificity of the receptor in their tumor cells. In this study, the effects of corticotropin-releasing factor (CRF), a newly isolated hypothalamic hormone, in addition to TRH and LH-RH, on plasma levels of GH and the other anterior pituitary hormones were evaluated in 6 patients with acromegaly. Synthetic ovine CRF (1.0 microgram/kg), TRH (500 micrograms) or LH-RH (100 micrograms) was given as an iv bolus injection, in the morning after an overnight fast. Blood specimens were taken before and after injection at intervals up to 120 min, and plasma GH, adrenocorticotropin (ACTH), thyrotropin, prolactin, luteinizing hormone, follicle-stimulating hormone and cortisol were assayed by radioimmunoassays. A non-specific rise in plasma GH was demonstrated following injection of TRH and LH-RH, in 5 of 6 and 2 of 5 patients, respectively. In all subjects, rapid rises were observed in both plasma ACTH (34.3 +/- 6.2 pg/ml at 0 min to 79.5 +/- 9.5 pg/ml at 30 min, mean +/- SEM) and cortisol level (9.1 +/- 1.3 micrograms/dl at 0 min to 23.4 +/- 1.2 micrograms/dl at 90 min). However, plasma levels of GH and the other anterior pituitary hormones did not change significantly after CRF injection. These results indicate that CRF specifically stimulates ACTH secretion and any non-specific response of GH to CRF appears to be an infrequent phenomenon in this disorder.     

Adrenocorticotropin and cortisol responses to ovine corticotropin-releasing factor in alcohol dependence disorder. Preliminary report

A 100-micrograms bolus of synthetic ovine corticotropin-releasing factor was administered intravenously to 10 nondepressed inpatients suffering from an alcohol dependence disorder. The test was performed during withdrawal and after 4 weeks of abstinence. During withdrawal, the plasma cortisol responses of alcoholic patients and 7 control subjects were similar, except for an earlier decrease of cortisol in the former group. However, after 4 weeks of abstinence, the cortisol response was significantly lower in alcoholic patients than in controls. These abnormalities observed during discontinuance of alcohol consumption may reflect adaptive mechanisms of the hypothalamic-pituitary-adrenal activity which may be previously altered by chronic alcohol intoxication.     

Solid-phase parallel synthesis of azarene pyrrolidinones as factor Xa inhibitors

A focused library (4 x 14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14 polymer supported 4-amido-2,3,5,6-tetrafluorophenyl (TFP) sulfonate esters inputs. Several compounds were identified as factor Xa inhibitors (IC50< or =0.1 microM) helping to establish the SAR among these four series of azarene pyrrolidinones.     

Solid-phase synthesis and hybridization properties of DNA containing sulfide-linked dinucleosides.

Oligodeoxyribonucleotides incorporating non-hydrolyzable dialkyl sulfide linked thymidine dimers (TsT) were synthesized chemically by the solid-phase approach. The sulfide dimer TsT was stable to degradation by snake-venom phosphodiesterase, calf spleen phosphodiesterase, Nuclease P1 and Nuclease S1. Thermal denaturation analysis indicated that the incorporation of TsT dimers into DNA weakened, but did not prevent, binding to complementary DNA and RNA over a wide range of salt concentrations (10 mM to 2 M NaCl).     

Structures of ovine corticotropin-releasing factor and its Ala32 mutant as studied by CD and NMR techniques

The corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide hormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1870-76]. Here, we have determined the secondary structure of this Ala-substituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the alpha-helix formation. In contrast to an earlier report, it was observed the alpha-helical structure extends to the C-terminus of oCRF. By analyzing the CalphaH and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of pH and temperature, and the alpha-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable alpha-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF.     

Effect of protein synthesis inhibition on brain corticotropin-releasing factor and plasma adrenocorticotropin

The effect of inhibiting protein synthesis on concentrations of corticotropin-releasing factor (CRF) in rat brain and plasma adrenocorticotropin (ACTH) was assessed following the administration of the general protein synthesis inhibitor anisomycin. Compared with vehicle-injected controls, protein synthesis inhibition resulted in significantly reduced CRF immunoreactivity (CRF-ir) in median eminence within 1 h (p less than 0.01), remained decreased after 4 h (p less than 0.025), and was nonsignificantly decreased after 24 h. Plasma ACTH levels were greatly increased within 1 h posttreatment (p less than 0.0005), continued elevated after 4 h (p less than 0.01), and returned to normal levels after 24 h. CRF-ir measured in other brain areas 24 h after anisomycin showed decreased levels in medulla-pons (p less than 0.025) and neurointermediate lobe of pituitary (p less than 0.05), with no change noted in frontal cortex, hippocampus, midbrain-thalamus, or cerebellum. Overall these data show that blockade of normal protein synthesis with anisomycin can elicit changes in CRF-ir and ACTH content.     

Solid-phase synthesis and biological evaluation of a parallel library of 2,3-dihydro-1,5-benzothiazepines

Solid-phase synthesis of a parallel library of 3'-hydroxy-2,3-dihydrobenzothiazepines has been carried out through [4+3] annulation of alpha,beta-unsaturated ketones with aminothiophenol, using Wang resin as solid support. The synthesized compounds were evaluated for their potential as antibacterial, tumor inhibitors as well as acetyl- and butyrylcholinesterase inhibitors. None of the compounds showed any significant antibacterial activity. However, quite a few compounds showed significant potential as crown gall tumor inhibitors. These results reflect a strong exploratory potential in search of new benzothiazepines as source of anticancer agents. The results of the inhibition of cholinesterase revealed that benzothiazepines have a greater potential as butyrylcholinesterase inhibitors as compared to acetylcholinesterase. Moreover, the substitution of hydroxy group at C-3 in ring A led to increased activity when compared to unsubstituted- and 2'-OH substituted benzothiazepines.