Two series of CRF antagonists with N alpha- and C alpha-methylated alanine and leucines were evaluated for their biological activities in vitro and in vivo in several systems. The poly-N-methylated analogue of alpha-helical-CRF9-41, [N alpha MeLeu10,15,27,37,N alpha MeAla22,32,41]-alpha-Hel-CRF9-41, was found to be considerably less potent than the parent non-N-methylated analogue. This result was expected on the basis that alpha-helicity was thought to be required for biological activity and the prediction that backbone substitutions on the nitrogen have a tendency to break alpha-helices (a hypothesis that was confirmed by circular dichroism). Next, a series of constrained analogues of the potent CRF antagonist, [DPhe12,Nle21,38]h/rCRF12-41, was synthesized that contained C alpha-methylleucine and/or C alpha-methylalanine (Aib) residues at selected positions. Because C alpha-methylation is recognized to increase alpha-helicity, and because there is now strong NMR data suggesting that residues 6-36 assume a well-defined alpha-helix, it was expected that these analogues would be more potent. Although usual solid-phase peptide synthesis procedures were followed, success in coupling the C alpha-methyl amino acids was obtained only with a 1:1 mixture of BOP/HOBt. In vitro potencies of the synthesized compounds were measured in a collagenase-dispersed anterior pituitary cell culture bioassay. Monosubstituted analogues were shown to be twice to one fourth as potent as the parent compound; while the pluri-substituted peptides were slightly less potent. This decrease in potency might be correlated to an unexpected lower helical content of the pluri-substituted compounds (as determined by CD spectroscopy), as it was suggested that the bioactive conformation of the CRF was predominantly alpha-helical. Interestingly, one analogue, [DPhe12,Nle21,38,C alpha-MeLeu37]h/rCRF12-41, was found to be more potent and longer acting than the parent compound in two in vivo assays measuring ACTH release after intravenous administration to adrenalectomized rats and reversal of stress-induced delay in gastric emptying in the rat after intracisternal administration. The molecular basis for this increased duration of action and potency is being investigated.
In an attempt to determine which conformational parameters are important for the biological activity of ovine corticotropin-releasing factor (oCRF), we have synthesized in significant amounts (50-200 mg) and characterized chemically, structurally (CD), and biologically, oCRF analogues with substitution of each amino acid by its corresponding D-isomer. Out of 37 of these analogues, three were found to be equipotent to, or twice as potent as, oCRF, 13 had potencies in the range from 10 to 60%, 17 had potencies ranging from 1 to 10%, and the four others had potencies less than 0.5%. None of the analogues antagonized oCRF-induced release of ACTH in vitro at concentrations > or = 1000 oCRF. Since antagonists to CRF action can be generated by deletion of the first 8-14 residues, a series of CRF antagonists which exhibit significantly higher in vitro and in vivo biological potency than [Met18,Lys23,Glu27,29,40,Ala32,41,-Leu33,3 6,38] h/rCRF, [alpha-helical-CRF9-41], is also described. [D-Phe12,Nle21,38,Arg36]h/rCRF, in particular, was found to be ca. 15 times more potent than alpha-helical-CRF9-41 in vitro. In the rat, however, this analogue was about as effective as alpha-helical-CRF9-41 in blocking CRF-induced decrease in mean arterial blood pressure and increase in heart rate. Its potency in blocking epinephrine release by CRF was not significantly different from that of alpha-helical-CRF9-41. In the adrenalectomized rat, [Lys36]alpha-helical-CRF(9-41) (1.7 mg/kg) blunted the effect of endogenous CRF over a 90-min period; by comparison, a similar dose of alpha-helical-CRF9-41 was effective for less than 1 h.
Four analogs of ovine somatostatin (SRIF, PSOMATOTROPIN RELEASE INhibiting factor), the sequence of which is H-Ala-Gly-Cys-Lys-Asn-Phe-Phe-trp-Lys-Thr-Phe-Thr-Ser-Cys-OH, have been synthesized by the solid-phase methodology. The compounds were assayed and were found to possess the following somatotropin release inhibiting potencies relative to pure synthetic somatostatin in vitro and in vivo, respectively: [Ala3,14]somatostatin, 0.6 and 2.0%; [SMe-Cys3,14]somatostatin, 4 and 0.6%; [NAc-Cys3]somatostatin, 39 and 105%; [des-Ala1-Gly2]somatostatin, 65 and 71%. The dihydrosomatostatin analogs [NAc-Cys3-H2]somatostatin and [des-Ala1Gly2-H2]somatostatin after two purifications by gel filtration were assessed to be at least 80% homogeneous and had respectively 99 and 89% of somatostatin potencied in vivo. Structure-activity relationships are discussed.
In both the rodent and primate, administration of progesterone elicits an acute surge-like release of LH in the setting of prior estrogen treatment. Whether these facilitative effects of estrogen and progesterone on gonadotropin secretion reside at pituitary or hypothalamic loci is not known. To further investigate the mechanisms by which estrogen combined with progesterone amplifies gonadotropin secretion, we studied the responses of seven estrogen-primed postmenopausal women to progesterone administration with or without cotreatment with a potent GnRH antagonist, [Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,DGlu6(AA), DAla10]GnRH. Repetitive blood sampling for the later measurement of serum concentrations of LH, FSH, and PRL was begun 4 h before the administration of progesterone and continued for 36 h. We observed that progesterone administration after 72 h of priming with ethinyl estradiol resulted in a surge-like release of LH and FSH in all subjects. Concomitant administration of the GnRH antagonist abolished the surge-like release of both gonadotropins in all subjects. In contrast, administration of the antagonist had no effect on PRL release. These results indicate that endogenous GnRH action is an obligatory component of the progesterone-induced surge-like release of both gonadotropic hormones in the estrogen-primed human.
No abstract
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.