Observation of Anomalous Internal Pair Creation in

Electron-positron angular correlations were measured for the isovector magnetic dipole 17.6 MeV (J^{π}=1^{+}, T=1) state→ground state (J^{π}=0^{+}, T=0) and the isoscalar magnetic dipole 18.15 MeV (J^{π}=1^{+}, T=0) state→ground state transitions in ^{8}Be. Significant enhancement relative to the internal pair creation was observed at large angles in the angular correlation for the isoscalar transition with a confidence level of >5σ. This observation could possibly be due to nuclear reaction interference effects or might indicate that, in an intermediate step, a neutral isoscalar particle with a mass of 16.70±0.35(stat)±0.5(syst) MeV/c^{2} and J^{π}=1^{+} was created.

Observation of low- and high-energy Gamow-Teller phonon excitations in nuclei.

Gamow-Teller (GT) transitions in atomic nuclei are sensitive to both nuclear shell structure and effective residual interactions. The nuclear GT excitations were studied for the mass number A = 42, 46, 50, and 54 "f-shell" nuclei in ((3)He, t) charge-exchange reactions. In the (42)Ca → (42)Sc reaction, most of the GT strength is concentrated in the lowest excited state at 0.6 MeV, suggesting the existence of a low-energy GT phonon excitation. As A increases, a high-energy GT phonon excitation develops in the 6-11 MeV region. In the (54)Fe → (54)Co reaction, the high-energy GT phonon excitation mainly carries the GT strength. The existence of these two GT phonon excitations are attributed to the 2 fermionic degrees of freedom in nuclei.

Reactor decay heat in 239Pu: solving the γ discrepancy in the 4-3000-s cooling period.

The ß feeding probability of (102,104,105,106,107)Tc, 105Mo, and 101Nb nuclei, which are important contributors to the decay heat in nuclear reactors, has been measured using the total absorption technique. We have coupled for the first time a total absorption spectrometer to a Penning trap in order to obtain sources of very high isobaric purity. Our results solve a significant part of a long-standing discrepancy in the γ component of the decay heat for 239Pu in the 4-3000 s range.

Potent and long-acting corticotropin releasing factor (CRF) receptor 2 selective peptide competitive antagonists.

We present evidence that members of the corticotropin releasing factor (CRF) family assume distinct structures when interacting with the CRF(1) and CRF(2) receptors. Predictive methods, physicochemical measurements, and structure-activity relationship studies have suggested that CRF, its family members, and competitive antagonists such as astressin [cyclo(30-33)[DPhe(12),Nle(21),Glu(30),Lys(33),Nle(38)]hCRF((12-41))] assume an alpha-helical conformation when interacting with their receptors. We had shown that alpha-helical CRF((9-41)) and sauvagine showed some selectivity for CRF receptors other than that responsible for ACTH secretion(1) and later for CRF2.(2) More recently, we suggested the possibility of a helix-turn-helix motif around a turn encompassing residues 30-33(3) that would confer high affinity for both CRF(1) and CRF(2)(2,4) in agonists and antagonists of all members of the CRF family.(3) On the other hand, the substitutions that conferred ca. 100-fold CRF(2) selectivity to the antagonist antisauvagine-30 [[DPhe(11),His(12)]sauvagine((11-40))] did not confer such property to the corresponding N-terminally extended agonists. We find here that a Glu(32)-Lys(35) side chain to side chain covalent lactam constraint in hCRF and the corresponding Glu(31)-Lys(34) side chain to side chain covalent lactam constraint in sauvagine yield potent ligands that are selective for CRF(2). Additionally, we introduced deletions and substitutions known to increase duration of action to yield antagonists such as cyclo(31-34)[DPhe(11),His(12),C(alpha)MeLeu(13,39),Nle(17),Glu(31),Lys(34)]Ac-sauvagine((8-40)) (astressin(2)-B) with CRF(2) selectivities greater than 100-fold. CRF receptor autoradiography was performed in rat tissue known to express CRF(2) and CRF(1) in order to confirm that astressin(2)-B could indeed bind to established CRF(2) but not CRF(1) receptor-expressing tissues. Extended duration of action of astressin(2)-B vs that of antisauvagine-30 is demonstrated in the CRF(2)-mediated animal model whereby the inhibition of gastric emptying of a solid meal in mice by urocortin administered intraperitoneally at time zero is antagonized by the administration of astressin(2)-B but not by antisauvagine-30 at times -3 and -6 h while both peptides are effective when given 10 min before urocortin.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor.

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Potent somatostatin undecapeptide agonists selective for somatostatin receptor 1 (sst1).

A family of analogues of des-AA(1,2,5)-[DTrp(8)/D2Nal(8)]-SRIF that contain a 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 was identified that has high affinity and selectivity for human somatostatin receptor subtype 1 (sst1). The binding affinities of des-AA(1,2,5)-[DTrp(8),IAmp(9)]-SRIF (c[H-Cys-Lys-Phe-Phe-DTrp-IAmp-Thr-Phe-Thr-Ser-Cys-OH], CH-275) (7), des-AA(1,5)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (CH-288) (16), des-AA(1,2,5)-[Tyr(7),DTrp(8),IAmp(9)]-SRIF (23), and des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-SRIF (25) are about (1)/(7), (1)/(4), (1)/(125), and (1)/(4) that of SRIF-28 (1) to sst1, respectively, about (1)/(65), (1)/(130), <(1)/(1000), and <(1)/(150) that of 1 to sst3, respectively, and about or less than (1)/(1000) that of 1 to the other three human SRIF receptor subtypes. A substitution of DTrp(8) by D2Nal(8) in 7 to yield des-AA(1,2,5)-[D2Nal(8),IAmp(9)]-SRIF (13) and in 16 to yield des-AA(1,5)-[Tyr(2),D2Nal(8),IAmp(9)]-SRIF (17) was intended to increase chemical stability, selectivity, and affinity and resulted in two analogues that were less potent or equipotent with similar selectivity, respectively. Carbamoylation of the N-terminus as in des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) increased affinity slightly as well as improved selectivity. Monoiodination of 25 to yield 26 and of 27 to yield 28 resulted in an additional 4-fold increase in affinity at sst1. Desamination of the N-terminus of 17 to yield 18, on the other hand, resulted in significant loss of affinity. Attempts at reducing the size of the ring with maintenance of selectivity failed in that des-AA(1,4,5,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (33) and des-AA(1,4,5,6,12,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (34) progressively lost affinity for all receptors. Both des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) and des-AA(1,2,5)-[DCys(3),DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (29) show agonistic activity in a cAMP assay; therefore, the structural basis for the agonist property of this family of analogues is not contingent upon the chirality of the Cys residue at position 3 as shown to be the case in 18-membered ring SRIF octapeptides. None of the high affinity structures described here showed receptor antagonism. We have prepared the radiolabeled des-AA(1,2,5)-[DTrp(8),IAmp(9),(125)ITyr(11)]-SRIF ((125)I-25) and des-AA(1,2,5)-[DTrp(8),IAmp(9), (125)ITyr(11)]-Cbm-SRIF ((125)I-27), used them as in vitro tracers, and found them to be superior to des-AA(1,5)-[(125)ITyr(2),DTrp(8),IAmp(9)]-SRIF ((125)I-16) for the detection of sst1 tumors in receptor autoradiography studies.

Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors.

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)(+) RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 microg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Constrained corticotropin-releasing factor (CRF) agonists and antagonists with i-(i+3) Glu-Xaa-DXbb-Lys bridges.

We hypothesized that covalent constraints such as side-chain to side-chain lactam rings would stabilize an alpha-helical conformation shown to be important for the recognition and binding of the human corticotropin-releasing factor (hCRF) C-terminal 33 residues to CRF receptors. These studies led to the discovery of cyclo(20-23)[DPhe12,Glu20,Lys23,Nle21,38]hCRF (12-41) and of astressin ¿cyclo(30-33)[DPhe12,Nle21,38,Glu30,Lys33]hCR F(12-41)¿, two potent CRF antagonists, and of cyclo(30-33)[Ac-Leu8,DPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(8-41), the shortest sequence equipotent to CRF reported to date (Rivier et al. J. Med. Chem. 1998, 41, 2614-2620 and references therein). To test the hypothesis that the Glu20-Lys23 and Glu30-Lys33 lactam rings were favoring an alpha-helical conformation rather than a turn, we introduced a D-amino acid at positions 22, 31, and 32 in the respective rings. Whereas the introduction of a D-residue at position 31 was only marginally deleterious to potency (ca. 2-fold decrease in potency), introduction of a D-residue at position 22 and/or 32 was favorable (up to 2-fold increase in potency) in most of the cyclic hCRF, alpha-helical CRF, urotensin, and urocortin agonists and antagonists that were tested and was also favorable in linear agonists but not in linear antagonists; this suggested a unique and stabilizing role for the lactam ring. Introduction of a [DHis32] (6) or acetylation of the N-terminus (7) of astressin had a minor deleterious or a favorable influence, respectively, on duration of action. In the absence of structural data on these analogues, we conducted molecular modeling on an Ac-Ala13-NH2 scaffold in order to quantify the structural influence of specific L- and DAla6 and L- and DAla7 substitutions in [Glu5,Lys8]Ac-Ala13-NH2 in a standard alpha-helical configuration. Models of the general form [Glu5,LAla6 or DAla6,LAla7 or DAla7,Lys8]Ac-Ala13-NH2 were subjected to high-temperature molecular dynamics followed by annealing dynamics and minimization in a conformational search. A gentle restraint was applied to the 0-4, 1-5, and 8-12 O-H hydrogen bond donor-acceptor pairs to maintain alpha-helical features at the N- and C-termini. From these studies we derived a model in which the helical N- and C-termini of hCRF form a helix-turn-helix motif around a turn centered at residue 31. Such a turn brings Gln26 in close enough proximity to Lys36 to suggest introduction of a bridge between them. We synthesized dicyclo(26-36,30-33)[DPhe12,Nle21,Cys26,Glu30 ,Lys33,Cys36, Nle38]Ac-hCRF(9-41) which showed significant alpha-helical content using circular dichroism (CD) and had low, but measurable potency ¿0. 3% that of 6 or ca. 25% that of [DPhe12,Nle21,38]hCRF(12-41)¿. Since the 26-36 disulfide bridge is incompatible with a continuous alpha-helix, the postulate of a turn starting at residue 31 will need to be further documented.

Astressin analogues (corticotropin-releasing factor antagonists) with extended duration of action in the rat.

In earlier reports we identified specific point substitutions (DPhe12,Nle21,38), cyclization strategies [in particular, introduction of lactam rings such as that of cyclo(Glu30,Lys33)], and deletions (residues 1-7) in the CRF molecule that led to agonists. We also noted that further deletions (residues 8-14) produced antagonists such as astressin ¿cyclo(30-33)[DPhe12,Nle21,38, Glu30, Lys33]hCRF(12-41)¿ (1). We hypothesized that the lactam ring promoted conformational stability to yield analogues with increased potency both in vitro and in vivo as compared to that of their linear counterparts. Additionally, we reported that cyclo(30-33)[DPhe12,Nle21,38, Glu30,DHis32,Lys33]hCRF(12-41) (3) and dicyclo(26-36,30-33)[Ac-Asp9,DPhe12,Nle21,38, Cys26, Glu30,Lys33, Cys36]hCRF(9-41) were ca. twice and 1/100 as potent as astressin, respectively, suggesting a putative turn that encompasses residues 30-33 (previous paper: Koerber et al. J. Med. Chem. 1998, 41). To increase the potency of 1 and/or 3 in vivo, we extended their chain length by one (5-8), two (9, 10), and three (11, 12) residues at the N-terminus and acetylated (6, 8, 10, 12). Of the compounds tested for duration of action (1, 3-6, 8), we found 6 and 8 to be slightly longer-acting than astressin or [DHis32]astressin, while their potencies in vitro were not significantly different from that of 3. Additionally, we introduced CalphaMe-leucine residues in lieu of leucine at positions 14, 15, 19, 27, and 37 in [DHis32]astressin. The analogue [CalphaMe-Leu27,DHis32]astressin (16) was more potent (although not statistically in all cases) than the other four analogues in vitro. While acetylation of the N-terminus of 16 (i.e., 18) or of [CalphaMe-Leu27]astressin (i.e., 19) did not have a significant effect on in vitro potency, elongation of the N-terminus by one or three residues in addition to acetylation resulted in cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27,Glu3 0,DHis32,Lys33, Nle38]Ac-hCRF(11-41) (21), cyclo(30-33)[DPhe12,Nle21,CalphaMe-Leu27, Glu30,Lys33,Nle38]Ac-hCRF(9-41) (22), and cyclo(30-33)[DPhe12, Nle21, CalphaMe-Leu27,Glu30,DHis32,Lys33,Nle38 ]Ac-hCRF(9-41) (23) that were longer-acting than 6 and 8 (ca. 2 h inhibition of ACTH secretion at 25 micrograms/adrenalectomized rat). Analogues 22 and 23 were also more potent than astressin at reversing intracisternal CRF- and abdominal surgery-induced delay of gastric emptying in conscious rats.

Minimal-size, constrained corticotropin-releasing factor agonists with i-(i+3) Glu-Lys and Lys-Glu bridges.

In three earlier publications (Miranda et al. J. Med. Chem. 1994, 37, 1450-1459; 1997, 40, 3651-3658; Gulyas et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10575-10579) we have hypothesized that covalent constraints such as side-chain-to-side-chain lactam rings would stabilize an alpha-helical conformation shown to be important for the recognition and binding of the CRF C-terminus 30 residues, to CRF receptors. These studies led to the discovery of useful CRF antagonists such as alpha-helical CRF (alpha-hel-CRF) and Astressin both in vitro and in vivo. To test the hypothesis that such lactam rings may also be modulating activation of the receptor when introduced at the N-terminus of CRF, we studied the influence of the successive introduction from residues 4 to 14 of a cyclo(i, i+3)[Lysi-Glu(i+3)] and a cyclo(i,i+3)[Glui-Lys(i+3)] bridge on the in vitro potency of the agonist [Ac-Pro4,dPhe12,Nle21,38]hCRF(4-41) and related compounds. We have also introduced the favored cyclo(Glu30-Lys33) substitution found to be remarkable in several families of antagonists (such as Astressin) and in a number of CRF agonists and investigated the role of residues 4-8 on receptor activation using successive deletions. Earlier studies had shown that in both oCRF and alpha-helical CRF, deletion of residues 1-6, 1-7, and 1-8 led to gradual loss of intrinsic activity (IA) (from 50% IA to <10% IA) resulting in alpha-hel-CRF being a potent competitive antagonist. We show that acetylation of the N-terminus of these fragments generally increases potency by a factor of 2-3 with no influence on IA. While cyclo(30-33)[Ac-Leu8,dPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(8-41) (30) is the shortest reported analogue of CRF to be equipotent to CRF (70% IA), the corresponding linear analogue (31) is 120 times less potent (59% IA). Addition of one amino acid at the N-terminus ¿cyclo(30-33)[Ac-Ser7,dPhe12,Nle21, Glu30,Lys33,Nle38]hCRF(7-41) (28)¿ results in a 5-fold increase in agonist potency and full intrinsic activity (113%). The most favored modifications were also introduced in other members of the CRF family including sauvagine (Sau), urotensin (Utn), urocortin (Ucn), and alpha-hel-CRF. Parallel and consistent results were obtained suggesting that the lactam cyclization at residues 29-32 and 30-33 (for the members of the CRF family with 40 and 41 amino acid residues, respectively) will induce (in the shortened agonists) a structural constraint (alpha-helix) that stabilizes a bioactive conformation similar to that shown in the Astressin family of CRF antagonists and that residue 8 (leucine or isoleucine) bears the sole responsibility for activation of the receptor since deletion of that residue leads to potent antagonists (Gulyas et al. Proc. Natl. Acad.Sci. U.S.A. 1995, 92, 10575-10579).

Inactivation of a serotonin-gated ion channel by a polypeptide toxin from marine snails.

The venom of predatory marine snails is a rich source of natural products that act on specific receptors and ion channels within the mammalian nervous system. A 41-amino acid peptide, final sigma-conotoxin GVIIIA, was purified on the basis of its ability to inactivate the 5-HT3 receptor, an excitatory serotonin-gated ion channel. final sigma-Conotoxin contains a brominated tryptophan residue, which may be important for peptide activity because the endogenous ligand for the 5-HT3 receptor is a hydroxylated derivative of tryptophan. final sigma-Conotoxin inactivates the 5-HT3 receptor through competitive antagonism and is a highly selective inhibitor of this receptor. Serotonin receptors can now be included among the molecular targets of natural polypeptide neurotoxins.

Constrained corticotropin-releasing factor antagonists with i-(i + 3) Glu-Lys bridges.

Hypothesis driven and systematic structure-activity relationship (SAR) investigations have resulted in the development of effective central nervous system (CNS) antagonists of corticotropin (ACTH)-releasing factor (CRF) such as alpha-helical CRF(9-41) and analogues of our assay standard [DPhe12,Nle21,38]hCRF(12-41). On the other hand, equally potent CRF antagonists that block the hypothalamic/pituitary/adrenal (HPA) axis had not been described until recently. Predictive methods, physicochemical measurements (nuclear magnetic resonance spectrometry and circular dichroism spectroscopy), and SAR studies suggest that CRF and its family members (urotensins and sauvagine) assume an alpha-helical conformation when interacting with CRF receptors. To further test this hypothesis, we have systematically scanned the hCRF(9-41) or hCRF(12-41) sequences with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown could maintain or enhance alpha-helical structure. From this series we have identified seven analogues that are either equipotent to, or 3 times more potent than, the assay standard; in addition, as presented earlier cyclo(30-33)[DPhe12,-Nle21,38,Glu30, Lys33]hCRF(12-41) (astressin) is 32 times more potent than the assay standard in blocking ACTH secretion in vitro (rat pituitary cell culture assay). In vivo, astressin is also significantly more potent than earlier antagonists at reducing hypophysial ACTH secretion in intact stressed or adrenalectomized rats. Since the corresponding linear analogues that were tested are significantly less potent, our interpretation of the increased potency of the cyclic analogues is that the introduction of the side chain to side chain bridging element (Glu30-Lys33, and to a lesser extent that of Glu14-Lys17, Glu20-Lys23, Glu23-Lys26, Glu26-Lys29, Glu28-Lys31, Glu29-Lys32, and Glu33-Lys36) induces and stabilizes in the receptor environment a putative alpha-helical bioactive conformation of the fragment that is not otherwise heavily represented. The effect of the introduction of two favored substitutions [(cyclo(20-23) and cyclo(30-33)] yielded 37 with a potency 8 times that of the assay standard but actually 12 times less than expected if the effect of the two cycles had been multiplicative. These results suggest that the pituitary CRF receptor can discriminate between slightly different identifiable conformations, dramatically illustrating the role that secondary and tertiary structures play in modulating biological signaling through specific protein-ligand interactions.

Bromocontryphan: post-translational bromination of tryptophan.

We demonstrate that post-translational bromination of a tryptophan residue occurs in the biologically active octapeptide bromocontryphan, purified and characterized from Conus radiatus venom. Clones encoding bromocontryphan were identified from a cDNA library made from C. radiatus venom ducts. The mRNA sequence obtained predicts a prepropeptide which has the mature peptide sequence at the C-terminal end, with the L-6-bromotryptophan residue encoded by UGG, the Trp codon. These data provide the first direct evidence for post-translational bromination of a polypeptide which is translated through the normal cellular machinery. In addition to bromination, the peptide, which induces a "stiff tail" syndrome in mice, has several other modifications as shown by the sequence [Formula: See Text] in which Hyp = hydroxyproline. Asterisks indicate post-translational modifications (left to right): proteolytic cleavage at the N-terminus; hydroxylation of Pro3; epimerization of Trp4; bromination of Trp7, and C-terminal amidation. Bromocontryphan appears to have the highest density of post-translational modifications known among gene-encoded polypeptides. The overall result is a molecule which closely resembles marine natural products produced through specialized biosynthetic pathways comprising many enzyme-catalyzed steps.

A novel post-translational modification involving bromination of tryptophan. Identification of the residue, L-6-bromotryptophan, in peptides from Conus imperialis and Conus radiatus venom.

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = gamma-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor.

Predictive methods, physicochemical measurements, and structure activity relationship studies suggest that corticotropin-releasing factor (CRF; corticoliberin), its family members, and competitive antagonists (resulting from N-terminal deletions) usually assume an alpha-helical conformation when interacting with the CRF receptor(s). To test this hypothesis further, we have scanned the whole sequence of the CRF antagonist [D-Phe12,Nle21,38]r/hCRF-(12-41) (r/hCRF, rat/human CRF; Nle, norleucine) with an i-(i + 3) bridge consisting of the Glu-Xaa-Xaa-Lys scaffold. We have found astressin [cyclo(30-33)[D-Phe12,Nle21,38,Glu30,Lys33]r/ hCRF(12-41)] to be approximately 30 times more potent than [D-Phe12,Nle21,38]r/hCRF-(12-41), our present standard, and 300 times more potent than the corresponding linear analog in an in vitro pituitary cell culture assay. Astressin has low affinity for the CRF binding protein and high affinity (Ki = 2 nM) for the cloned pituitary receptor. Radioiodinated [D-125I-Tyr12]astressin was found to be a reliable ligand for binding assays. In vivo, astressin is significantly more potent than any previously tested antagonist in reducing hypophyseal corticotropin (ACTH) secretion in stressed or adrenalectomized rats. The cyclo(30-33)[Ac-Pro4,D-Phe12,Nle21,38,Glu30,Lys33++ +]r/hCRF-(4-41) agonist and its linear analog are nearly equipotent, while the antagonist astressin and its linear form vary greatly in their potencies. This suggests that the lactam cyclization reinstates a structural constraint in the antagonists that is normally induced by the N terminus of the agonist.

Conformationally restricted competitive antagonists of human/rat corticotropin-releasing factor.

Corticotropin releasing factor (CRF) is a 41-peptide amide which stimulates the release of ACTH (Vale et al. Science 1981, 213, 1394). CRF has been postulated to assume an alpha-helical conformation upon binding to its pituitary receptor (Hernandez et al. J. Med. Chem. 1993, 36, 2860). We have exploited this hypothesis in the design of a limited series of cyclic analogues and have taken into consideration the effects of side-chain deletion (Alanine scan, Kornreich et al. J. Med. Chem. 1992, 35, 1870) as well as of changes in chirality (Rivier et al. J. Med. Chem. 1993, 36, 2851), with the rationale that side chains necessary for binding could also be replaced by side-chain bridges. In particular, we have used computer modeling to predict likely side chain bridging opportunities and evaluated the effects of such replacements by correlating biological results with those derived from CD spectroscopy. We have synthesized 38 monocyclic peptide amides, competitive antagonists of human/rat CRF, using solid-phase methodology on MBHA resin. After purification by preparative RP-HPLC, the peptides were analyzed by RP-HPLC and capillary zone electrophoresis and characterized by mass spectroscopy and amino acid analysis. CRF antagonists were tested for their ability to interfere with CRF-induced release of ACTH by rat anterior pituitary cells. In most cases, one of the bridge heads was located at a position where substitution by a D-residue was tolerated (i.e., positions 12 and 20). It has become clear that careful optimization of bridge length and chirality is critical. This is best exemplified by the fact that out of the 38 analogues that were synthesized and tested, only two, [cyclo(20-23)[DPhe12,Glu20,Lys23, Nle21,38]h/rCRF12-41 and cyclo(20-23)[DPhe12,Glu20,Orn23,Nle21,38] h/rCRF12-41], were found to be more potent (3 and 2 times, respectively) than [DPhe12,Nle21,38]h/rCRF12-41, the parent compound. Six analogues belonging to two different families were found to be half as potent as the standard, 18 had 2-20% of the potency of the standard, and the others were significantly less potent. CD results of all analogues in 50% TFE (a concentration of TFE that induced nearly maximum helicity of [DPhe12,Nle21,38]h/rCRF12-41) suggest that while helicity may be an important factor for CRF analogue recognition, little correlation is found between percent helicity as determined by spectral deconvolution and biological activity in vitro.

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides.

A number of L-glutamyl and L-aspartyl dipeptides, glutathione, gamma-D-glutamylglycine and gamma-D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, the L enanthiomorphs of gamma-glutamyl peptides were more potent displacers than gamma-D-glutamylglycine and -taurine but the latter were more specific for the quisqualate type of receptors. gamma-L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas gamma-L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. gamma-L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent gamma-L-glutamyl peptides (glutathione, gamma-L-glutamyl-L-glutamate, -L-aspartate, and -glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.

Analogs of growth hormone-releasing hormone induce release of growth hormone in the bovine.

Biological potencies of three 29 amino acid growth hormone-releasing hormone analogs (GHRH[1-29]) were determined in the bovine and compared to synthetic human GHRH (44 amino acids; hGHRH[1-44]NH2) for their ability to increase serum growth hormone (GH) concentrations. Four prepubertal Holstein heifers (179 +/- 10 kg) received hGHRH(1-44)NH2 or analogs (D-Ala2, Nle27, Agm29 GHRH[1-29], [JG-73]; D-N-MeAla2, Nle27, Agm29 GHRH[1-29], [JG-75]; and desamino-Tyr1, D-Ala2, Nle27, Agm29 GHRH[1-29], [JG-77]) at the following doses: 0, 6.25, 25, 100 and 400 micrograms/animal. All treatment-dose combinations were administered to each heifer with at least a 1-d interval between treatments. Sixteen blood samples were collected via jugular cannulas 20 min before and up to 6 h after treatment injection. There was a linear dose-dependent GH release in response to hGHRH(1-44)NH2 and the three analogs. Growth hormone peak amplitudes for the three analogs were similar to those observed after administration of the hGHRH(1-44)NH2 (P greater than .05). However, when total area under the GH response curves for each treatment was averaged over all the doses, JG-73 stimulated greater GH release than hGHRH(1-44)NH2 (P less than .05) Heifers injected with the 400-microgram dose of hGHRH(1-44)NH2 or the three analogs showed a primary release of GH followed by a secondary release 1 h later. At all other doses, only a primary GH release was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

An evaluation of intravenous, subcutaneous, and in vitro activity of new agmatine analogs of growth hormone-releasing hormone hGH-RH (1-29)NH2.

The effects of new Agmatine (Agm) analogs of human growth hormone-releasing hormone (GH-RH) were compared to GH-RH (1-29)NH2 and to (D-Ala2)GH-RH(1-29)NH2 after intravenous (IV) and subcutaneous (SC) administration to pentobarbital-anesthetised male rats and in vitro using superfused rat pituitary cell system. After IV administration, the analogs: (D-MeAla2,Nle27)GH-RH(1-28)Agm(JG-75), (desamino-Tyr1,D-Ala2,Nle27)GH-RH(1-28)Agm(JG-77), (D-Ala2,Nle27)GH-RH(1-28)Agm(JG-73) and (D-Ala2)GH-RH(1-29)NH2 showed a potency 2.6-3.9 times greater than GH-RH(1-29)NH2 at 5 min and 1.6-2.7 times higher at 15 min. After SC administration these analogs were 30-74 times more potent than GH-RH(1-29)NH2. The ratio between the IV and SC GH-releasing activity of the analogs ranged from 2 to 5, while GH-RH(1-29)NH2 was about 50 times more active IV than SC. This indicates that 20-50% of the analogs can be absorbed from SC tissues, but only 2% of GH-RH(1-29)NH2. The in vitro activity of the agmatine analogs on GH release closely paralleled their IV potency and was 2.8-3.9 times greater than that of GH-RH(1-29)NH2. No significant difference in potency was found between (D-Ala2)GH-RH(1-29)NH2 and JG-75 after IV administration and in vitro, although JG-75 contained only 28 amino acids. We conclude that the reason for the large discrepancies between the previously reported activities of (D-Ala2)GH-RH(1-29)NH2 was simply due to the different ways of administration of this analog, SC vs IV, and not to species specificity. The replacement of Arg29 by Agmatine in (D-Ala2,Nle27)GH-RH(1-29)NH2 causes a 3 fold increase in SC potency, but the replacement of D-Ala2 with D-MeAla2 reduces the SC, but not the IV and in vitro activity in half.