Nicole Bernad

Comparative Study of In Vitro and In Vivo Activities of Bombesin Pseudopeptide Analogs Modified on the C-Terminal Dipeptide Fragment

Analogs of bombesin in which the peptide bond between the two last amino acid residues were replaced by a pseudopeptide bond mimicking the transition state analog were evaluated. These compounds were able to recognize the bombesin receptor on isolated rat pancreatic acini with high potency (Ki from 0.60 +/- 0.27 nM to 4.3 +/- 2.3 nM). Although they were devoid of agonist activity, they were able to antagonize bombesin-induced amylase secretion in this model, with potencies in accordance with their affinities (IC50 from 1.6 +/- 0.3 nM to 10.0 +/- 1.7 nM). When tested in vivo in the anesthetized rat, these bombesin receptor antagonists exhibited high potency in inhibiting bombesin-induced pancreatic secretion (H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-CH[CH2-CH(CH3)2]-CHOH-(CH 2)3-CH3, JMV845, was among the most potent compounds with ED50 of 7.82 +/- 2.89 nM in inhibiting bombesin-induced protein secretion). The results of this study showed that replacing the peptide bond between the two last amino acid residues in bombesin by mimicking the transition state analog resulted in in vitro and in vivo potent bombesin receptor antagonists.

A new biological contribution of cyclo(His-Pro) to the peripheral inhibition of pancreatic secretion

The tripeptide pyro-Glu-His-Pro-NH2[thyrotropin-releasing hormone (TRH)] was isolated from the hypothalamus as a thyrotropin-releasing factor. It has a broad spectrum of central nervous system-mediated actions, including the stimulation of exocrine pancreatic secretion. TRH is also synthesized in the endocrine pancreas and found in the systemic circulation. Enzymatic degradation of TRH in vivo produces other bioactive peptides such as cyclo(His-Pro). Because of the short half-life of TRH and the stability of cyclo(His-Pro) in vivo, we postulated that at least part of the peripheral TRH effects on the exocrine pancreatic secretion may be attributed to cyclo(His-Pro), which has been shown to have other biological activities. This study determines in parallel the peripheral effects of TRH and cyclo(His-Pro) as well as the putative contribution of other TRH-related peptides on exocrine pancreatic secretion in rats. TRH and its metabolite cyclo(His-Pro) dose dependently inhibited 2-deoxy-D-glucose (2-DG)-stimulated pancreatic secretion. TRH and all the related peptides tested had no effect on the basal and cholecystokinin-stimulated amylase release from pancreatic acinar cells in vitro. These data indicate that cyclo(His-Pro) mimics the peripheral inhibitory effect of TRH on 2-DG-stimulated exocrine pancreatic secretion. This effect is not detected on isolated pancreatic acini. Our findings provide a new biological contribution for cyclo(His-Pro) with potential experimental and clinical applications.The tripeptide pyro-Glu-His-Pro-NH2[thyrotropin-releasing hormone (TRH)] was isolated from the hypothalamus as a thyrotropin-releasing factor. It has a broad spectrum of central nervous system-mediated actions, including the stimulation of exocrine pancreatic secretion. TRH is also synthesized in the endocrine pancreas and found in the systemic circulation. Enzymatic degradation of TRH in vivo produces other bioactive peptides such as cyclo(His-Pro). Because of the short half-life of TRH and the stability of cyclo(His-Pro) in vivo, we postulated that at least part of the peripheral TRH effects on the exocrine pancreatic secretion may be attributed to cyclo(His-Pro), which has been shown to have other biological activities. This study determines in parallel the peripheral effects of TRH and cyclo(His-Pro) as well as the putative contribution of other TRH-related peptides on exocrine pancreatic secretion in rats. TRH and its metabolite cyclo(His-Pro) dose dependently inhibited 2-deoxy-d-glucose (2-DG)-stimulated pancreatic secretion. TRH and all the related peptides tested had no effect on the basal and cholecystokinin-stimulated amylase release from pancreatic acinar cells in vitro. These data indicate that cyclo(His-Pro) mimics the peripheral inhibitory effect of TRH on 2-DG-stimulated exocrine pancreatic secretion. This effect is not detected on isolated pancreatic acini. Our findings provide a new biological contribution for cyclo(His-Pro) with potential experimental and clinical applications.

A synthetic glycine-extended bombesin analogue interacts with the GRP/bombesin receptor.

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.

New general strategy of dimerization of bioactive molecules

Abstract We describe a new way of obtaining dimeric structures based on intermolecular diketopiperazine formation. The bioactive substance to dimerize was first linked to a glycine moiety. Then a coupling step using DMAP in stoichiometric quantity resulted in the cyclization involving both C-terminal carboxylic functions and the amide nitrogen. This general strategy has been applied to peptide and non-peptide bioactive molecules.

Biological Evaluation of JMV180 Cholecystokinin Analogs

The dose-response curve for cholecystokinin (CCK)-stimulated enzyme secretion in isolated pancreatic acini from different species is biphasic. With increasing doses of CCK, amylase secretion increases to a maximum and then decreases at higher concentrations. The CCK analog Boc-Tyr(SO3H)-Nle-G1y-Trp-NIe-Asp-O-CH2CH2C6H5 (JMVISO), lacking the C-terminal amide function, in which the phenylalanine residue was replaced by 2-phenylethyl alcohol’ and both methionines in position 28 and 31 were substituted by norleucines, exhibited “partial agonist activity” of CCK in rat pancreatic acini. C-terminal JMV180 did not produce a decrease in amylase stimulation at supramaximal concentrations in rat pancreatic acini,2 but it showed a plateau of maximal stimulation. It has been hypothesized that compound JMVlSO interacts with both lowand high-affinity peripheral CCK binding sites. It acts as an agonist at CCK high-affinity binding sites and as an antagonist at low-affinity binding sites3 Compound JMV180 has been used extensively to determine the relations between occupation of each class of the CCK binding sites and their linkage to various transduction systems and resulting biological activities and it is still widely used in various studies.”’ Interestingly, replacing L-tryptophan by D-tryptophan in compound JMVlSO produced Boc-Tyr(S03H)-Nle-Gly-D-Trp-Nle-Asp-0-CH2-CH2CbH5 (JMV179) which proved to be a full and potent CCK receptor antagonist.x When compound JMV180 or the corresponding D-tryptophan analog was used in in vivo studies, larger doses than expected from in vitro results had to be used to obtain biological a c t i ~ i t y . ~ J ~ We also observed that although the corresponding 2-phenylethylamide analog of compound JMV180, such as Boc-Tyr( S03H)-Nle-Gly-Trp-N1e-AspNH-CH2CH2-C6H5, was less potent in vitro than JMV180, it was equally potent in in vivo studies. These observations suggested that compounds JMVlSO and JMV179 were probably not very stable in vivo; we were particularly concerned about the stability of the ester linkage. One of our goals was to synthesize an analog of compound JMV180 that would have enhanced stability, particularly for in vivo studies, and maintain high in vitro activity. In the first part of this work, we report on the biological activity of analogs of compound JMV180 in which the ester bond was replaced by a “carba” linkage (CH2-CH2) (FIG. 1). This modification implied the synthesis of 3-amino 7-phenyl heptanoic acid (P-homo-Aph) with the R configura-

Modification of receptor selectivity and functional activity of cyclic cholecystokinin analogues

Abstract We reported earlier on the synthesis and biological activity at the CCK-B receptor of cyclized derivatives of CCK. These peptides, in which the positions 28 and 31 were replaced by lysine residues, were bridged by a succinyl moiety. To determine the importance of the nature and size of the cyclic structure, cyclic analogues were synthesized in which: (i) the lysine residues were replaced by ornithine and diaminobutyric acid and (ii) the succinic moiety was replaced by a malonic, adipic and glutaric moiety. They were tested for their ability to inhibit the specific binding of 125 I-BH-CCK-8 to CCK receptors in rat pancreatic acini and guinea pig brain membranes. They were also evaluated for their ability to stimulate amylase secretion from rat pancreatic acini. The potency and selectivity of these analogues were compared with those obtained with CCK-4 and compound JMV320, a potent and selective CCK-B receptor ligand synthesized earlier in our laboratory.

Synthesis and biological activities of peptidomimetic analogues of compound A71623, a potent and selective CCK-A receptor agonist

The involvement of cholecystokinin receptors in the phenomenon of satiety has been the impetus for significant research efforts, leading to the design and synthesis of CCK-A selective agonists for the possible treatment of obesity. The Abbott laboratories have described a novel series of pseudotetrapeptides represented by compound A71623, a highly potent and selective peripheral receptor agonist, but possessing very poor bioavailability. Starting from the structural requirements of this series of compounds, a peptidomimetic study was investigated, especially focusing on the N-terminal part of A71623. Using standard coupling methods, introduction of unnatural aromatic amino acids bearing a 2-carboxyethyl side chain on their α-amino group, along with backbone length modulation, afforded selective analogues, presenting a highly modified peptidic backbone. From our two lead compounds, further optimization is under development, tending towards nonpeptidic structures.