Identification of critical microRNAs in gastrointestinal stromal tumor patients treated with Imatinib.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. Imatinib mesylate was considered to be a breakthrough drug in clinical treatment of GIST, but GIST patients showed resistance against it. We aimed to identify critical microRNAs (miRNAs) related to imatinib resistance in imatinib-treated GIST patients. Microarray datasets under the accession number of GSE63159 and GSE45901 were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed miRNAs (DEMs) that are related to imatinib resistance were identified. GO function and KEGG pathway enrichment analyses were performed, and lncRNA-miRNA-target gene regulatory networks were constructed. Finally, the critical miRNAs and their target genes that are related to imatinib resistance or sensitivity were identified. In total, 20 DEMs in the GSE63159 dataset (7 significantly up-regulated and 13 down-regulated) and 23 DEMs in the GSE45901 dataset (8 up-regulated and 15 down-regulated) were identified. In lncRNA-miRNA-target gene regulatory networks, five critical miRNAs and 109 target genes were identified. GO function and KEGG pathway enrichment analysis showed that the target genes of DEMs were mainly involved in several signaling pathways, such as focal adhesion and the GnRH signaling pathway. Among the five miRNAs, the overexpression of hsa-miR-28-5p and hsa-miR-125a-5p had significant correlation to imatinib resistance or imatinib sensitivity in GIST patients. Hsa-miR-28-5p and hsa-miR-125a-5p may be involved in the development and progression of GIST, and they may be able to serve as prognostic markers for imatinib-response in GIST patients.

Pharmacology study of the neutral myocardial imaging agent technetium-99m-N(NOEt)2.

OBJECTIVE: The biological properties of a new neutral myocardial imaging agent 99mTcN(NOEt)2 were evaluated. METHODS: Blood clearance in rabbits, biodistribution in rats, and initial myocardial imaging in dogs were performed. RESULTS: Radiochemical purity of 99mTcN(NOEt)2 was more than 90% and stable for 6 h at room temperature. Blood disappearance was analyzed with a biexponential model and T1/2(alpha) = 2.53 min, T1/2(beta) = 330 min and CI = 378 ml/h were obtained. Biodistribution studies demonstrated that 99mTcN(NOEt)2 localized selectively in the rat myocardium. Cardiac uptakes were 4.69, 4.20, 3.95 and 3.43% ID/g at 5, 30, 60 and 90 min postinjection, respectively. The mean heart-to-lung activity ratios were 1.69, 2.40 and 2.55 at 10 min, 30 min and 60 min postinjection, respectively. CONCLUSION: Technetium-99m-N(NOEt)2 exhibited favorable stability and biological properties. Further study in humans is required.

Examination of somatostatin involvement in the inhibitory action of GIP, GLP-1, amylin and adrenomedullin on gastric acid release using a new SRIF antagonist analogue.

1. The effect of a new type 2 selective somatostatin (SRIF) receptor antagonist (DC-41-33) on somatostatin-induced inhibition of pentagastrin-stimulated gastric acid secretion in conscious, chronic gastric fistula equipped rats was studied. 2. Infused intravenously, DC-41-33 dose-dependently inhibits SRIF-induced inhibition of pentagastrin-stimulated gastric acid secretion with an IC50 of 31.6+/-1.2 nmol kg(-1) versus 10 nmol kg(-1) SRIF and blocks the inhibitory effects of SRIF when simultaneously co-infused. Its effectiveness provides additional evidence that SRIF-inhibition of gastric acid release is a SRIF type 2 receptor-mediated process. 3. DC-41-33 is able to completely reverse the inhibitory effect of glucose-dependent insulinotropic polypeptides, GIP and GIP-(1-30)NH2, and glucagon-like polypeptide, GLP-1(7-36)NH2, on pentagastrin-stimulated gastric acid secretion thus confirming that they exert these effects through stimulation of endogenous SRIF release. 4. DC-41-33 only partially blocks potent amylin and adrenomedullin-induced inhibition of gastric acid secretion, therefore suggesting that somatostatin may not function as a primary mediator in the action of these peptides. 5. Our results indicate that DC-41-33, is a potent in vivo inhibitor of exogenous and endogenous SRIF in rats. It represents a new class of SRIF analogues which should eventually provide excellent tools for further evaluating the many physiological roles of SRIF and its five receptor subtypes.

Adrenomedullin: a hypotensive hormone in man.

1. Adrenomedullin, a recently discovered 52-amino-acid peptide hormone, circulates in plasma at low picomolar levels in man. Animal studies and studies in vitro indicate that it has diverse biological actions, including vasodilatation, natriuresis and diuresis, and positive inotropism as well as anti-proliferative effects. We investigated the bioactivity of two doses of adrenomedullin in healthy human subjects. 2. Human adrenomedullin was given intravenously to eight male subjects at 2 and 8 ng min-1 kg-1, and haemodynamic, hormonal, renal and biochemical responses were recorded in a placebo (vehicle)-controlled, randomized study. 3. Compared with vehicle, adrenomedullin reduced mean arterial pressure (P = 0.05 for duration of infusion, mean difference at end of infusion 7.7 mmHg), systolic arterial pressure (P = 0.04 for duration of infusion, mean difference at end of infusion 10.7 mmHg) and at the lower dose reduced diastolic arterial pressure (P = 0.05 for lower dose, mean difference at end of infusion 6.3 mmHg) in the absence of compensatory responses in sympathetic activity or renin release. Urine volume and electrolyte excretion were unaffected. 4. The threshold for biological activity of adrenomedullin in man is lower, for arterial pressure than for renal or hormonal responses, and is evident at plasma concentrations seen in disorders of the circulation. Adrenomedullin may be an important hormone under pathophysiological circumstances.

Adrenomedullin, amylin, calcitonin gene-related peptide and their fragments are potent inhibitors of gastric acid secretion in rats.

Adrenomedullin, amylin and calcitonin gene-related peptides (CGRP) share close sequence homology and have overlapping spectra of biological activities, particularly with respect to cardiovascular and gastrointestinal functions. Comparisons of the effects of these three peptides on gastric acid release have been made by i.v. infusions in conscious rats equipped with gastric fistulae. All peptides were extremely potent inhibitors of basal, pentagastrin- and 2-deoxy-D-glucose-stimulated gastric acid secretion with IC50 values in the subnanomolar to nanomolar range. These effects were not inhibited by C-terminal extra-cyclic fragments of the peptides which often act as competitive receptor antagonists in other biological systems. At high concentrations C-terminal fragments of human adrenomedullin and rat alpha-calcitonin gene-related peptide displayed some receptor agonist activity. Furthermore, the N-terminally situated disulfide-bridged ring fragments, human adrenomedullin-(15-22), rat amylin-(1-8) and rat alpha-calcitonin gene-related peptide-(1-8), retained significant gastric acid inhibitory potencies thus suggesting involvement of receptor(s) with significantly differing ligand binding profiles than those characterized previously.

Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo.

Adrenomedullin is a 52-amino acid vasodilator peptide produced in many tissues, including bone. It has 20% sequence identity with amylin, a regulator of osteoblast growth, and circulates in picomolar concentrations. The present study assesses whether adrenomedullin also acts on osteoblasts. At concentrations of 10(-12) M and greater, adrenomedullin produced a dose-dependent increase in cell number and [3H]thymidine incorporation in cultures of fetal rat osteoblasts. This effect was also seen with adrenomedullin-(15-52), -(22-52), and -(27-52), but adrenomedullin-(40-52) was inactive. These effects were lost in the presence of amylin blockers, suggesting they were mediated by the amylin receptor. Adrenomedullin also increased [3H]thymidine incorporation into cultured neonatal mouse calvaria but, unlike amylin, did not reduce bone resorption in this model. Adrenomedullin stimulated phenylalanine incorporation into both isolated osteoblasts and calvaria. When injected daily for 5 days over the calvariae of adult mice, it increased indexes of bone formation two- to threefold (P < 0.0001) and increased mineralized bone area by 14% (P = 0.004). It is concluded that adrenomedullin regulates osteoblast function and that it increases bone mass in vivo. The potential of this family of peptides in the therapy of osteoporosis should be further evaluated.

A chimeric VIP-PACAP analogue but not VIP pseudopeptides function as VIP receptor antagonists.

The ability to assess the importance of VIP in different physiological processes is limited by the lack of specific potent antagonists. In the present study, we have adopted two different approaches used successfully with other peptides in an attempt to identify new VIP receptor antagonists. One involves the formation of pseudopeptides by insertion of reduced peptide bonds in the NH2-terminus from position 2 to 8 of VIP. The other methodology involves the formation of a COOH-terminal chimeric analogue by combining VIP(6-28) and PACAP(28-38). The ability of each of these peptides to function as an antagonist was compared with reported VIP antagonists. All of the peptides inhibited [125I]VIP binding to VIP receptors on guinea pig pancreatic acini. For the pseudopeptides the affinities were: [psi 3-4]VIP (0.2 microM) = 4 x [psi 4-5]VIP = 8 x [psi 8-9]VIP = 14 x [psi 6-7]VIP, [psi 2-3]VIP = 25 x [psi 5-6]VIP. Each nonpseudopeptide analogue also inhibited VIP binding with relative potencies of VIP(6-28)-PACAP(28-38) (1 microM) = 2.5 x [4-Cl-D-Phe6,Leu17]VIP, VIP(10-28), neurotensin(6-11)-VIP(7-28) = 6 x [Ac-Tyr1,D-Phe2]GRF. All pseudopeptides were agonists with relative potencies: [psi 3-4]VIP > [psi 6-7], [psi 4-5]VIP > [psi 5-6] > [psi 8- 9]VIP > [psi 2-3]VIP. The reported VIP receptor antagonist, neurotensin(6-11)-VIP(7-28), was also an agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Galanin: structure-dependent effect on pancreatic amylase secretion and jejunal strip contraction.

Rat and porcine galanin and their fragments inhibited cholecystokinin-8 (CCK-8)-stimulated amylase secretion with the following activities: rat galanin-(1-29) = porcine galanin-(1-29) = galanin-(1-15) = rat galanin-(3-29) > rat galanin-(2-29) = porcine galanin-(2-29) > galanin-(1-10). Fragments of rat galanin-(9-29) and N alpha-acetyl-galanin-(9-29) were able to inhibit CCK-8-stimulated pancreatic amylase secretion but only at higher dose levels. Porcine galanin-(15-29) and rat galanin-(21-29) were unable to produce significant inhibition. Rat and porcine galanin-(1-29), galanin-(1-15) and rat N alpha-acetyl-galanin-(9-29) also inhibited basal pancreatic amylase secretion. In the rat jejunal strip contraction model, rat galanin-(1-29) and porcine galanin-(1-29) have similar potencies. Galanin-(1-15) and galanin-(1-10) stimulate rat jejunal strip contraction with decreasing potencies. Elimination of Gly1 from the N-terminus of both rat and porcine galanin had no significant effect either on pancreatic amylase secretion or on jejunal strip contraction. The rat galanin-(3-29) and (9-29) are not active in the stimulation of rat jejunal strip contraction. Acetylation of porcine galanin-(9-29) created a peptide that was a powerful stimulator of rat jejunal strip contraction. The present data indicate that N-terminal rat galanin amino acid residues are crucial for rat jejunal strip contraction but are not required for inhibition of pancreatic amylase. These results suggest that the galanin amino acid sequence contains several specific domains, which can be recognized by specific galanin receptor subsets.

Tyrosine 67 in the epidermal growth factor-like domain of tissue-type plasminogen activator is important for clearance by a specific hepatic receptor.

Human tissue-type plasminogen activator (t-PA) is cleared rapidly from the circulation by hepatic receptors, one of which recognizes a site in the epidermal growth factor-like domain of the molecule. To define this site more precisely, we have used oligonucleotide-mediated mutagenesis to introduce amino acid substitutions at specific positions located in turns that connect antiparallel beta-sheets in the epidermal growth factor-like domain. Mutated t-PA proteins with amino acid substitutions of the tyrosine residue at position 67 showed markedly lower rates of endocytosis and degradation by cultured cells of the rat hepatoma (H4) line that express a specific receptor for t-PA, and their half-life in the circulation of rats was extended significantly because of a reduction in the rate of the rapid alpha-phase of clearance. The enzymatic properties and fibrinolytic activity of these mutants in vitro were not significantly different from those of wild-type t-PA. We conclude that tyrosine 67 comprises a key determinant in the clearance of t-PA by a specific hepatic receptor.

Reduced gastric acid inhibitory effect of a pGIP(1-30)NH2 fragment with potent pancreatic amylase inhibitory activity.

Gastric inhibitory polypeptide (GIP) strongly stimulates insulin secretion in the presence of glucose and also stimulates somatostatin release from gastric mucosa. It was reported recently that both stimulatory activities can be dissociated by removing the C-terminal 12 amino acid residues. Since insulin and somatostatin are involved in regulation of exocrine pancreatic and gastric secretion in rats, we compared the inhibitory effects of pGIP and the pGIP(1-30)NH2 fragment on pancreatic amylase and gastric acid secretion. pGIP(1-30)NH2 displayed full activity on inhibition of bombesin (BN)-stimulated amylase release relative to GIP itself, but was about 10-fold less potent in inhibiting gastric acid secretion. These results suggest that the receptors involved in these two events have quite different ligand binding requirements and that more specific analogues of GIP can be designed which should be of value in elucidating the physiological roles of this hormone.

Systematic development of bombesin/gastrin-releasing peptide antagonists.

Several families of very potent bombesin (Bn) receptor antagonist analogues have recently been developed and their biological potencies evaluated in a number of in vitro systems including guinea pig and rat pancreatic acini and Swiss 3T3 cells. These studies showed that analogues can exhibit diverse properties ranging from full antagonists, partial agonists, or full agonists depending on the assay system and animal species employed. We have developed two classes of more potent, shorter chain antagonists based on [psi CH2NH(13-14)]Bn(6-14) and desMet14Bn(6-13)NH2 structures. [D-Phe6 psi Leu13-Leu14] Bn(6-14)NH2 was a potent antagonist (Ki 6nM) in Swiss 3T3 cells and guinea pig acini but exhibited 10% partial agonist activity and lower binding affinity (Ki 60 nM) in rat acini. The partial agonism could be eliminated by using p-Cl-Phe or D-Phe at the C-terminus and partially eliminated using D-4-Cl-Phe in position 6. With the antagonist [D-Phe6]Bn(6-13)NH2 (Ki 96 nM), alkyl substituents on the amide group increased affinity 25-fold with the propylamide being the most potent peptide (Ki 4 nM) in 3T3 cells or guinea pig acini. It did, however, have high 40% partial agonist activity in rat acini. Alkyl esters or hydrazide derivatives were, in contrast, pure antagonists in all systems tested with [D-Phe6]Bn(6-13)OMe having the highest affinity in all systems and also excellent in vivo properties. All of the potent antagonists examined had little affinity for neuromedin B--preferring bombesin receptors, which had entirely new ligand structure-activity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)

New reduced peptide bond substance P agonists and antagonists: effects on smooth muscle contraction.

Following the recent discovery of a new substance P (SP) competitive pancreatic acini cell receptor antagonist containing a reduced peptide bond in place of the C-terminal peptide bond, a new series of full chain and short chain (heptapeptide and hexapeptide) substance P analogues have been prepared in which one of the C-terminal-region peptide bonds has been replaced by CH2NH or CH2O groups. They were compared for their ability to recognize NK1 and/or NK2 tachykinin receptor binding sites on guinea pig ileum and rat duodenum smooth muscle preparations, respectively. It was found that all full sequence SP pseudopeptides were agonists with much reduced bioactivity in both tested systems and, in addition, [Gly9 psi(CH2NH)Leu10,Leu11]SP was found to be a relatively selective agonist for NK1 binding sites. Substitution of leucine at position 11 of SP heptapseudopeptides with phenylalanine generated a pseudopeptide with weak agonist activity when Gln at position 5 was replaced by D-Phe, or antagonists when this residue was replaced by D-Nal or D-Cpa. [Leu10 psi(CH2NH)Leu11]SP-(6-11) with Gln at position 6 substituted by D-Phe was a relatively stronger antagonist in both assay systems. These results suggest that, as with several other peptide systems of late, manipulation of the peptide bonds in SP can produce receptor antagonists which in some cases approach the potency of the classic spantide series and, furthermore, that the approach might be used to induce NK receptor specificity in both agonist and antagonist analogs.

Covalently cyclized agonist and antagonist analogues of bombesin and related peptides.

During a search for possible cyclization points in shortened, potent bombesin agonists and antagonists, it was found that the joining of amino acid residues in positions 6 and 14 by various means resulted in retention of significant binding affinity for rat pancreatic acini and murine Swiss 3T3 cells. In one series of analogues, Cys residues in these positions were used for bridging via a disulfide bond. (D)-C-Q-W-A-V-G-H-L-C-NH2 retained significant binding affinity for rat pancreatic acini cells and was a full amylase releasing agonist (EC50 187 nM). Potency was markedly increased by substituting D-Ala for Gly (EC50 67 nM compared to 10 nM for its linear counterpart) and was decreased by substituting L-Cys for D-Cys in this analogue (EC50 214 nM), thus strongly suggesting stabilization of peptide folding by the D residues. Elimination of the COOH-terminal amino acid produces competitive antagonists in the linear analogues; however, (D)-C-Q-W-A-V-G-H-C-NH2 was devoid of activity. Likewise, cyclization to position 13 with the 14 amino acids intact to give (D)-C-Q-W-A-V-G-H-C-L-NH2 resulted in an almost inactive peptide. On the other hand, as in the linear series, the reduced peptide bond analogue, (D)-C-Q-W-A-V-(D)-A-H-L-psi (CH2NH)-C-NH2, was a receptor antagonist (IC50 5.7 mM), albeit much weaker than the corresponding linear analogues, but with no residual agonist activity. Direct head-to-tail cyclization was also tried. Both cyclo[(D)-F-Q-W-A-V-G-H-L-L] (EC50 346 nM) and the shorter cyclo [Q-W-A-V-G-H-L-L] (EC50 1236 nM) were full agonists. Elimination of the COOH-terminal residue in cyclo[(D)-p-Cl-F-Q-W-A-V-(D)-A-H-L] produced an agonist (EC50 716 nM) rather than an antagonist. These results provide support for the proposal that both bombesin agonists and antagonists adopt a folded conformation at their receptor(s). Furthermore, the retention of appreciable potencies using several cyclization strategies and chain lengths suggests that further optimization of these structures both in terms of potency and ring size is possible. Since these peptides have increased conformational restriction, they should begin to serve as useful substrates for NMR and molecular modeling studies aimed at comparing the obviously subtle differences between agonist and antagonist structures.

Comparison of prolonged in vivo inhibitory activity of several potent bombesin (BN) antagonists on BN-stimulated amylase secretion in the rat.

New BN analogues designed to be competitive receptor antagonists at the BN/gastrin releasing peptide receptor(s) can exhibit diverse properties ranging from full antagonist, partial agonist or weak agonist activity, depending on the assay system and animal species employed. Here we evaluate the following 3 antagonists which have the most potent receptor affinities in several in vitro assay systems and are representative of 3 main classes of BN antagonists for their in vivo effects on pancreatic amylase secretion in the rat: [D-Cpa6,Phe14,psi 13-14]BN(6-14), [D-Phe6]BN(6-13) propylamide, and [D-Phe6]BN(6-13) methyl ester. After injection in the rat, the methyl ester was clearly the most potent antagonist and completely inhibited BN-stimulated amylase release at the 20 nmol/kg (IV bolus) for about 2 h. In contrast, the propylamide analogue at the 200 nmol/kg (IV bolus) dose produced incomplete inhibition of amylase release. Inhibition was transient and lasted for only about 1 h, possibly reflecting the significant agonist activity of this latter peptide in the rat pancreatic amylase secretion test in vitro. The psi-analogue, while being the longest acting analogue, was also incapable of lowering amylase to basal level at 50 times the BN dose, suggesting that it is a mixed agonist-antagonist in vivo as was also previously shown in vitro in the rat.

Short chain bombesin pseudopeptides with potent bombesin receptor antagonist activity in rat and guinea pig pancreatic acinar cells.

A series of potent bombesin antagonists based on the reduced C-terminal peptide bond modification which in the past resulted in the first really potent antagonists are compared for effects on bombesin-stimulated amylase release from and binding to rat and guinea pig pancreatic acini. It was found that the original member of this series, [Leu13 psi (CH2NH)Leu14] bombesin, displayed partial agonist activity with 11% efficacy in the rat. More recent analogues of this type which were found previously to be even more potent pure antagonists in the guinea pig pancreas or 3T3 cells, exhibited similarly higher binding affinity for rat acini but displayed even higher residual partial agonist activity in the rat. For instance, [D-Phe6,Leu13 psi (CH2NH)Phe14]bombesin-(6-14) was one of the most potent bombesin antagonists known in the guinea pig and 3T3 cell systems but has 40% partial agonist activity in the rat. Several structural modification strategies were developed to remove rat partial agonist properties with retention of high antagonist potency in all systems tested. The most effective of these was the substitution of a Cl on the aromatic ring of the Phe residue (p-Cl-Phe, Cpa) in position 14 to give [D-Phe6,Leu13 psi (CH2NH)Cpa14]bombesin-(6-14). This had higher binding affinities for both rat and guinea pig pancreatic acini and was a pure antagonist on both cell types. Another effective method was alteration of the stereochemistry of the position 14 amino acid in [D-Phe6,Leu13 psi (CH2ND)D-Phe14]bombesin-(6-14) which had somewhat lowered binding affinities but pure antagonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)

des-Met carboxyl-terminally modified analogues of bombesin function as potent bombesin receptor antagonists, partial agonists, or agonists.

In the present study we examined the effect of carboxyl-terminal modifications of des-Met14-bombesin (Bn) on Bn receptor affinity in murine 3T3 cells, rat and guinea pig pancreatic acini, and the ability to initiate biologic responses by synthesizing 18 des-Met14-Bn(6-13) analogues. With guinea pig acini and 3T3 cells, affinity was affected by the chain length of the alkyl moiety (R) added to [D-Phe6]Bn(6-13)NH2R with relative potencies: propyl greater than ethyl greater than butyl = hexyl greater than heptyl greater than free amide, whereas in rat acini affinity was not increased by the chain length. In each cell system the affinity of the alkylamide was not increased by insertion of a phenyl group in the alkyl side chain, by making the analogue more neuromedin B-like or by addition of a reduced peptide bond. The affinity in each cell system was increased by additions of other electron releasing groups to the COOH-terminal carboxyl group such as [D-Phe6]Bn(6-13)ethyl or methyl ester, or hydrazide. In guinea pig pancreas and 3T3 cells, 12 analogues were antagonists, 1 a full and 5 partial agonists. In rat pancreas, 8 were antagonists, 5 full agonists, and 5 partial agonists. Potent antagonists in each cell system were the methyl and ethyl ester, hydrazide, and ethylamide analogues. In 3T3 cells or guinea pig pancreas, agonist activity of the alkylamide was critically dependent on the chain length, whereas with rat pancreatic Bn receptors any alkylamide longer than the ethylamide had agonist activity. In all three cell systems any alteration that made the alkylamide more neuromedin B-like caused agonist activity. These results demonstrate that the nature of the substitution on the carboxyl terminus of des-Met14-Bn analogues is critically important, not only for determining Bn receptor affinity, but also for determining the ability to initiate a biologic response. In contrast to previous studies, the present results demonstrate that the presence of the COOH-terminal amino acid in position 14 of Bn is not essential for initiating a biologic response. Several des-Met14-Bn analogues were potent partial agonists, whereas others such as the hydrazide or ethyl ester are very potent antagonists.

Desmethionine alkylamide bombesin analogues: a new class of bombesin receptor antagonists with potent antisecretory activity in pancreatic acini and antimitotic activity in Swiss 3T3 cells.

Bombesin-related peptides have a large number of physiological functions as well as having an autocrine growth mechanism for the regulation of small cell lung cancer cells. In the present study we have synthesized 21 des-Met amide or alkylamide analogues of bombesin and compared their abilities to function as bombesin receptor antagonists in guinea pig pancreatic acini and Swiss 3T3 cells with those of the previously most potent antagonist described, [Leu13 psi(CH2NH)Leu14]bombesin (analogue I). All des-Met analogues functioned as antagonists. Bn(1-13)NH2 was approximately equipotent to I (Ki = 60-80 nM) whereas Bn(6-13)NH2 was 30-fold less potent (Ki = 1800 nM). Formation of an ethylamide, Bn(6-13)ethylamide, increased the potency 30-fold such that this octapeptide was equipotent to I. The addition of a D-Phe6 moiety to I did not change potency but caused a 30-fold increase in potency of Bn(6-13)NH2 and a 8-fold increase in the potency of Bn(6-13)ethylamide (Ki = 16 nM). Additional studies of both NH2- and COOH-terminal alterations in Bn(6-13)NH2 demonstrated that the most potent antagonist was [D-Phe6]Bn(6-13)propylamide (PA), having IC50's of 1.6 nM and 0.8 nM for bombesin-stimulated amylase release and Swiss 3T3 cell growth, respectively. Detailed studies of the most potent amide analogue, [D-Phe6]Bn(6-13)NH2, and alkylamide analogue, [D-Phe6]Bn(6-13)PA, demonstrated that these analogues functioned as competitive antagonists and that their action was selective for the bombesin receptor. These results demonstrate that, as with CCK- and gastrin-related peptides, the C-terminal amino acid is important for initiating a biologic response but not essential for determining receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced peptide bond pseudopeptide analogues of substance P. A new class of substance P receptor antagonists with enhanced specificity.

Each of the last 6 peptide bonds in the COOH terminus of [Leu11]substance P [( Leu11]SP) and [Nle11]spantide were replaced with [CH2NH], and each analogue was tested for SP agonist or antagonist activity by determining its ability to interact with SP receptors on dispersed acini from guinea pig pancreas. Each of the 6 spantide and 5 of the 6 SP analogues had no agonist activity, whereas [psi 9-10]SP was an agonist. For the spantide pseudopeptides, the psi 10-11 analogue (Ki,2.8 microM) was equipotent as an antagonist to spantide itself, whereas the psi 9-10, psi 8-9, psi 7-8, and psi 6-7 analogues were 2.5, 7, 5, and 3 times less potent. For the SP pseudopeptides, the psi 10-11 analogue was the most potent antagonist (Ki, 6.2 microM), whereas the psi 8-9, psi 7-8, and psi 6-7 analogues were 7-, 36-, and 39-fold less potent. There was a close correlation between the ability of each pseudopeptide to inhibit binding of 125I-Bolton-Hunter-SP and to affect amylase secretion. [psi 10-11]SP inhibited SP-stimulated amylase release in a competitive manner, and its inhibitory ability was specific for the SP receptor. Despite [psi 10-11]SP, spantide, and [psi 10-11]spantide having similar affinities for the SP receptor (Ki, 2-6 microM), for inhibition of binding of 125I-[Tyr4]bombesin, the analogues differed with [psi 10-11]SP having a 50-fold lower affinity than for the SP receptor, whereas [psi 10-11]spantide had a 4-fold lower affinity and spantide a 1.5-fold lower affinity for the SP receptor. These results demonstrate that SP pseudopeptides represent a new class of SP receptor antagonists and, in contrast to the currently described SP receptor antagonists, are more specific for SP receptors.

Short-chain pseudopeptide bombesin receptor antagonists with enhanced binding affinities for pancreatic acinar and Swiss 3T3 cells display strong antimitotic activity.

The high inhibitory potency of the previously developed bombesin antagonist [Leu13, psi CH2NHLeu14]bombesin (analogue I) (IC50 values of 30 and 18 nM for inhibition of bombesin-stimulated amylase secretion from guinea pig acinar cells and Swiss 3T3 cell growth, respectively) diminished considerably when shorter chain lengths were examined. For instance, [Leu13, psi CH2NHLeu14]bombesin-(5-14),[Leu13, psi CH2NHLeu14] bombesin-(6-14), and [Leu9, psi CH2NHLeu10]neuromedin C had IC50 values of 150, 150, and 280 nM, respectively. Incorporation of a D-Phe residue at position 6 of [Leu13, psi CH2NHLeu14] bombesin did not significantly change the various biological parameters. However, its presence in [Leu13, psi CH2NHLeu14]bombesin-(6-14) and at position 2 of psi-neuromedin C-(2-10) resulted in about 10-fold increases in potency up to and above that of the original antagonist. For instance, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) and des-Gly1-[D-Phe2,Leu9,psi CH2NHLeu10]neuromedin C exhibited IC50 values of 5 and 28 nM, respectively. Analogues based on the litorin sequence which contains an NH2-terminal pyroglutamic acid residue at the bombesin position 6 equivalent were also quite potent. The ability of various analogues to interact with bombesin receptors on pancreatic acini correlated reasonably well with potencies derived from inhibition of bombesin-stimulated growth of Swiss 3T3 cells. Additional studies of NH2- and COOH-terminal structure-activity relationships resulted in the synthesis of [D-Phe6,Leu13,psi CH2NHPhe14]bombesin-(6-14), which was particularly effective in inhibiting 3T3 cell growth at high picomolar concentrations (IC50 = 0.72 nM and Ki = 3.1 nM for 3T3 cells; IC50 = 7.5 nM and Ki = 9.9 nM for acini). Detailed investigations with one of the most potent antagonists, [D-Phe6,Leu13,psi CH2NHLeu14]bombesin-(6-14) (Ki = 14 nM for acini cells and 7.1 for 3T3 cells), demonstrated that this analogue was a competitive inhibitor of bombesin and that this activity was specific for the bombesin receptor. Thus, inhibitory potencies have been improved generally up to 25 times over previously reported structures; and, given that bombesin itself has a Ki of 1.2 nM for 3T3 cell binding, some of these analogues are extraordinarily high affinity receptor antagonists. They can also be synthesized more readily and offer fewer proteolytic degradation sites than the original pseudopeptide and should be excellent candidates for in vivo studies aimed at inhibition of bombesin-dependent human small cell lung carcinoma growth.