ACE-like hydrolysis of gastrin analogs and CCK-8 by fundic mucosal cells of different species with release of the amidated C-terminal dipeptide.

Various gastrin analogues and CCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) are hydrolyzed in vitro by angiotensin-converting enzyme (ACE), the main and initial cleavage occurring at the Met-Asp (or Leu-Asp) bond, releasing the C-terminal dipeptide amide Asp-Phe-NH2. Tetragastrin analogues (e.g., Boc-Trp-Leu-Asp-Phe-NH2) are degraded by a vesicular membrane fraction from rat gastric mucosa, yielding the C-terminal dipeptide Asp-Phe-NH2. We report here on the degradation of gastrin analogues and CCK-8 by a gastric mucosal cell preparation containing specific gastrin receptors. We have shown that gastrin analogues were specifically degraded by gastric mucosal cells from different species (e.g., rabbit and dog) at 37 degrees C (pH 7.4), releasing the C-terminal dipeptide Asp-Phe-NH2, similarly to ACE. This cleavage was found to be temperature and pH sensitive, and was inhibited by metalloproteinase inhibitors and by captopril, strongly suggesting that this enzymatic system closely resembles ACE. We have also demonstrated that a close correlation seems to exist between the apparent affinity of the gastrin analogues for gastrin receptors on gastric mucosal cells, and their ability of being hydrolyzed by this cell preparation. Moreover, all gastrin analogues which have been demonstrated to act as gastrin antagonists remained unaffected in the incubation conditions.

Synthesis of gastrin antagonists, analogues of the C-terminal tetrapeptide of gastrin, by introduction of a beta-homo residue.

A series of analogues of Boc-Trp-Leu-Asp-Phe-NH2, a potent gastrin agonist, were synthesized by introducing a beta-homo residue in the sequence. These compounds were tested in vivo on acid secretion, in the anesthetized rat, and for their ability to inhibit binding of labeled gastrin to its receptors on gastric mucosal cells. These analogues behaved as gastrin antagonists. The most potent compounds in this series were Boc-Trp-Leu-beta-homo-Asp-NHCH2C6H5 (10) (IC50 = 1 microM, ED50 = 0.2 mg/kg), Boc-Trp-Leu-beta-homo-Asp-NHCH2CH2C6H5 (11) (IC50 = 0.75 microM, ED50 = 0.5 mg/kg), Boc-Trp-Leu-beta-homo-Asp-Phe-NH2 (12) (IC50 = 1.5 microM, ED50 = 0.1 mg/kg), and Boc-Trp-Leu-beta-homo-Asp-D-Phe-NH2 (13) (IC50 = 2 microM, ED50 = 0.1 mg/kg). We could demonstrate the importance of the region of the peptide bond between leucine and aspartic acid and of the structure of the C-terminal dipeptide Asp-Phe-NH2, for exhibiting biological activity on acid secretion.

Synthesis and biological activities of pseudopeptide analogues of the C-terminal heptapeptide of cholecystokinin. On the importance of the peptide bonds.

A series of pseudopeptide analogues of the C-terminal heptapeptide of cholecystokinin in which each peptide bond, one at a time, has been replaced by a CH2NH bond were synthesized: Z-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp psi-(CH2NH)Phe-NH2 (1), Z-Tyr(SO3-)-Nle-Gly-Trp-Nle psi (CH2NH)Asp-Phe-NH2 (2), Z-Tyr(SO3-)-Nle-Gly-Trp psi-(CH2NH)Nle-Asp-Phe-NH2 (3), Z-Tyr(SO3-)-Nle-Gly psi(CH2NH)Trp-Nle-Asp-Phe-NH2 (4), Z-Tyr(SO3-)-Nle psi-(CH2NH)Gly-Trp-Nle-Asp-Phe-NH2 (5), Z-Tyr(SO3-)-Met-Gly-Trp-Nle-Asp psi (CH2NH)Phe-NH2 (6), Z-Tyr-(SO3-)-Met-Gly-Trp-Nle psi (CH2NH)Asp-Phe-NH2 (7), Z-Tyr(SO3-)-Met-Gly-Trp psi (CH2NH)Nle-Asp-Phe-NH2 (8). These derivatives were studied for their ability to stimulate amylase release from rat pancreatic acini and to inhibit the binding of labeled CCK-9 to rat pancreatic acini and to guinea pig brain membrane CCK receptors. They were compared to the potent CCK-8 analogue Boc-Asp-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2. All of these pseudopeptides were able to stimulate amylase secretion with the same efficacy as CCK-8 but with varying potencies. These compounds were also potent in inhibiting the binding of labeled CCK-9 to CCK receptors from rat pancreatic acini and from guinea pig brain membranes.

Biological effects of human gastrin I and II chemically modified at the C-terminal tetrapeptide amide.

Binding to gastrin receptors and gastric acid secretion experiments were performed with gastrin derivatives modified at the C-terminal tetrapeptide amide from HG-13 sequence. 1. When the ultimate phenylalanine amide was replaced by a phenethylester or a phenetylamide moiety, the resulting compound bound to gastrin receptors (Kd approximately 10 nM) and exhibited antagonist activity on gastrin-induced acid secretion in the anesthetized rat. 2. Changing the peptide bond between Trp and Leu residues to a -omega(CH2-NH)- bond resulted in a compound which also bound to gastrin receptors (Kd approximately 10 nM) but presented agonist activity on acid secretion in the rat. In contrast, when the peptide bond between Leu and Asp residues was replaced by a -omega(CH2-NH)- bond, the resulting compound was devoid of any affinity for gastrin receptor (Kd greater than 10(-6) M) and of any biological activity. 3. The HG-13 derivatives were synthesized in sulfated and unsulfated forms: O-sulfation of the HG-13 tyrosine residue did not change its intrinsic in vivo activity but enhanced its affinity for gastrin receptors (Kd approximately 0.3 nM). On the contrary, O-sulfation of the various chemically modified HG-13 had no significant effect in either in vitro or in vivo experiments. 4. Finally, no significant difference between binding on parietal (F3) and nonparietal (F1) cells was observed, in agreement with the presence of a gastrin-type receptor in these two cell populations.

Cathepsin D displays in vitro beta-secretase-like specificity.

The formation of A beta and A beta-containing fragments is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of A beta and its C-terminally extended sequences is liberated from the beta-amyloid precursor protein (beta APP) by beta-secretase(s). This activity appears highly increased by the presence (N-terminally to Asp-1) of a double-mutation (KM-->NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the "normal' (N peptide) and mutated (delta NL peptide) sequences targeted by beta-secretase(s), we have detected a human brain protease displaying preferred efficiency for the delta NL peptide than for the non-mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PC12 cells and primary cultured astrocytes. The pepstatin sensitivity and pH maximum of the brain activity that appeared reminiscent of those displayed by the acidic protease cathepsin D led us to examine this enzyme as a putative beta-secretase-like candidate. Purified cathepsin D displays higher catalytic parameters for the delta NL peptide than for the non-mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. However, cathepsin D does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block A beta secretion by transfected cells. Furthermore, cathepsin D hydrolyses recombinant baculoviral delta NL beta APP751 at a 6-fold higher rate than beta APP751 and gives rise to a 12-kDa C-terminal product that is recognized by antibodies fully specific of the N-terminus of A beta. Altogether, our study indicates that cathepsin D displays several in vitro beta-secretase-like properties that suggests that this protease could fulfill such a role, at least in the Swedish genetic form of Alzheimer's disease.

Gastrin13 and the C-terminal octapeptide of cholecystokinin are differently coupled to G-proteins in guinea-pig brain membranes.

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we synthesized and characterized a labelled gastrin ligand, [125I]BH[Leu15]gastrin-(5-17) (3-(3-[125I]iodo-4-hydroxyphenyl)propionyl[Leu15]gastrin-(5-17)). On isolated canine fundic mucosal cells and human Jurkat lymphoblastic cell line, known to express CCKB/gastrin receptors, the binding experiments performed indicate that [125I]BH[Leu15]gastrin-(5-17) provides a convenient biologically active ligand for cholecystokinin/gastrin receptor studies. We showed in this study that, on guinea-pig brain membranes known to possess CCKB and CCKA receptors, [125I]BH[Leu15]gastrin-(5-17) binds to a single class of high-affinity binding sites in a saturable and specific manner. [125I]BH[Leu15]gastrin-(5-17) interacts with guinea-pig brain membranes with a maximal binding capacity that is about three-fold lower than that of [125I]BHCCK8 (CCK8, the C-terminal octapeptide of cholecystokinin). The apparent affinities of CCK analogues and selective CCK receptor antagonists L-365,260 and MK-329 for the sites labelled by both probes were in accordance with a CCKB-like profile. Association-dissociation kinetics of [125I]BH[Leu15]gastrin-(5-17) and [125I]BHCCK8 were performed and compared. They showed that [125I]BHCCK8 equilibrated more slowly than [125I]BH[Leu15]gastrin-(5-17). The effects of pH, monovalent and divalent cations on binding of both probes were investigated. The results obtained did not indicate strong differences between [125I]BH[Leu15]gastrin-(5-17) and [125I]BHCCK8 binding. Binding experiments in the presence of stable analogues of GTP showed a different behavior between [125I]BH[Leu15]gastrin-(5-17) and [125I]BHCCK8. GTP gamma S strongly decreased [125I]BH[Leu15]gastrin-(5-17) binding whereas it weakly affected [125I]BHCCK8 binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Are C-terminal octapeptide of cholecystokinin and [Leu11]gastrin-(5-17) different in stimulating acid secretion in isolated rabbit gastric glands?

In the present study we compared various CCK(B) receptor antagonists and tried to detect a difference in biological activity between the C-terminal octapeptides of cholecystokinin (CCK-8) and [Leu11]gastrin-(5-17) in isolated rabbit gastric glands. Binding experiments showed that different CCK(B)/gastrin receptor agonists bound with high affinity and that antagonists inhibited this binding in accordance with a CCK(B)/gastrin pharmacological profile. [Leu11]gastrin-(5-17), CCK-8 and cionin were found to induce [14C]aminopyrine accumulation to 25% above the basal level. Under the same experimental conditions, histamine induced a response twice as great as the response obtained with [Leu11]gastrin-(5-17) or CCK-8. [Leu11]gastrin-(5-17) (10(-7) M), CCK-8 (10(-8) M) and cionin (10(-8) M) appeared to be full agonists. CCK(B)/gastrin receptor antagonists including L-365,260 (3R-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-N-(3-methylphenyl) urea), L-364,718 (3S-(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-1H-indole-2-carboximide) (a selective CCK(A) receptor antagonist), PD-135,158 (4([2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[[1.7.7-trimethyl-bicyclo[2. 2.1]hept-2-yl)oxy]carbonyl]amino]propyl]amino]-1-phenylethyl] amino-4-oxo-[1S-1alpha.2beta[S*(S*)]4alpha]]-butano nate N-methyl-D-glucamine) (bicyclo system 1S-endo), YM-022 ((R)-1-[2,3-dihydro-1-(2'-methylphenacyl)-2-oxo-5-phenyl-1H-1,4-++ +benzodiazepin-3-yl]-3-(3-methylphenyl)urea) and JMV-180 (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-O-CH2-CH2-C6H5) exhibited the same profile for inhibition of [Leu11]gastrin-(5-17) or CCK-8-induced [14C]aminopyrine accumulation in rabbit gastric glands. These results suggested that [Leu11]gastrin-(5-17) and CCK-8 induced [14C]aminopyrine accumulation by the same mechanism. [Leu11]gastrin-(5-17)- or CCK-8-induced [14C]aminopyrine accumulation was inhibited by about 40% by the histamine H2 receptor blocker cimetidine. These results are consistent with there being cooperativity between [Leu11]gastrin-(5-17) (or CCK-8) and histamine in the acid secretory pathway. Similarly, the CCK(B)/gastrin receptor antagonists were tested against histamine-induced [14C]aminopyrine accumulation and surprisingly, only compound L-365,260 appeared active and even more potent than cimetidine.

Cholecystokinin and gastrin are not equally sensitive to GTP gamma S at CCKB receptors: importance of the sulphated tyrosine.

We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and cholecystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCKB receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCKB receptors were measured by inhibition of [125I]Bolton Hunter-CCK-8 (3-[125I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTP gamma S (guanosine 5'-O-(3-thio)triphosphate) or aluminum tetrafluoride (AlF4-). Activation of the G proteins by GTP gamma S or AlF4- led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCKB receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCKB receptors and their related intracellular events.

Synthesis and biological activities of some human gastrin analogs.

The synthesis of analogs of the C-terminal tridecapeptide of gastrin is described. These pseudopeptide analogs were obtained either by replacing the C-terminal phenylalanine amide with 2-phenylethylalcohol or with 2-phenylethylamine, or by replacing the peptide bond between Trp and Leu, or between Leu and Asp with an aminomethylene (CH2NH). The ability of these compounds to stimulate gastric acid secretion in anesthetized rats and to inhibit binding of labeled CCK-8 to isolated cells from rabbit fundic mucosa was tested. [desPhe13, Leu11]-HG-12-I-beta-phenylethylester 33, [desPhe13, Leu11]-HG-12-II-beta-phenylethylester 38, [desPhe13, Leu11]-HG-12-I-beta-phenylethylamide 32, and [desPhe13, Leu11]-HG-12-II-beta-phenylethylamide 37 acted as gastrin receptor antagonists, while [Trp10-psi(CH2NH)-Leu11]-HG-13-I 31 and [Trp10-psi(CH2NH)-Leu11]-HG-13-II 36 acted as agonists. Unexpectedly, [Leu11-psi(CH2NH)-Asp12]-HG-13-I 30 and [Leu11-psi (CH2NH)-Asp12]-HG-13-II 35 were almost devoid of affinity for the gastrin receptor.

[Strategies for identification of secretases implicated in Alzheimer's disease]. / Stratégies d'identification des sécrétases impliquées dans la maladie d'Alzheimer.

In Alzheimer's disease, cortical areas of affected patients are invaded by extracellular proteinous deposits called senile plaques, the main component of which is called amyloid beta-peptide or A beta. This peptide derives from the proteolytic attack of a precursor, the beta-amyloid precursor protein, by two enzymes called beta- and gamma-secretases. Alternatively, beta APP can be cleaved by an additional activity named alpha-secretase that occurs inside the A beta sequence, thereby precluding its formation, and concomitantly liberating a secreted fragment, namely APP alpha. Therefore, secretases seem to play a key role in the control of physiological and potentially pathogenic beta APP catabolites and could be envisioned as possible therapeutic targets in Alzheimer's disease. Here, we describe possible experimental approaches to identify such proteolytic activities.

[Structure-activity relationship of cystokinins: analogs exhibiting selective activity]. / Relations structure-activité de la cholécystokinine: analogues présentant des activités sélectives.

Structure-activity relationship on cholecystokinin is presented. C-terminal heptapeptide analogues of cholecystokinin exhibiting selective agonist or antagonist activities were synthesized and their biological and pharmacological properties studied. We showed that: 1) Suppression of the C-terminal phenylalanine residue leads to peripheral as well as central cholecystokinin receptor antagonists; 2) Suppression of the C-terminal amide function produces "partial agonists" exhibiting interesting biological and pharmacological activities; 3) Replacement of L-tryptophan residue by D-tryptophan in such "partial agonist analogues" resulted in potent CCK receptor antagonists; 4) The peptide bond between methionine28 and glycine29, as well as the glycine residue are quite significant for the central biological activity; 5) It is possible to obtain highly potent and selective CCK analogues for the central receptor (CCK-B) by cyclization including the C-terminal tetrapeptide. Synthesis and pharmacological studies with these analogues have allowed to precise the significance of some amino acid residues as well as of some peptide bonds. They are significant pharmacological tools for the study of CCK-A (peripheral) and CCK-B (central) receptors, their biological actions and their associated intracellular messengers.

Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide.

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.

Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment-1. [Tyr47, Nle53] preprorenin-(47-60) peptide methyl ester.

The [Tyr47, Nle53] preprorenin (47-60) peptide methyl ester, a tetradecapeptide related to the human renin prosegment, has been synthesized using a three-segment coupling strategy. Selective deprotection of the segments before coupling allowed an easy removal of the final tetradecapeptide side chain-protecting groups by acidolysis and an easy purification. Antibodies raised against this peptide bound the plasmatic inactive renin.

Solution synthesis of antigenic and inhibiting peptides of the human renin prosegment-2. [Tyr40] preprorenin-(40-50) peptide methyl ester.

The [Tyr40] preprorenin (40-50) peptide methyl ester, an undecapeptide related to the human renin prosegment, has been synthesized using a stepwise strategy with hydrogenolisable protections on the side chains. The final deprotection was very difficult as observed by 1H NMR and reversed phase HPLC. 2D 1H NMR spectroscopy of the purified peptide allowed the assignment of all protons.

Peptides mimicking the flap of human renin: synthesis, conformation, and antibody recognition.

Four peptides related to human renin flap region have been synthesized. Two of them are ring closed through appropriately designed disulfide bridges. Structure analysis involving IR and NMR techniques and recognition by polyclonal human renin antibodies provides support for a beta-hairpin secondary structure of the cyclized peptides identical with that presented by the flap section in the speculative human renin model [Blundell, T., Sibanda, B. L., & Pearl, L. (1983) Nature (London) 304, 273-275; Sibanda, B. L., Blundell, T., Hobart, P. M., Fogliano, M., Bindra, J. S., Dominy, B. W., & Chirgwin, J. M. (1984) FEBS Lett. 174, 102-111].

Structure-activity relationship studies on cholecystokinin: analogues with partial agonist activity.

In the present study, hepta- and octapeptide analogues of the C-terminal part of cholecystokinin, modified on the C-terminal phenylalanine residue, were synthesized. CCK analogues were prepared in which the peptide bond between aspartic acid and phenylalanine had or had not been modified and were lacking the C-terminal primary amide function. These CCK derivatives were able to cause full stimulation of amylase release from rat pancreatic acini but without a decrease in amylase release at supramaximal concentrations. There was a close relationship between the abilities of these derivatives to stimulate amylase release and their abilities to inhibit binding of 125I-BH-CCK-9 to CCK receptors on rat and guinea pig pancreatic acini. These CCK analogues were also able to recognize the guinea pig brain CCK receptors, some of them being particularly potent. The findings indicate that the aromatic ring of phenylalanine is important for the binding to brain and pancreatic CCK receptors, whereas the C-terminal primary amide function is not essential for the binding to pancreatic CCK receptors but is crucial for biological activity of rat pancreatic acini.