Abilities of some tryptophan and phenylalanine derivatives to inhibit gastric acid secretion

158

Biochimica et Biophysica Acta 845 (1985) 158-162

Elsevier BBA 11458

A b i l i t i e s o f s o m e tryptophan and p h e n y l a l a n i n e derivatives to inhibit gastric acid secretion

R. M a g o u s

a,

j. M a r t i n e z b,,, M . F . L i g n o n a n d J.P. Bali a

a,

D. N i s a t o c, B. C a s t r o b

a E.R. C N R S 228, Ecole Nationale Sup~rieure de Chimie et Facultb de Pharmacie, 8 Rue de l'Ecole normale, 34075, Montpellier, b Centre de Pharmacologie-Endocrinologie mixte C N R S - I N S E R M , B.P. 5055, 34033 Montpellier Cedex, and c Centre de Recherches Clin-Midy-Sanofi, Avenue du Professeur Blayac, 34000 Montpellier (France)

(Received October 31st, 1984)

Key words: Gastric acid secretion; Phenylalanine; Tryptophan; Amino acid derivative

Benzotript (N-p-chlorobenzoyI-L-tryptophan) has been shown to be a receptor-antagonist in vivo and in vitro for peptides from the gastrin family. In the present study, we examine tryptophan, and some of its N- and C-acylated derivatives, as well as some phenylalanine derivatives, to show their ability to inhibit gastrin-induced acid secretion in the rat in vivo and to compete for the binding of [t25l]-(Leu-15)-HG-17 to its cellular receptor on rabbit isolated gastric mucosal cells. N- and C- derivatives of tryptophan and phenylalanine were found to inhibit gastrin-induced acid secretion and binding of [tzsI]-(Leu-15)-HG-17 to its mucosal cell receptors. By either criterion, the relative antagonistic potencies of the compounds tested were: tert-butyloxycarbonyI-L-tryptophan-p-nitrophenyl ester = tert-butyloxycarbonyI-L-tryptophan-carbamoyimethyl ester > tert-butyloxycarbonyl-L-tryptophyl-L-methionyl-carbamoylmethyl ester = tert-butyloxycarbonyI-Lphenylalanine-carbamoyimethyl ester --- tert.butyioxycarbonyl-L-tryptophyl-L-methionyi-amide > tertbutyloxycarbonyl-L-tryptophan > tert-butyioxycarbonyl-L-phenylalanine > benzyloxycarbonyi-L-tryptophan = benzotript, with minor differences between the in vivo and the in vitro experiments. These results demonstrate that both the nature of the amino acid residue and the N- and C- substitutions are important in determining antagonist activity and affinity for gastrin receptors.

Introduction

In a recent study, we have shown that benzotript (N-(p-chlorobenzoyl)-L-tryptophan) was able to inhibit both gastrin binding and acid production from isolated gastric mucosal cells. This inhibition was shown to be competitive for gastrin and non-competitive for acetylcholine-stimulated cells. Proglumide, an analogue of the C-terminal dipeptide t-aspartyl-t-phenylalanine-amide of * To whom correspondence should be addressed. Abbreviations: HG-17, human gastrin 17; Hepes, 4-(2-hy-

droxyethyl)-l-piperazineethanesulfonic acid

gastrin, also inhibited acid production, but with a 10-fold lower potency than benzotript [1]. These two compounds were also found to be antagonists of gastrin-stimulated acid secretion in vivo, with the same ratio of potency. These results and others obtained by Morley [2], by Yabe et al. [3] and by ourselves [4,5] suggest an important role for the tryptophan residue in the binding of gastrin to the cell receptors. Similarly, Rajh et al. [6] suggested that the charge donor capacity of this amino acid residue is of primary importance to the biological activity of cholecystokinin-pancreozymin. More recently, Jensen and coworkers [7-9] demonstrated that some N-acyl tryptophan derivatives,

0167-4889/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

159

TABLE I STRUCTURES AND NAMES OF VARIOUS TRYPTOPHAN AND PHENYLALANINE DERIVATIVES

/ CO_R1 ~ - ~ - C H

2-CH\ NH-R 2

H R1 = OH R' = O H R' = O H R1 = OCH 3

R2 = R2 = R2 = RE=

R1 = O - ~ ) - N 0 2

R 2 = Boc

Boc-Tryptophan-ONp

R1=

R 2 = Boc R 2 = Boc

Boc-tryptophan-Cam Boc-Trp-Met-NH 2

O-CH2-CONH 2 R1 = M e t - N H 2 R1 = OH

H Boc Z H, H C l

R2 = CI-~-CO-

Tryptophan Boc-tryptophan Z-tryptophan Tryptophan-OMe-HCl

Benzotript

including benzotript, can function as cholecystokinin-receptor antagonists, competitively inhibiting cholecystokinin-induced amylase release, Ca 2+ efflux and [125I]cholecystokinin binding to isolated pancreatic acinar cells. Because of the chemical similarity of .cholecystokinin and gastrin, we investigated the effect of some N- and C- substituted derivatives of tryptophan and phenylalanine (Table I) on gastrin binding to isolated gastric mucosal cells, and gastrin-induced acid secretion. Material and Methods

Materials. Rabbits were from INRA, Montpellier, France, and male Wistar rats (300 g) from "Centre d'Elevage", Montpellier, France. Collagenase was from Serva, F.R.G. Earle's balanced salt medium was from Biomerieux, France. [1251]Leu-15 human gastrin 17 (2000 Ci/mmol) was from New England Nuclear. (Nle-ll)-HG-13 was a gift from Professor E. Wunsch, Max-Planck Institut ftir Biochemie, Munich, F.R.G. Unless otherwise stated, the standard incubation medium contained: medium A, 132 mM NaCI, 5.4 mM KC1, 5 mM Na2HPO 4, 1 mM NaH2PO 4, 1.2 mM MgSO4, 1 mM CaC12, 25 mM Hepes, 0.2% glucose, 0.2% bovine serum albumin, 0.02% phenol red, pH 7.4; medium B, Earle's balanced salt

medium without bicarbonate and containing 10 mM Hepes and 0.2% bovine serum albumin, pH 7.4. All the incubations were performed with 95% 02/5% CO2 as the gas phase. The propionate-succinate solution contained 1.6 mM propionic acid, 1.65 mM succinic acid, 3.3 mM NaOH, pH 5.5. Amino acid and peptide derivatives. L-Tryptophan, tert-butyloxycarbonyl-L-tryptophan (BocTrp), benzyloxycarbonyl-L-tryptophan (Z-Trp), tert-butyloxycarbonyl-L-phenylalanine (Boc-Phe) and L-tryptophan methyl ester hydrochloride (LTrp-OMe-HCl) were from Bachem, Bubendorf, Switzerland. Tert-Butyloxycarbonyl-L-tryptophan p-nitrophenyl ester (Boc-Trp-ONp) was prepared according to the procedure of Bodanszky and Natarajan [10]. Tert-Butyloxycarbonyl-L-tryptophan carbamoylmethyl ester (Boc-Trp-Cam) and tert-butyloxycarbonyl-L-phenylalanine carbamoylmethyl ester (Boc-Phe-Cam) were synthesized as previously described [11,18]. The syntheses of the peptides were performed according to the procedures published previously [12]. The details of these syntheses will be published as a separate report. Benzotript was a gift from Professor A. Rovati, Monza, Italy. Abbreviations used are in accordance with the IUPAC-IUB recommendations (Eur. J. Biochem. (1984) 138, 5-7). Gastric acid secretion in oivo. Gastrin-induced acid secretion was determined in anaesthetized rats according to the experimental procedure of Ghosh and Schild [13] as modified previously [14,15]. Fasted male rats were anaesthetized with urethane (intraperitoneally). After tracheotomy, the oesophagus and duodenum were canulated. A closed-loop was established through a constant-rate pump with an output of 3 ml-rain - 1 and the stomach was washed at 30°C with 30 ml of a propionate-succinate solution. The cumulative pH of the perfusate was recorded continuously with time. (NIe-11)-HG-13 dissolved in 0.15 M NaCI was bolus-injected intravenously and the amount of H ÷ secreted was determined by the pH difference between stimulated and basal recorded traces. 80 pmol of (Nle-11)-HG-13 per injection, which induced submaximal stimulation, were usually employed as the stimulant, and the mean of H ÷ secreted was 1 6 + 2 /~mol H ÷ (number of separate experiments= 17). The inhibitory effect of the various compounds was measured after

160

simultaneous bolus injection of different concentrations of each compound and (NIe-11)-HG13. Before a new bolus injection of the peptides, the stomach was washed until stabilization of basal acid secretion occurred. No more than three injections were made on the same animal. The inhibition of H ÷ secretion was expressed as percentage of that secreted after gastrin alone. Isolation of gastric mucosai cells. The collagenase/EDTA procedure employed to dissociate gastric mucosa was as previously described [15]. The fundic mucosa was scraped from a rabbit, chopped into small cubes and dispersed in medium A containing 0.18 U.I./ml collagenase (gassed O2//CO2). After 15 rain incubation at 37°C, tissue fragments were allowed to settle and the medium was discarded. The fragments were washed in CaE÷/MgE+-free medium A containing 2 mM EDTA, then incubated in the same medium for 10 rain. The fragments were transferred to medium A containing 0.18 U.I./ml fresh collagenase and incubated for 15 rain at 37°C with continuous gassing (O2/CO2). The cell suspension was centrifuged for 15 min at 200 × g, then washed twice with medium A. This procedure gave about 5 • 107 cells per g of wet mucosa with 95% viability (trypan blue exclusion). The mixed population contained 45% parietal cells (determined by electron microscopy).

Binding Studies. Gastrin binding was determined by incubation for 30 min at 37°C, in medium B with 20 pM [125I]-(Leu-15)-HG-17 (40000 cpm/ml), of 5- 106 cells/ml and various concentrations of the compounds or unlabelled (NIe-ll)-HG-13. All values reported in this paper are for specific binding, i.e., binding measured with tracer alone or in the presence of various compounds (total binding) minus binding measured with 1/~M (NIe-ll)-HG-13 (nonspecific binding). Results and Discussion

Tryptophan and phenylalanine are component parts of the C-terminal tetrapeptide of gastrin. Some of their derivatives were able to inhibit gastrin-induced acid secretion in the reperfused rat stomach (Ghosh and Schild model) (Table II). Acylation of the amino function of tryptophan increased both the potency with which tryptophan inhibited gastrin-induced acid secretion and gastrin binding to cell receptors: tert-butyloxycarbonylL-tryptophan and benzyloxycarbonyl-L-tryptophan were much more potent in inhibiting these parameters than was L-tryptophan. As was previously observed [16,17], acylation by a tert-butyloxycarbonyl group yielded a product of better potency than did acylation by a benzyloxycarbonyl group:

T A B L E II A N T A G O N I S T IN VIVO ACTIVITIES A N D I N H I B I T I O N OF B I N D I N G O F [125I]-(LeulS)-HG-17 TO G A S T R I N R E C E P T O R S BY T R Y P T O P H A N A N D P H E N Y L A L A N I N E DERIVATIVES Boc is

tert-butyloxycarbonyt; Z

is benzyloxycarbonyl.

Tryptophan and phenylalanine derivatives

In vivo activity EDso ( p m o l / k g ) a

Binding ICso (M) b

Boc-L-tryptophan Z-L-tryptophan L-Tryptophan methyl ester HC1 Boc-L-tryptophan p-nitrophenyl Boc-L-tryptophan carbsmoylmethyl e s t e r Boc-L- tryptophyl-L-methionine amide Boc-L-phenylalanine Boc-L-tryptophyI-L-methionine carbamoylmethyl e s t e r Boc-L-phenylalanine carbamoylmethyl e s t e r L-Tryptophan Benzotript

82.5 150 550 23.5 35 57 150 45 49 880 175

5'10 -4 3-10 - 4 > 5.10 -3 3-10 -5 5"10 - 4 1.10 - 3 1.5.10- 3 3.10- 3 1-10 -3 > 10- 2 2.5.10- 4

a Each value was determined with 3 doses of antagonist in duplicate. b Each value was determined from binding inhibition curves drawn with 4 concentrations in duplicate in 3 separate experiments.

161

tert-butyloxycarbonyl-L-tryptolShan

was 2-times more potent than benzyloxycarbonyl-L-tryptophan in inhibiting gastrin-induced acid secretion. Acylation of the amino function of tryptophan by the p-chlorobenzoyl group yielded a compound (benzotript) with almost the same antagonist activity as benzyloxycarbonyl-L-tryptophan. Tryptophan-derivatives with C- and N-terminal blocked presented interesting inhibitory activity. In this series,

tert-butyloxycarbonyl-L-tryptophan-p-nitrophenyl ester and tert-butyloxycarbonyl-L-tryptophancarbamoylmethyl ester were the most potent: they were 3-times more potent than tert-butyloxycarbonyl-L-tryptophan and 30-times more potent than L-tryptophan. Accordingly, these two compounds were the most potent in inhibiting binding of [125I]-(Leu-15)-HG-17 to its receptors, in vitro. Extension of the carboxylic group of tryptophan b y L - m e t h i o n i n e a m i d e or L - m e t h i o n i n e carbamoylmethyl ester (giving one more amino acid residue of the C-terminal gastrin sequence) yielded active inhibitors which were less potent than tert-butyloxycarbonyl-L-tryptophan-pnitrophenyl ester or tert-butyloxycarbonyl-L-tryptophan-carbamoylmethyl ester. These two derivatives were not very potent in inhibiting binding of [125I]-(Leu-15)-HG-17 to its receptors. Surprisingly, addition of the methionine residue did not increase either the in vivo antagonist activity or the inhibition of gastrin binding to its receptors. With the phenylalanine derivatives, similar results were observed but the inhibitory activity of these compounds was lower, as well as their binding to gastrin receptors. Although in vivo and in vitro results were in accordance, some discrepancies occurred. They might be due to differences in the lipophilicity of compounds or to species differences, or due to the presence in the gastric mucosal cell preparations of gastrin receptors not related to acid secretion. Our results are in accordance with those of Jensen et al. [9], who reported a cholecystokinin-antagonist activity of tryptophan and phenylalanine derivatives on pancreatic acini. These findings are another example of compounds able to interact with both cholecystokinin and gastrin receptors.

Conclusion In a previous study [1] we found that benzotript and proglumide can function as gastrin receptor antagonists. In the present report, we have examined the action of some tryptophan and phenylalanine derivatives on gastrin binding and on inhibition of gastrin-induced acid secretion. We found that some of them, particularly N- and Cprotected derivatives, were potent inhibitors of gastrin-induced acid secretion in vivo. In terms of their ability to antagonize the actions of gastrin in vivo, the relative potencies of the compounds tested were: tert-butyloxycarbonyl-t-tryptophan-p-nitrophenyl ester = tert-butyloxycarbonyl-t-tryptophan-carbamoylmethyl ester > tert-butyloxycarbonyl-L-tryptophan-L-methionyl-carbamoylmethyl ester = tert-butyloxycarbonyl-L-phenylalanine-carbamoylmethyl ester = tert-butyloxycarbonyl-t-tryptophan-L-methionyl°amide > tertbutyloxycarbonyl-L-tryptophan > tert-butyloxycarbonyl-L-phenylalanine > benzyloxycarbonyl-Ltryptophan = benzotript > L-tryptophan-methylester-HCl > L-tryptophan. In terms of inhibiting gastrin binding to its receptors on mucosal cells, almost the same order of potency has been observed.

Acknowledgements The authors would like to thank CNRS, INSERM and P I R M E D for their support in this work, and J. Laur and F. Michel for their skilful technical assistance.

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