Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones

!eurcocptides

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173481,

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IRREVERSIBLE LABELLING ENKEPHALIN CHLOROMETHYL Benyhe, S. Medzihradszky

J. *

OF RAT BRAIN KETONES

Hepp *, M. Szucs, and M. Wollemann

OPIOID

J. Simon,

RECEPTORS

A.

BY

Borsodi,

K.

of Biochemistry, Biological Research Center of the Institute Hungarian Academy of Sciences, 6701 Szeged, P.O.B. 521. * Central Research Institute Hungary; and for Chemistry Hungarian Academy of Sciences, 1025 Budapest, P.O.B. 17. Hungary (reprint request to S.B.) ABSTRACT Chloromethy$ ke,$one derivatives of leucine ertkephalfn (LE), D-Ala -Leu -enkephalin (DALE) and D-Ala -D-Leu enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of membrane with the fraction enkephalin chlor methyl ketones causes a significant 9 inhibition of / H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition nM) S'H/S~~~~:~~~ f~~nd~~~ t:tt affinity (KD C 1 (putative mu-l site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids opioid peptides, suggesting that and nonspecific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was found that the covalegt inhibition of mu al30 sites (/ H/-dihydromorphine and / H/-DAGO binding) 3s more effective than that of delta sites (/ H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels the for opioid receptors, allowing us to study the structure of the mu receptor subtype. ---------_____-_____~~~~~-~-~~-~-_~-_--~________-____________ Abbreviations used LE = Leucine enkephalin; LECK D_;la4";;;3' enkephalin chloromethyl keton9; DALE = enkephalin; DALECK _; LE;$la -Leu5-enkephalin chlorome5hyl ketTne; DADLE = D-Ala2 -enkephalin; DADLECK 2 D-Ala -tLeu5 enkephalin chloromethyl ketone; DAGO = D-Ala -(Me)Phe Gly -01 enkephalin; Phe-Leu-CK = Phe-Leu chloromethyl ketone; TPCK = N-tosyl-L-Phenylalanine chloromethyl ketone

173

INTRODUCTION Chloromethyl ketone derivatives of peptides may serve as selective inhibitors of proteolytic enzymes (l), and may react selectively with corresponding peptide the receptors. Such derivatives of enkephalins seemed to be good candidates for affinity labelling of opioid receptors. Structure-affinity relationship studies showed that the Cterminal end of enkephalins is the relatively free for chemical modification without essential loss of their biological activity (2). Recently leucine enkephalin and D-Ala2-Leu5 -enkephalin were elongated by a chloromethyl ketone group on the C terminus (to yield LECK and DALECK, respectively) were and tested by several laboratories (3-5). It is clear from some of these studies (4,5) that LECK and DALECK bind irreversibly to the opioid receptors in vitro, but hinging selectivity was not proved. However, in a later study / H/-DALECK irreversible binding was reported to be selective for the mu receptor and a single subunit was shown to be labelled (6). The synt9esis Sf another such enkephalin chloromethyl ketone, D-Ala -D-Leu -enkephalin chloromethyl ketone (DADLECK) described here, and the binding potencies of DADLECK, ZLECK and LECK are compared to those enkephalins and opiate alkaloids. MATERIALS

AND METHODS

C:emicals: /3H/- naloxone (3.1 TBq/mmol = 83.7 Ci/mmol), / H/-dihydromorphine (2.51 TBq/mmol = 67.8 Ci/mmol) and 13H/DALE (1.37 TEJq/mmol = 37.3 Ci/mmol) were synthesized by Dr. G. Toth (7-9). H-DAGO (1.67 TBq/mmol = 45 Ci/mmol) was purchased from Amersham. Naloxone hydrochloride was kindly donated by Levorphanol and dextrorphan tartrate were Endo Laboratories. hydrochloride kindly provided Morphine by Hofmann-La-Roche. was a commercial TPCK and DADLE were product. Bacitracin, obtained from Sigma. All other chemicals were of analytical grade. dipeptides Preparation of chloromethyl ketones: The C-terminal (BOC-Phe-Leu-OH, BOC-Phe-Dprotected by tert-butyloxycarbonyl Leu-OH) were each transformed into the mixed anhydride with isobutyl chloroformate and reacted with diazomethane in ether BOC-Phe-Leu-CHN D-Leu-CHN . to BOC-PheThe yield ketones were c$nvei:ed into BOC-Phe- Leu-CH*Cl or diazomethyl Deprotectign HCl at 0 'C. was BOC-Phe-D-Leu-CH2C1 with effected by acidolysis. The dipeptide chloromethyl ketones were acylated with BOCmixed BOC-Tyr-D-Ala-Gly-OH, through the Tyr-Gly-Gly-OH or prepared with isobutyl chloroformate in the presence anhydride and the resulting products (BOC-LECK, of N-methylmorpholine, BOC-DADLECK) were crystallized from acetone-ether. BOC-DALECK, was of the BOC-pentapeptide chloromethyl ketones Deprotection was with HCl in methanol and hydrochloride performed the sealed precipitated anhydrous ether, dried, and kept in with

174

ampoules at 4 OC. compounds were homogeneous by TLC, and All crystalline Details showed the correct composition on elemental analysis. of the synthesis were published elsewhere (10). Membrane preparation: A crude membrane fraction of rat (PVG/C the strain) brain was prepared according to (11). Briefly, were cerebella animals were decapitated, the brains without and homogenized in 30 volumes of ice-cold 50 rapidly removed mM Tris/HCl (pH 7.4) buffer by a teflon-glass homogenizer. at 40,000 x g for 20 min the pellet was After centrifugation resuspended in fresh buffer, incubated at 37 'C for 30 min, then recentrifuged and the final pellet was suspended in 80 volumes (for affinity (for displacement studies) or 5 labelling) of buffer and freshly used. Protein content was determined according to (12). Binding assay: For determining the binding affinity of opioid 50 mM ligands the membrane suspension (300-500 ug protein, Tris/HCl (pH 7.4) buffer containing 50 ug/ml gacitracin) was co-incubated with she radioactive compounds (/ HI-naloxone for 60 min at 0 'C; / H/- dihydromorphine, 20 min, 23 'C in the 45 min, 23 'C) and unlabelled dark; /3~/-~~L~, ligands (in cases of non-specific binding) in a final volume of 1 ml. All assays were performed in triplicate. Non-specific binding was defined as the bound radioactivity in the presence of 10 uM naloxone, morphine or DALE. Incubation was started by the addition of membrane prosein and was stopped by filtration GF/B through Whatman (/ H/-naloxone) GF/C (other radioligands) were glass fibre filters under vaczzm. Filters of ice-cold washed twice with 10 ml buffer and dried. Radioactivity was determined in a toluene-based cocktail by a LKB Minibeta liquid scintillation spectrometer. Affinity reaction: The incubation mixture contained 500 ul of rat brain membranes (2-3 mg protein), 200 ul of the ligand at different concentrations, in 50 mM Tris/HCl (pH 7.4) buffer in a final volume of 2 ml. After 90 min incubation at 30 'C, the samples were diluted with 28 ml ice-cold buffer and centrifuged at 25,000 x g for 10 min. The pellets were suspended in 30 ml buffer, incubated for 10 min at room temperature and centrifuged again. This washing step was repeated 4 times. The final pellets were homogenized in 8 ml of the 50 mM Tris/HCl buffer and used immediately for binding assays. RESULTS AND DISCUSSION The affinity of the chloromethyl ketone derivatives in binding reversibly to rat brain membranes was compared to mu and delta agonists as well as antagonists. Table I shows the calculated IC values of the different compounds toward multiple opio59 binding sites. LECK, DALECK 3nd DADLECK exhibit a high affinity in displacement at the / H/-naloxone binding sites. Addition of sodium ions to 100 mM (13) caused a loss 4n the affinity of the chloromethyl ketones in competing with / H/-naloxone, indicating their agonist character. 175

TABLE I. Relative

potencies

of

various

ligsnds

in colpeting binding

Unlsb~ll~d

T

capounds

in

;.eine

enkephslin

NSloxonS

or

/3H/-dih,drmrphine

unlsb~lkl Displscevnt of

specific

compounds in c”rvSs

wire

3% 1sbcllSd

agonist

and antagonist

ratio

‘%o btm

NaCl

T

1) )&DALE

(4

(delta)

17 20 17

70

6

z

:

60 50 60

3000 loo0 2500

50 20 ha

20

3OOw lOa@ 8 2 50000 2

zoo00

or

vith

200 150 8OOOO

constructed

of

I-naloxone

; 0.5

increasing

50 us/ml becitrscin.

wsre

values

determined.

All (ligand

Vsluas

1 0.7 1

0.03 0.01

20 or

conccntrstions SSSSJS rere concentration reprssent

12 10 12

4 40

1

(2 “kl) in the absence

“M) with

and the IC M

binding)

2; 75

presence

0.01 of

(16”-lo4

perforrd

in

producing

the mss” of

;Pu/delts ratio

20 15 20

:8

0.5 /

/3K/-DALE (3

the presence

ligsnd

ma and delta

2000 2000 1500

incubated (3 “H)

with

msmbrsnes

120 100 90

LECK DNZCK DADLECK

membranes rere

Ha+

~100 d

buffer

DADLE

Rst brain

brain

ICso w9

(LE)

TFCK Phe-LSU-CK ‘k.rphine k*O~phS”Ol ktrorphs” YSlOXO”S

reversibly

to rat

2-4

sodium

ions.

H ) of triplicate. 50 % inhibition independent

experimsnts.

ketone chloromethyl enkephalin the binding of The derivatives was extremely high in comparison with TPCK and Pheof the C terminal carboxyl group Leu-CK (Table I). Replacement produced an increase the chloromethyl ketone 3'n enkephalins by binding sites, ligand affinity to mu (/ H/-dihydromosphine) of was their affinity to delta sites (/ H/-DALE binding) whereas reduced. When membranes were preincubated at 30 'C for 40 min (at enkephalin concentrations of an pH 7.4) with different dosewashed, a extensively then ketone and chloromethyl inhibition of /3H/-naloxone binding was observed. A dependent of about 10 uM of eac3 compound was required for concentration 50 % irreversible blockade of the / H/-naloxone binding sites. native the pretreatment was carried out with same When the the with naloxone a complete restoration of enkephalins or observed after the washing procedures (Fig.1). A binding was high concentration of Phe-Leu-CR (50 uM) was used as a control the opioid specificity of enkephalin chloromethyl ketones. for was found, In this case, no inhibition The resylts of saturation studies over wide concentration freshly Both Fig.2. shown in are ranges of / H/-naloxone prepared rat brain membranes and membrangs preincubated with showing H-Naloxone, sites for binding two buffer had curvilinear Scatchard plots. However, in membranes treated 176

c._

b:

,pplOO

LECK

DALECX

DADLECK

Ligand concentration

(J&l 1

Fig:1 Concentration dependence of the irreversible interaction Rat opioid receptors with enkephalin chloromethyl ketones. of membranes were incubated with various concentrations of brain with the 100 uM of chloromethyl ketones, or enkephalin for 90 min at 30 OC in 50 mM reversibly-binding ligands Tris/HCl (pH 7.4) buffer containing 50 ug/ml bacitracin. After fgur washes the binding sites remaining was measured with 2 nM 0 'C. The remaining specific binding per mg / H/-naloxone at expressed as a percentage of the control protein is (preincubation in buffer and four washes) binding. ketones, high affinity chloromethyl the with enkephalin after component was diminished extensive washing, binding this site was accessible to that the alkylation. indicating with on LECK and DADLECK are in good agreement These results our previous.observations with DALECK (5). subtype specificity of the irreversible blockade was The as follows: after the affinity reaction (carried out as tested were detailed in Mat3rials and Methods) the washed membran5s with / H/-DAGO (mu-selective ligand) and / H/-DALE labelled (delta-selective ligand) in the p;;;;ncEn;f 10 nM DAGO (14). seen As can be from Fig.3, DALECK caused a significant inhibition of the mu binding sites. Inhibition of the delta receptor was much less pronounced. Similar results with DALECK were found by Newma 3 and Barnard (6). A partial protection of / H/- naloxone binding was found when mu and delta agonists or antagonists were added 10 min before the affinity reaction was started (Fig.4). Moreover the mu agonist DAGO produced always greater protection than the delta-preferring agonist DADLE.

177

1

5

10

I

5

Bound b1031 cpm

Fig:2 Scatchard plots for 13H/-naloxone (0.05-25 nM) binding with preincubated to rat brain membranes (w) or membranes (0) enkephalin chloromethyl ketones and washed extensively. Panel A shows the effect of 10 uM LECK, and Panel B 10 uM DADLECK. alone In control experiments (0) the membranes were incubated and washed similarly to the treated and then centrifuged represent the means of triplicate values of samples. Points specific binding (B) in a single experiment.

3H-DAG0

3H-NX

Fig:3

Irreversible

inhibition

178

3H-DALE (d)

of mu and delta

receptors

by

enkephalin chloromethyl ketones. Membranes were incubated with 50 uM LECK or DALECK, washed as described in 3the text and case of mu receptor binding / H/-DAGO in a In the asseyed. final concentration of 3 nM was added to washed membranes with levorphanol and (for non-specific binding) or wiihout 10 uM 40 min at 37 C3 Remaining delta subtype was incubated for activity was determined with 3 nM / H/-DALE in the presence of 10 nM unlabelled DAGO that supresses cross-reaction between mu and delta binding. Non-specific binding was measured with 10 uM DALE. binding was expressed as percentage of Specific 100 % control. mu activity was 4780 cpy/mg protein, (100 % binding delta binding was 3620 cpm/mg protein). I H/-naloxone (100 % = 7023 cpm/mg protein) is also shown.

%I-Na10xtne

3H-Nabxme

+ DALECK

Fig:4 Protection of /3H/-naloxone binding sites by opiates. naloxone, DAGO and DADLE (100 uM each) were added to the reaction 10 min mixtures before addition of enkephalin chloromethyl ketones (50 uM). After 90 min jncubation unbound ligands were washed out and the remaining / H/-naloxone (2 nM) binding was determined. Control membranes were preincubated with are buffer. Results the average of two parallel incubations and carried out in triplicate. Control specific binding was 6240 cpm/mg protein. In these experiments the mu specificity of the chloromethyl derivatives was confirmed. These ketone ligands has been found to be good tools in studying opiate receptors and their subunit structure (6,16) and more experiments are currently in progress in this direction.

179

ACKNOWLEDGEMENT We would technical

like to assistance.

thank

Mrs.

Ildiko

Nemeth

for

excellent

REFERENCES 1

Powers, J. (1977). Ch emistry acids, peptides and proteins 178.

2

relationships of Morley, J. (1980). Structure-activity enkephalin like peptides. Ann. Rev. Pharmacol. Toxicol. 20: 81-110.

3

Pelton, J., Johnston, R., Balk, J., Schmidt, C. and Roche, E. (1980). Synthesis and biological activity of chloromethyl ketones of leucine enkephalin. Biochem. Biophys. Res. Commun. 97: 1391-1398.

4

Venn, R. and Barnard, E. (1981). A potent affinity reagent for the opiate receptor. 256: 1529-1532.

and biochemistry (ed. Weinstein,

of amino B.). 4: 65-

peptide J. Biol.

Chem.

5

Szucs, M. Benyhe, S., Borsodi, A. et al. (1983)2 Binging characteristics and analgesic activity of D-Ala -Leu enkephalin chloromethyl ketone. Life Sci. 32: 2777-2784.

6

Newman, L. and Barnard, E. (1984). Identification of an opioid receptor subunit carrying the mu binding site. Biochemistry 23: 5385-5389.

7

Toth, G., Kramer, M., Sirokman, F., Borsodi,3A. and Ronai, A. (1982). Preparation of (7,8,19,20H)-Naloxone of high specific activity. J. Label. Comp. Radiopharm. 19: 1021-1029.

8

Toth, G., Kramer, M., Szucs, M., Benyhe, S3 and Sirokman, F. (1982). Preparation of 1,7,8- HDihydromorphine of high molar activity and its application in opiate receptor binding experiments. Radiochem. Radioanal. Letters 56: 209-216.

9

Benyhe, S. Toth, G. Kevei, J. et al. (1985). Char ct rizatiqn of rat brain opioid receptors by (TyrRes. 3,5,'H)',D-Ala ,Leu5-enkephalin binding . Neurochem. 10: 627-635.

10

Medzihradszky, K., Szecsi, J., Csanady, G. and Hepp, J. (1983) Enkephalin diazomethyl and chloromethyl ketones: synthesis and biological activity. In: Blaha, K. and '1982. Walter de Gruyter, Malon, P. (eds). Peptides Berlin; 623-628.

180

11

Pasternak, G., Wilson, H. and Snyder, S. (1975). Differential effects of protein-modifying reagents on receptor bindings of opiate agonists and antagonists. Mol. Pharm. 11: 340-351.

12

Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.

13

Pert, C. and Snyder, S. (1974). Opiate receptor binding of agonists and antagonists affected diferentially by sodium. Mol. Pharm. 10: 868-879.

14

Kosterlitz, H., Paterson, S. and Robson, L. (1981). Characterizationof the k-subtype of opiate receptor in the guinea pig brain. Br. J. Pharmacol. 73: 939-949.

15

Szucs, M., Simon,J. Belcheva, M. et al. (&985). A$finisy labelling of opioid receptor subunits by H-D-Ala -Leu enkephalin chloromethyl ketone. J. Neurochem. Suppl. 44: S87D

Accepted 3/7/86

181