inhibited by serotonin agonists or antagonists but not by histaminergic compounds. On the contrary, in the guinea pig cerebellum, inactivation of histamine recep ...
VOl. 260, No , 14,, Issue of July 15. pp. 8423-8429,1985 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1985 by The American Society of Biological Chemists, Inc.
Photoaffinity Probes for Serotonin and Histamine Receptors SYNTHESIS AND CHARACTERIZATION OF TWO AZIDEANALOGUES
OF KETANSERIN* (Received for publication, December 26,1984)
Walter Wouters, Jacky Van Dun, J o s b E. Leysen, andPierre M. Laduron From the Department of Biochemical Pharmacology,Janssen Pharmaceutical Research Laboratories, E-2340 Beerse, Belgium
Two azide analoguesof ketanserin (6- and 7-azido- lecithin (7, 8). [3H]Ketanserin was also shown to label S23-[2-[4-(4-fluorobenzoyl)-l-piperidinyl]ethyl]- receptors on blood platelets (9) and evidence was obtained 2,4(lH,3H)-quinazolinedione) were synthesized and that phosphatidic acid-phosphatidylinositol turnover forms tested as possible photoaffinity probes for serotonin-Sz part of the signal transducing system coupled to S2-receptor and histamine-HI receptors. In reversible binding ex- sites (10). periments, the azides showed high affinity for both Besides its high affinity binding to serotonin-S2 receptor receptor types. sites, ketanserin also binds to histamine-H1 receptors but When membrane preparations were incubated with with a lower affinity. Histamine-H1 receptors were first idennanomolarconcentrationsof7-azidoketanserinand tified in in vitro binding studies on the guinea pig ileum (11). subsequently irradiated with UV light, both serotonin Later on, [3H]pyrilamine, which appeared to bind selectively and histamine receptors became irreversibly blocked.to HI receptors, was used to investigate H1-receptors in difThis irreversible binding was dependent on azide conferent peripheral tissues and in the central nervous system centrations and time of irradiation and did not change in the presenceof the scavenger p-aminobenzoic acid. (12,13). In peripheral tissues, histamine-H1 receptors mediate In contrast, irreversible blockade at low concentra- the antagonism of several physiological processes (e.g. contions of 6-azidoketanserin was only obtained for his- traction of the guinea pig ileum). Although the precise role of tamine receptors. However, this blockade was abol- brain histamine-H1 receptors is far from being clear, they probably play an important role in sedation, one of the promished by addition of the scavenger p-aminobenzoic acid inent central effects of H1-antagonists. indicating that it was not due to a real photoaffinity Up to date, molecular characterization and purification of mechanism. In the rat prefrontalcortex, irreversible blockingof serotonin-Szandhistamine-HI receptors has been greatly serotonin receptors with 7-azidoketanserin could be hampered by the lack of suitable techniques. inhibited by serotonin agonists or antagonists but not Photoaffinity probes have been developed for several brain by histaminergic compounds. On the contrary, in the and peripheral receptor systems (14-18) and havelargely guinea pig cerebellum, inactivationof histamine recep-contributed to their characterization. Photoaffinity probes tors could be inhibited by histamine antagonists and have the advantage over other affinity probes that they are histamine itself butnotby serotonergic compounds. first bound reversibly to the receptor, making it possible to This provides a way for differential photolabeling of study the reversible ligand-receptor interactions; subseeither of these receptors. quently they are irreversibly bound to the receptor byUV irradiation (19, 20). LSD was tested as a naturally photolabile probe for serotonin receptors (21). However, LSD binding is not selective Serotonin (5-HT) receptors have been identified in various for serotonin-S2 sitessince it also binds to serotonin-S1 sites brain areas ini n vitro tests using different kinds of 3H ligands and [3H]LSD is incorporated in several different proteins (1). [3H]Serotonin, used at nanomolar concentrations labels upon UV irradiation. A histamine analogue has been used to a site which is presently known as the5-HT1 orserotonin-S1 irreversibly antagonize histamine-induced contractions in isobinding site, the functional role of which is, however, still lated peripheral tissues (22, 23). However, the extremely low rather unclear (2). 5-HT2 or serotonin-S2receptors were first affinity of this compound makes it not suitable to use it as a identified in rat frontal cortex by the binding of [3H]spiperone photoaffinity probe for receptor studies. Just recently we (3). The role of these sites is much better understood. They reported that serotonin-S2 receptors couldbe irreversibly were shown to mediate the antagonism of various serotonergic blocked by 7-azidoketanserin (24). We now describe the syneffects measured both in vitro and in vivo such as serotonin- thesis of two azide derivatives of ketanserin andthe complete induced contractions in the isolated rat caudal arteries (l), characterization of these new compounds as photoaffinity tryptamine-induced clonic seizures (3,4),and 5-hydroxytryp- probes for both serotonin-S* and histamine-H1 receptors. tophan-induced head twitches (2). Recently, the more selective ligand [3H]ketanserin was MATERIALS AND METHODS shown to selectively label brain S2-receptors both in vitro (5) Drugs and Chemicals and in vivo (6) as well as S2-receptors, solubilized with lyso~
~~~
* This work was supported by a grant from Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in de Nijverheid en de Landbouw (Brussels). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
Drugs were obtained from their respective companies of origin. [3H]Spiperone (22.9 Ci/mmol), [3H]WB-4101(20.7 Ci/mmol), [3H] clonidine (66.8 Ci/mmol), [3H]dihydroalprenolo1(39.7 Ci/mmol), and [3H]pyrilamine (26.7 Ci/mmol) were obtained from New England Nuclear. [3H]Dexetimide (16.3 Ci/mmol) was supplied byI.R.E. (Fleurus, Belgium). [3H]Sufentanil(15 Ci/mmol), [3H]ketanserin(15 Ci/mmol), and [3H]haloperidol (22 Ci/mmol), were obtained from
8423
Photoaffinity Labeling of Serotonin Histamine and
8424
Receptors
TABLEI Conditions for photolabeling experiments and subsequent binding assays in eight different receptor models ~
Serotonin-
Histamine-
s2
HI
Animal species Rat Guinea pig Brain region Frontal cortex Cerebellum +L P M L P M Membrane fraction 1:lOO 1:lOO Dilution Buffer A 50 mM Tris- 50 mM TrisHCI, pH 7.7 HCl, pH 7.7
+
+ +
Buffer B
Tris salt"
Tris salt"
3H-Ligand
Spiperone
Pyrilamine
4 nM Concentration 2 nM Blank comMethysergide Astemizole pound Concentration 2 y M 11 y M
1
DopamineDP
~~
a1-
a2-
P-
Adrenergic
Adrenergic
Adrenergic
Rat Rat Rat Rat Striatum Forebrain Total cortex Total cortex Total particu- Total partic- Total particu- Total particlate ulate late ulate 1:80 1:80 1:80 1:lOO 50 mM Tris- 50 mM Tris- 50 mM Tris- 50 mM TrisHCl, pH 7.7 HCl, pH HCl, pH 7.7 HCl, pH 8 7.7 Tris salt" 50 mM Tris- 50 mM Tris- 50 mM TrisHC1, pH HCl, pH 7.7 HCl, pH 8 7.7 Clonidine Haloperidol WB4101 Dihydroalprenolol 2 nM 1 nM 3 nM 2 nM (dl)(+)-Butacla- Prazosin Noradrenaline Propranolol
Rat Forebrain M+L +P
1'2;
12yM
11yM
1IyM
12pM
Muscarinic
Opiate
Rat Striatum Total particulate
1:200 1:200 50 mM Tris-HC1, 50 mM Na-K phospH 7.4 phate, pH 7.4 50 mM Tris-HC1, 50 mM Na-K phosphate, pH 7.4 pH 7.4 Sufentanil
Dexetimide
1 nM 2 nM Dextromoramide Dexetimide 11oyM
"Tris salt: 50 mM Tris-HC1, pH 7.6, 120 mM NaCl, 5 mM KC1, 2 mM CaCll, 1 mMMgC12, 0.1% ascorbic acid, and 10 y~ pargyline.
Janssen Life Sciences Products. Radioactive purity of 3H-ligandswas regularly checked by thin layer chromatography and was required to be at least 98%.p-Aminobenzoic acid, serotonin, and histaminewere from Janssen Chimica. Bufotenine was obtained from Serva (Heidelberg, Germany). All common reagents were from different suppliers and wereof the highest purity available. 6-Aminoketanserin (R 64 048) and 7-aminoketanserin(R 63 234) are original Janssen Research compounds and were obtained through Janssen Chimica (Beerse, Belgium). Synthesis and Chemical Analysis of Azides
equatorial), 2.52 (m, 2H, "CHZ), 2.15 (m, 2H, piperidine axial), 1.72 (m, 2H, piperidine equatorial), 1.51 (m, 2H, piperidine axial). 7-Azidoketanserin-Starting with 410 mg (1 mmol) of 7-amino-3[2-[4-(4-fluorobenzoyl)-l-piperidinyl]ethyl]-2,4(1H,3H)-quinazolinedione (7-aminoketanserin, R 63 234), the synthesis was carried out as described above for 6-azidoketanserin. Recrystallization from acetonitrile afforded 240 mg (55%)of 7-azido-3-[2-[4-(4-fluorobenzoyl)l-piperidinyl]ethyll-2,4(lH,3H)-quinazolinedione (7-azidoketanserin, R 63 836); m.p. 193.7 "C. Thin layer chromatography revealed single spots with RFvalues of 0.64 and 0.85 in solvent systems A and B, respectively. Elemental analysis:
Analytical Techniques-Melting points were taken on a Mettler CzzHz1FN603 melting point apparatus. Elemental analysis was performed using a Calculated C 60.54 H 4.85 N 19.26 Carlo Erba element analyzer 1106. Mass spectra were obtained with Found C 59.99 H 4.76 N 19.04 a Finningham 4500 mass spectrometer. NMR analysis was carried out using a Bruker AM 360 spectrometer; spectra were recorded in M/S: M+ found 436 (m/e 100% abundance; 220). 'H NMR (dimethyl dimethyl sulfoxide-d6.Ultraviolet spectra were obtained using a Beck- sulfoxide-ds): d 11.4 (s, l H , " N H ) , 8.05 (n-, 2H, aromatic), 7.93 (d, mann Model 25 spectrophotometer. Thin layer chromatography was lH, quinazoline), 7.34 (m, 2H, aromatic), 6.95 (d, lH, quinazoline), performed on Silica Gel 60 Fpsaplates (Merck, Darmstadt, Germany). 6.80 (d, lH, quinazoline), 4.00 (t, 2H, "CH,), 2.95 (m, 2H, piperidine Chromatograms weredeveloped in chloroform/methanol/acetoniequatorial), 2.13 (m, 2H, piperidine axial), 1.72 (m, 2H, piperidine trile/acetic acid (9/9/9/2, v/v) (solvent system A)or in ethanol/ equatorial), 1.50 (m, 2H, piperidine axial). ammonium hydroxide (29/1, v/v) (solvent system B). Spots were Tissue Preparation detected by exposure to short wavelength UV light. 6-Azidoketanserin-410 mg (1 mmol) of 6-amino-3-[2-[4-(4-fluoWistar rats and pirbright guine pigs were killed by decapitation robenzoyl)-l-piperidinyl]ethyl]-2,4(1H,3H)-quinazolinedione (6-ami- and their brainswere rapidly dissected. Freshly dissected tissues were noketanserin, R 64 048) were dissolved in 50 mlof 1 N acetic acid immediately homogenized in ice-cold buffer A (see Table I) to prepare and the solution was cooled to 4 "C in an ice bath. 345 mg of sodium a total particulate membrane preparation or in0.25 M sucrose for the nitrite (5 mmol) dissolved in 2 ml of water was added and themixture preparation of a (M + L + P ) fraction (M + L P fraction, the total was stirred for 20 min at 4 "C under vacuum. Then a solution of 325 mitochondrial plus microsomal subcellular fraction) as previously mgof sodium azide (5 mmol) dissolved in 2 ml of water was added described (5). Membrane preparations were immediately used for and themixture was further stirred at 4 "C for 15 min. The pH of the binding assays. mixture was adjusted to about 9 with 10 N sodium hydroxide. The precipitate was filtered off and washed with a large amount of water. Binding Assays I t was dried under vacuum at 50 "C over phosphorus pentoxide. The For serotonin-S2 receptors, assay conditions were as described in product was recrystallized from acetonitrile, yielding 263 mg (60%) Ref. 25 when [3H]spiperone was used or as in Ref. 5 when [3H] of 6-azido-3-[2-[4-(4-fluorobenzoyl)-l-piperidinyl]ethyl]-2,4(1H,3H)quinazolinedione (6-azidoketanserin, R 64 288); m.p. 245.8 "C. Thin ketanserin was used as the ligand. For histamine-HI, dopamine-D2, layer chromatography showed a single spot with a RFvalue of 0.64 in al-adrenergic, a,-adrenergic, @adrenergic, and muscarinic receptors solvent system A and a value of 0.81 in solvent system B. The product assay conditions were as in Ref. 25 and for opiate receptors conditions was further characterized by elemental analysis, mass spectrometry, were as in Ref. 26. Binding to membrane fractions was assayed with a rapid filtration and NMR analysis. Elemental analysis: technique using WhatmanGF/B glass fiber filters and a 40-well Multividor filtration manifold (Janssen Scientific Instruments DiviC&LFNe03 sion). Filters were equilibrated in Insta-Gel I1 (Packard) and radioCalculated C 60.54 H 4.85 N 19.26 activity was counted in a Packard Tri-Carb 460 liquid scintillation Found C 60.32 H 4.72 N 19.19 counter. MS: M+ found 436 (m/e 100% abundance; 220). 'H NMR (dimethyl Photoaffinity Labeling sulfoxide-ds): 6 11.5 (s, l H , " N H ) , 8.05 (m, 2H, aromatic), 7.55 (d, Aliquots of the membrane preparations for different receptor syslH, quinazoline), 7.45 (d, lH, quinazoline), 7.35 (m, 2H, aromatic), 7.22 (d, lH, quinazoline), 4.02 (t, 2H, "CH,), 2.96 (m, 2H, piperidine tems in buffer A (see Table I) were preincubated in quartz test tubes
+
Photoaffinity Labeling of Serotonin Histamine and
0.50
.
0.10
0.30 W
u z
I
8
Q
m 0.20
i
\ \
0.10
\\\\\ Y
RESULTS
-4
0
I
1
220
210
260
$00
2io
rio
310
WAVELENGTH I nm 1
\ \
\ 0.10
-
\\\\
\ 1
0.
'LO
d
WAVELENGTH l n m l
FIG. 1. UV spectra of 6- and 7-azidoketanserin and their M solutions parent amino derivatives (see insets). Spectra of and after (---) 10 min of in 1%ethanol were recorded before (-) irradiation with long wavelength (366 nm) UV light. for 15 min at 37 "C with different concentrations of the azidoderivatives of ketanserin in the presence or absence of other drugs. The samples were cooled to 4 "C and subsequently irradiated during different Deriods of time with lone waveleneth (366 nm) UV lieht at Y
I
8425
an average distance of 4 cmfrom a Camag Universal UV lamp (Camag-Muttenz, Switzerland). During photolysis, samples were constantly mixed using a multiaxle rotating mixer. Followingphotolysis, samples were filtered under vacuum through Whatman GF/B glass fiber filters. To remove free and reversibly bound ligands, filters were incubated with 5 mlof buffer B (see Table I) at 37 "C for 5 min. Incubation was stopped by vacuum application. This wash procedure was repeated four times in total. Then unoccupied receptor sites were determined using a new filter assay procedure as described by Leysen et al. (27) with modifications. In short, filters left on the filtration manifold with the vacuum released were incubated with 400 ~1 of buffer B at 37 "C containing the appropriate concentration of 3Hligand in thepresence or absence of a displacer to determine nonspecific binding (see Table I). Incubation was stopped by vacuum application and filters were rinsed twice with 5 ml of ice-cold buffer A. Filters were counted as described above. The tissue, adsorbed to glass fiber filters was found to retain its original receptor binding properties and serotonin-Sz or histamine-H1receptors were specifically labeled by the above described filter assay procedure (28).
0.50
0
Receptors
.
Y
Synthesis and Photosensitivity of Azides-Both 6- and 7azidoketanserin were obtained in good yield from the corresponding amines by direct diazotization and subsequent reaction with sodium azide.Their photosensitivity was checked by UV spectroscopy. Fig. 1 shows the spectra of both azides M in 1% ethanol) before and their amine precursors irradiation and after irradiation for 10 min with long wavelength (366 nm) UV light. The spectra of the azides showed a decrease for most of the absorption maxima, indicating not only the disappearance of the azide functional moiety but also dramatic changes in the quinazoline system. The spectra of the amines, however, did not markedly change after irradiation. Characterization of the Reversible Binding of the AzidesBoth new azido-compounds were tested for their reversible binding affinities in eight different receptor binding models including serotonin-Sz, histamine-H1, dopamine-D,, al-adrenergic, az-adrenergic, 0-adrenergic, opiate and muscarinic receptors. Table I1 summarizes the IC, values (concentration producing 50% inhibition of the specific binding of the 3Hligand) for inhibition of binding of the 3H-ligands to these different receptors compared to the values for theparent compound ketanserin, obtained in similar experiments. Both azides showed almost equallyhigh affinity for binding to serotonin and histamine receptors. They also showed binding with weaker affinity to al-adrenergic anddopamine-D, receptors and were inactive at concentrations up to 10 p~ in the other receptor systems. Irreversible Binding of 6- and 7-Azidoketanserin-In order to examine whether the new azides would be able to undergo irreversible binding, the eight different receptor systems were preincubated with different concentrations of the azides and subsequently photolysed during 10 min. After the washing procedure, unoccupied receptors were determined and IC50 values were calculated as theconcentration of the azide which irreversibly blocked 50% of the specific binding sites (see Table 11). 7-Azidoketanserin showed photolabeling of serotonin re= 5.6 nM) and of ceptors at nanomolar concentrations histamine receptors at somewhat higher concentrations (IC50 = 12 nM). In contrast, 6-azidoketanserin showed photolabeling of histamine receptors at nanomolar concentrations (IC50 = 5.0 nM) but in order to photolabel serotonin receptors much higher concentrations were needed (IC50 = 158 nM). There was no or only very weak photolabeling of al-adrenergic and dopamine receptors at micromolar concentrations of both azides. Without photolysis both azides dissociated completely from all receptors tested during the washing procedure. Pho-
Photoaffinity Labeling of Serotonin and Histamine Receptors
8426
TABLE I1 IC, values of 6- and 7-azidoketanserin and the parent compound ketanserin for inhibition of binding of 3H-ligands to eight different receptors and for photolnbelinf with bothazides in the same receptor systems ICm (nM) reversible binding Receptor system
%Ligand
6-Azidoketanserin"
IC60 (nM) photolabeling
7-Azido-
6-Azidoketanserin'
Ketanserinb ~
~
7-Azidoketanserin'
~~
2.9 2.3 1.3 5.9 2.9 6.3 158 5.6 5.0 18.6 5.0 1.2 12.0 42.2 22.4 631 28.2 1,600 398 3,500 5,012 549 >10,000 >10,000 210,000 >10,000 >10,000 >10,000 >10,000 210,000 >10,000 >10,000 >10,000 >10,000 210,000 >10,000 >10,000 >10,000 >10,000 210,000 >10,000 >10,000 >10.000 Binding assays were performed as described under "Materials and Methods." Values are means of at least two determinations performed in duplicate. * Values taken from Refs. 5 and 24. e Photolabeling for 10 min was performed as described in the text and under the conditions as summarized in Table I. Values are means of two determinations performed in duplicate.
Serotonin-Sp Serotonin-S2 Histamine-HI al-Adrenergic Dopamine-Dp ap-Adrenergic @-Adrenergic Opiate Muscarinic acetylcholine
Ketanserin Spiperone Pyrilamine WB-4101 Haloperidol Clonidine Alprenolol Sufentanil Dexetimide
v"
10 -7 M
20
0 10 u 0
a Z m m
0 0
100
gOk 80
@ I
n
1
2
3
L
5
6
7
8
9
1
0
1O"'M A
IO-~M lo-* M
FIG. 2. Kinetics of photolabeling with 6-and 7-azidoketanserin. Membrane preparations of rat prefrontal cortex (for serotonin-Sz receptors) or guinea pig cerebellum (for histamine-HI receptors) were preincubated with the different concentrations of the azides and subsequently photolysed for different periods of time. After photolysis, receptor binding assays were performed as described under "Materials and Methods." A , photolabeling of HIreceptors with 6-azidoketanserin; B , photolabeling of Sz-receptors with 7-azidoketanserin; C , photolabeling of HIreceptors with 7-azidoketanserin.
Photoaffinity Labeling of Serotonin and Histamine Receptors PIPAMPERONE
BUFOTENINE
SEROTONIN
8427
DROPERIDOL
(3
I -1 -1
W
m 4
-
9 DRUGCONCENTRATION
W
m U
7
s2 : 10000
52: 1122
S2: 12.6
52: 25.1
s2: 11.2
H i : >10000
H i : >10000
H i : > 1000
H i : > 1000
H i : 31.6
6
5
1
HALOPERIDOL
WB-&lOl
P
?
ImS
PYRILAMINE
CHLORPHENIRAMINE
HISTAMINE
-1
8
(-log M )
I I I
d’ I
I I
I
I
I
I I
I
i
-
I
I
I I
I I
d’ 9
8
7
6
9
I I
? + F + n PPPFll
5 1 9 8 7 6 5 1 9 8 DRUGCONCENTRATION (-log M )
7
6
5
1
9
8
7
6
5
1
s2 : > 10000
s2 : >1000 s2: >loo0 s2 :> 1000 s2 :> 1000 HI: 1.0 HI: 19.9 H>i :l o 0H0i : > 1000 H i : 8900 FIG. 3. Inhibition by various compounds of photolabeling of serotonin-S2and histamine-HIreceptors with 7-azidoketanserin. Membrane preparations of rat prefrontal cortex (Sz-receptors) (-) or guinea pig cerebellum (HI-receptors) (---) were preincubated with 10 nMof 7-azidoketanserin and increasing concentrations of the testcompounds. After photolysis during 10 min, membranes were washed and assayed for remaining specific receptor binding as described under “Materials and Methods.” Percentage inhibition of photolabeling was defined as [(B, - B2)/(B3- B2)] X 100 whereby B1 = binding after photolysis in the presence of azide and the test compound; B, = binding after photolysis in the presence of azide alone; and Ba = binding after photolysis in the presence of the test compound alone. IC, values were defined as the concentration of the compound producing 50% inhibition of photolabeling.
tolysis in the absence of azide did not inhibitbinding to either of the eight receptor systems. Kinetics of Photolabeling-Membrane preparations of rat prefrontal cortex were preincubated with 7-azidoketanserin and preparations of guinea pig cerebellum with either 6- or 7azidoketanserin at different concentrations. They were subsequently irradiated with UV light during different periods of time and after washing the remaining specific binding was determined. Fig. 2 shows the decrease of specific binding as a function of the time of irradiation at several different concentrations of the azides. With 7-azidoketanserin the photolabeling reaction of both serotonin and histamine receptors was rapid, reaching a plateau value after about 2 min of irradiaM) about 65% tion. At the highest concentration used of both receptors was irreversibly blocked. With 6-azidoketanserin, photolabeling of histamine receptors was extremely rapid, reaching a plateau afteronly 60-90 s, dependent on the
concentration of 6-azidoketanserin used. At M 6-azidoketanserin about 85% of the specific binding on histamine receptors was irreversibly blocked. Influence of the Scavenger p-AminobenzoicAcid on Photolabeling with 6- and 7-Azidoketanserin-The mechanism of photolabeling of serotonin receptors with 7-azidoketanserin and of histamine receptors with 6- and 7-azidoketanserin was examined using the chemical scavenger p-aminobenzoic acid. This scavenger will react readily with the activated azide free in solution. Table I11 summarizes the results of experiments in which membrane preparations of rat prefrontal cortex were incubated with 10 nM of 7-azidoketanserin and preparations of guinea pig cerebellum were incubated with 10 nMof 6- or 7-azidoketanserin in the presence or absence of 10 mM p aminobenzoic acid. Following photolysis during 10 min, the membranes were washed and the remaining specific binding sites were determined. Photolabeling of serotonin and hista-
Photoaffinity Labeling Serotonin of
8428
TABLEI11 Influence of the scavenger p-aminobenzoicacid (PABA) on photolabeling of histamine-H1 receptors with 6-azidoketanserinand of serotonin-& and histamine-Hl receptors with 7"atidoketanserin Preincubation, photolysis, and subsequent determination of remaining specific binding were performed as described in the textand under the conditions as summarized in Table I. Values are means of two experiments, performed in duplicate. Specific binding (% of control) PABA Azide (10nM)
Photolysis (10 m ~ ) (10 min)
6-Azidoketanserin, HI
-
-
+ + + +
-
-
+ + -
-50
+
+
96
++-59 +
100 100 100 100 10295 39 103 95
7-Azidoketanserin
SI
98 94 95 57
HI
100 105 102
89 53
mine receptors with 7-azidoketanserin was not significantly altered by the addition of the scavenger, indicating a true photoaffinity mechanism. Photolabeling of histamine receptors with 6-azidoketanserin, however, was dramatically reduced by the addition of p-aminobenzoic acid. Addition of the scavenger alone did not reduce or inhibit the binding of the "-ligands to both receptor systems. ReceptorSpecificity of Photolabeling with7-Azidoketanserin-Several compounds were tested for their ability to inhibit photolabeling of serotonin and histamine receptors with 7-azidoketanserin. Membrane preparations of rat prefrontal cortex and guinea pig cerebellum were preincubated with 10 nMof the azide and increasing concentrations of the test compounds. After photolysis during 10 min, membranes were washed and remaining specific binding of the radioligand was measured. Fig. 3 shows the inhibition curves and the corresponding values. Photolabeling of serotonin-Sz receptors from rat prefrontal cortex could be inhibited both by serotonin agonists (serotonin and bufotenine) and by compounds with potent serotonin antagonisticproperties (pipamperone, ketanserin, and droperidol); selective histaminergic (pyrilamine), dopaminergic (haloperidol); and al-adrenergic (WB4101) antagonists were completely inactive. Photolabeling of histamine receptors from guinea pig cerebellum could be inhibited by histamine itself and by several compounds with histamineantagonisticproperties (chlorpheniramine, pyrilamine, and ketanserin) but notby selective serotonergic, dopaminergic, or al-adrenergic antagonists. In control experiments, in the absence of azide, all compounds tested were shown to be completely washed out using the above described procedure. DISCUSSION
In the present paper, the synthesis of 6- and 7-azidoketanserin is described. Both azides were readily obtained by diazotization of their respective amine precursors and subsequent reaction with sodium azide. In reversible binding experiments the new compounds showed almost equally high affinity for binding to both serotonin-Sz and histamine-HIreceptors. This is in contrast with the parent compound ketanserin whose affinity for serotonin receptor binding is 15 times higher than that for histamine receptor binding. The addition of an electron dense azide moiety to the quinazoline ring system thus improved the affinity for histamine-HI receptors leaving the affinity for serotonin-S, receptors unaffected. The same phenomenon was
and Histamine Receptors seen with the amino precursors. Both 6- and 7-aminoketanserin showed almost equally high affinity for both receptor systems.' In photolabeling experiments, 6-azidoketanserin irreversibly blocked H1-receptors at nanomolar concentration whereas much higher concentrations were neededto block Szreceptors and cui-adrenergic receptors. However, whenp-aminobenzoic acid was added to the incubation mixture as a scavenger for reactive molecules, photolabeling of H1-receptors was dramatically reduced. This suggests that irreversible inactivation of Hl-receptors by 6-azidoketanserin is not due to a real photoaffinity mechanism (19). With 7-azidoketanserin, both serotonin-Sz and histamine-HI receptors could be irreversibly blocked at nanomolar concentrations of the azide. Labeling of cul-adrenergic and dopamine-Dz receptors was totally absent at this concentration. In contrast to what was seen with 6-azidoketanserin, the scavenger p-aminobenzoic acid did not affect photoinactivation with 7-azidoketanserin, indicating that a truephotoaffinity mechanism was involved. Although both azides of ketanserin show quite similar reversible binding characteristics, there are important differences in their irreversible binding properties. For the photoinactivation of ser0t0nin-S~receptors, the position of the azide substitution on the quinazoline ring seems to be crucial. In contrast, for histamine-H1 receptors the presence of the azide moiety is necessary in order to increase the binding affinity (see above) but the position of the substitution does not seem to influence photoinactivation. Furthermore, 7azidoketanserin shows irreversible labeling of Sz- and Hlreceptors by atrue photoaffinity mechanism, whereas 6azidoketanserin does not. The exact reason for this is not clear. Onepossible explanation is that photoactivated 6azidoketanserin would dissociate very rapidly from the receptor site,giving the free radical scavengerp-aminobenzoic acid the opportunity to bind to the activated molecules, thereby lowering the degree of photolabeling. Dissociation experiments with the nonactivated azides showed no differences between the dissociation rate of 6- and 7-azidoketanserin' and could therefore not confirm this possibility. Photolysis in the presence of 7-azidoketanserin was shown to be a rapid process and the amount of labeled sites was dependent on azide concentrations. At the highest azide conM) about 65% of serotonin-Sz and centrations used histamine-H1 receptors were irreversibly blocked. The fact that not all receptors can be photolabeled is a common feature of most photoaffinity systems and can be explained in several different ways: possibly part of the bound photoaffinity ligand is shielded from irradiation and can therefore not be photolysed or some of the ligand maybe bound in a sterically unfavorable way so that formation of a covalent linkage is impossible. To examine the receptor specificity of photolabeling with 7-azidoketanserin, several compounds were tested for their ability to inhibit the photoinactivation. The compounds were selected on the basis of their dissociation rate from the receptor site. Indeed, they need to show rapid dissociation in order to be washed out completely during the washing steps following irradiation. Labeling of ser0t0nin-S~receptors could be inhibited by nanomolar concentrations of compounds with potent serotonin antagonistic properties (pipamperone, ketanserin, and droperidol) and micromolar concentrations of serotonin agonists (bufotenine andserotonin)but not by compounds showing another pharmacological profile. On the other hand, photolabeling of histamine-Hl receptors was inhibited by nanomolar concentrations of typical histamine W. Wouters, unpublished results.
Photoaffinity Labeling of Serol!onin and Histamine Receptors antagonists (chlorpheniramine and pyrilamine) and by histamine itself at high micromolar concentrations but not by other compounds. Photoaffinity labelingwith7-azidoketanserin was also shown to be tissue-specific.Indeed, inthe rat prefrontal cortex (an area that is highly enriched in S2-receptors)the small amount of H1-sitescan be blocked withan excess of pyrilamine, therebydirectingphotolabelingexclusively to the Szreceptors. In guinea pig cerebellum (an area enriched in HIreceptors) pipamperonecan be used to block the few SB sites present so that selective labeling of histamine receptors is possible. The foregoingdemonstrates that 7-azidoketanserin is a potent photoaffinity probe for both serotonin-S2and histamine-HIreceptors,offering the possibility for differential selective photolabeling of either ofboth receptor systems. The synthesis of 3H- or lZ5I-labeled7-azidoketanserinis presently beingstudied. This newradioactivephotoaffinityligand shouldenable us to makerapidprogress in the molecular characterization,localization,andpurification of the serotonin and histamine receptorproteins. Acknowkdgments-We wish to thank Dr. J. Van Rompay and Dr. W. Lauwers and their co-workers for elemental analysis, NMR, and mass spectroscopy. The skillful technical assistance of J. Van Hertrooy was highly appreciated. REFERENCES 1. Leysen, J. E. (1981) J. Physiol. (Paris) 7 7 , 351-362 2. Peroutka, S. J., Lebovitz, R. M., and Snyder, S. H. (1981) Science 212,827-829 3. Leysen, J. E., Niemegeers, C. J. E., Tollenaere, J. P., and Laduron, P. M. (1978) Nature 2 7 2 , 168-171 4. Leysen, J. E., and Laduron, P. M. (1977) Arch. Int. Pharmacodyn. Ther. 230,337-339 5. Leysen, J. E., Niemegeers, C. J. E., Van Nueten, J. M., and Laduron, P. M.(1982) Mol. Pharmacol. 2 1 , 301-314 6. Laduron, P. M., Janssen, P. F. M., and Leysen, J. E. (1982) Eur. J. Pharmacol. 81,43-48
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