[46] Assessment of leukotriene D4 receptor antagonists

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PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS

[46]

dopropyl)dimethylammonio]-l-propane sulfonate], pH 7.4. The suspension is then homogenized on ice with a Teflon homogenizer, and centrifuged (4°) at 100,000 g for 30 min. The clear, slightly yellow supernatant is then diluted with 12 ml of ice-cold buffer (25 mM Tris-HC1, 5 mM MgCI2, pH 7.4) and contains approximately 0.8 mg/ml of solubilized membrane protein. Binding of the antagonist [3H]SQ29,548 to solubilized receptor protein is measured by rapid filtration using glass fiber filters (GF/B, Whatman) which have been presoaked for 1 hr in 0.3% polyethyleneimine (in distilled H20). Aliquots (200 /zl) of the solubilized protein are incubated with [3H]SQ29,548 (1.0 nM) at 25° for 30 min in the presence and absence of competing agents. The samples are then filtered according to the methods described above for platelet membranes.

[46] A s s e s s m e n t o f L e u k o t r i e n e D4 R e c e p t o r A n t a g o n i s t s By DAVID AHARONY Introduction Leukotriene C4 [5(S)-hydroxy-6(R)-S-glutathionyl-7(E),9(E), 1I(Z), 14(Z)-eicosatetraenoic acid, LTC4] is a metabolite of arachidonic acid generated via the 5-1ipoxygenase pathway in lung tissues and certain leukocytic cell types. ~In most tissues or organs, LTC4 is rapidly metabolized to leukotriene D4 [5(S)-hydroxy-6(R)-S-cystinylglycyl-7(E),9(E),ll(Z), 14(Z)-eicosatetraenoic acid, LTD4] which is subsequently converted to leukotriene E4 [5(S)-hydroxy-6(R)-S-cystinyl-7(E),9(E),ll(Z),14(Z)eicosatetraenoic acid, LTE4]. The facile conversion of LTC4 suggests that LTD4 and LTE4 may actually be the species that exert most of the observed pharmacology. LTD4, a component of the "slow-reacting substance of anaphylaxis" (SRA), is a potent constrictor of lung and vascular smooth muscles and also induces mucus hypersecretion and vascular permeability. ~ Numerous studies, utilizing functional and radioligandbinding receptor assays have demonstrated that LTD4 binds with high affinity to specific membrane receptors, which in most tissues examined, are distinct from those activated by its metabolic precursor LTC4. 2'3 In i B. S a m u e l s s o n , Science 220, 568 (1983). 2 S. T. Crooke, S. M o n g , M. A. Clark, G. K. H o g a b o o m , M. A. Lewis, a n d J. G. Gleason, Biochem. Actions Harm. 14, 81 (1987). D. W. S n y d e r a n d R. D. Krell, J. Pharmacol. Exp. Ther. 231, 616 (1984).

METHODS IN ENZYMOLOGY,VOL. 187

Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.

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ASSESSMENT OF LEUKOTRIENE D4 RECEPTOR ANTAGONISTS

415

contrast with LTC4, LTE4 binds exclusively to LTD4 receptors 4'5 and exerts actions similar to LTD4 via activation of a subset of LTD4 receptors. Since the discovery of the first antagonist FPL 55,712, 6 significant progress has been made in the past 5 years in developing several novel, potent, and selective LTD4 antagonists. These include: (a) structural analogs of FPL 55,712 (generally termed hydroxyacetophenones), such as the orally active, but modestly potent, LY 171,883 (Ref. 7) and YM 16638 (Ref. 8); (b) potent antagonists that resemble the structure of LTD4, such as SKF 104,353 (Ref. 9) and MK 571 (Ref. 10); (c) highly potent antagonists containing amine rings that bear little apparent resemblance to the above compounds such as ONO-RS-411 (Ref. 11), SR 2640 (Ref. 12), and ICI 198,615 (Fig. 1) (Refs. 13 and 14). The following describes receptor binding assays utilizing either a radiolabeled agonist (i.e. [3H]LTD4) or antagonist ([3H]ICI 198,615), which can be used to determine the potency and competitiveness of binding of LTD4 antagonists.S' 14,15Some notable differences as well as agreements between data obtained from ligand binding experiments versus that obtained in functional receptor assays will be emphasized.

4 S. Mong, M. O. Scott, M. A. Lewis, H.-L. Wu, G. K. Hogaboom, M. A. Clark, and S. T. Crooke, Eur. J. Pharmacol. 109, 183 (1985). 5 D. Aharony, C. A. Catanese, and R. C. Falcone, J. Pharmacol. Exp. Ther. 248, 581 (1988). 6 j. Augstein, J. B. Farmer, T. B. Lee, P. Sheard, and M. L. Tattersall, Nature (London) 245, 215 0973). 7 j. H. Fleisch, L. E. Rinkema, K. D. Haisch, D. Swanson-Bean, T. Goodsen, P. K, P. Ho, and W. S. Marshall, J. Pharmacol. Exp. Ther. 233, 148 (1985). 8 K. Tomioka, T. Yamada, T. Mase, H. Hara, and K. Murase, Arz. Forsch. Drug Res. 38, 682 (1988). 9 D. W. P. Hay, R. M. Muccitelli, S. S. Tucker, L. M. Vickery-Clark, K. A. Wilson, J. G. Gleason, R. F. Hall, M. A. Wasserman, and T. J. Torphy, J. Pharmacol. Exp. Ther. 243, 474 (1987). lo T. R. Jones, R. Zamboni, M. Bailey, E. Champion, L. Charette, A. W. Ford-Hutchinson, F. Frenette, J. Y. Gauthier, S. Leger, P. Masson, C. S. McFarland, H. Piechuta, J. Rokah, H. Williams, R. N. Jones, R. N. DeHaven, and S. S. Pong, Can. J. Physiol. Pharmacol. 67, 17 (1989). i1 H. Nakai, M. Konno, S. Kosuge, S. Sakuyama, M. Toda, Y. Arai, T. Obata, N. Katsube, T. Miyamoto, T. Okegawa, and A. Kawasaki, J. Med. Chem. 31, 84 (1988). ~z I. Anfeit-Ronne, D. Kirstein, and C. Kaergaard-Nielsen, Eur. J. Pharmacol. 155, l l7 (1988). ~3 D. W. Snyder, R. E. Giles, R, A. Keith, Y. K. Yee, and R. D, Krell, J. Pharmacol. Exp. Ther. 243, 548 (1987). 14 D. Aharony, R. C. Falcone, and R. D. Krell, J. Pharmacol. Exp. Ther. 243, 921 (1987). 15 D. Aharony, R. C. Falcone, Y. K. Yee, B. Hesp, R. E. Giles, and R. D. Krell, Ann. N. Y. Acad. Sci. 52,4, 162 (1988).

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PHARMACOLOGY" ANTAGONIST/SYNTHESIS INHIBITORS

[46]

0

o

o

\OH a FIG. 1. Structure o f l C I 198,615.

Assay Methods

Principle. The potency of a given LTD4 antagonist is determined by its ability to compete, in a concentration-dependent manner, against binding of [3H]LTD4 or [3H]ICI 198,61515 to guinea pig lung membranes (GPLM). Scatchard analysis of saturation of ligand binding, conducted with multiple concentrations of radioligand in the presence of a single concentration of antagonist, is utilized to determine competitiveness. Reagents Receptor preparation buffer (A): 20 mM Tris-HC1 containing 0.25 M sucrose and the protease inhibitors: bacitracin, I00 /xg/ml; benzamidine, 157/zg/ml; phenymethylsulfonyl fluoride, 87/zg/ml; and soybean trypsin inhibitor, 100 tzg/ml, pH 7.4 Assay buffer (B) : 10 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], pH 7.4, containing 10 mM CaCI2, 10 mM MgCI2, 2 mM cysteine, and 2 mM glycine Radioligands: [3H]LTD4 and [3H]ICI 198,615 with specific activities of -40 and -60 Ci/mmol, respectively, purchased from New England Nuclear (Boston, MA).

Preparation of Lung Membranes Guinea pig lung membranes (GPLM) are prepared as previously described: Male albino, Hartley-strain guinea pigs "(300-500 g) are decapitated, lungs perfused with Tyrode's buffer (pH 7.4), and immediately excised. Large blood vessels and all visible necrotic tissue are removed and the remaining tissue is flash-frozen under nitrogen and stored at -70 ° until processed into membranes. The typical yield from 50 animals is approximately 150 g of lung tissue. A batch of 50 g frozen lung is thawed, chopped with a Mcllwain tissue chopper, and washed several times with

[46]

ASSESSMENT OF LEUKOTR1ENE D4 RECEPTOR ANTAGONISTS

417

ice-cold phosphate-buffered saline (PBS) (0.1 M, pH 7.4), followed by homogenization with a Brinkman PT-20 Polytron (six pulses of 20 seconds each at setting of 6) in buffer A. The homogenate is centrifuged at 15,000 g for 10 min at 4° to remove cell debris. The supernatant is carefully poured through four layers of gauze and is then centrifuged at 40,000 g for 30 min at 4°. The pellet is resuspended in 50 ml 20 mM Tris-HC1 buffer, pH 7.4, utilizing a glass-Teflon motorized homogenizer and centrifuged again as above. This procedure is repeated and the pellets resuspended in 50100 ml of Tris buffer. The membranes can be stored in convenient 5 to 10-ml aliquots (containing 1-2 mg protein per milliliter) at - 7 0 ° for periods over a year with no loss of receptor binding activity. This procedure typically yields 150-200 mg membrane protein/50 g lung tissue.

Receptor Binding Assays The assay composition that allows competition by both agonists and antagonists is identical for the [3H]ICI 198,615 and [3H]LTD4 binding assays. In competition experiments, 150 ~1 of buffer B are mixed with 20/xl of either [3H]ICI 198,615 or [3H]LTD4 (0.5 nM final concentration) along with 15/zl of varying concentrations of tested compound. The incubation is initiated by addition of 125/zl of diluted (1:4-1:6) membranes (150-200/~g protein per milliliter) to a final volume of 310/~1 and the incubations carried out at 25° for 30 min. Nonspecific binding is defined by respective unlabeled ligand in 2000-fold excess of labeled ligand. Saturation (Scatchard) experiments are similar to the competition experiments with the exception that the concentrations of the 3H-labeled ligands are varied between 0.006 and 3 nM for [3H]ICI 198,615 and 0.05 and 2.5 nM for [3H]LTD4. The incubation is terminated by dilution with 3 ml of ice-cold 10 mM Tris/100 mM NaC1 followed immediately by vacuum filtration with a total of 16 ml of the Tris/NaC1 buffer, utilizing a Brandel Cell Harvester Model M-30 and Whatman GF/C filters. The radioactivity retained on the filters is determined with a scintillation counter. Data from competition or Scatchard experiments are analyzed as published in detail elsewhere. 15 Both ligands display good specific binding (i.e., -90%) to lung membranes and bind with high affinity (Kd values are 0.2-0.5 nM) and in a saturable manner (Bmax values are 1000-2000 fmol/mg) to GPLM. ~4,15Figure 2 illustrates that several selective and structurally diverse LTD4 antagonists inhibit the specific binding of [3H]LTD4. Antagonists exert even higher potency against [3H]LTE4, which preferentially binds (under the same conditions as described above) to a subset of high-affinity LTD4 receptors) Figure 3 illustrates the inhibition of [3H]ICI 198,615 binding by LTD4 antagonists, gi values for many antagonists obtained against

418

PHARMACOLOGY: ANTAGONIST/SYNTHESIS INHIBITORS '

!

I

,

,

0.10

1.0

~b,

~

100

90 80=:

I

I

[46]

I

L'Y 71,883

7o-

10

0

A\ -

0.01

~

10

.O..~ .m...~__. I00

[Ligand]nM

1000 10000 100000

FiG. 2. Inhibition of [3H]LTD4 (0.5 nM) binding to guinea pig lung membranes by structurally diverse antagonists. Nonspecific binding was defined with I p.M LTD4. Results are mean of duplicate determinations from a typical experiment.

[3H]LTD4 binding correlate well (r = 0.962) with those obtained against [3H]ICI 198,615 (Ref. 16). Moreover, an excellent correlation (r = 0.95) also exists between Ki values from these binding assays and KB values obtained in functional receptor assays.14 Similar results with LTD4 antagonists, have also been demonstrated in membranes from human lung. ,7 The results from several such experiments are summarized in Table I which also demonstrates the ability of leukotriene agonists to inhibit binding. In contrast to antagonists, agonists exert significantly less affinity in competing against the antagonist as compared with the agonist, and they inhibit only 60-70% of the specific binding defined with 2 /xM of ICI 198,615 or FPL 55,712. However, when nonspecific binding is defined by 100 nM ICI 198,615, 300 nM SKF 104,353, or by 3/xM LTD4, both agonists and antagonists completely displace the specifically bound [3H]ICI 198,615 in a manner compatible with a single class of binding sites. Scatchard analysis demonstrates that selective LTD4 antagonists, such as ICI 198,615, inhibit [3H]LTD4 binding in a competitive manner. '4 Similarly, Fig. 4 illustrates that FPL 55,712 is a competitive antagonist of [3H]IC1 198,615 binding to LTD4 receptors on GPLM. Using similar methods, ,6 D. Aharony, R. C. Falcone, Y. K. Yee, and R. D. Krell, Biotechnol. Update 3, 1 (1988). ,7 D. Aharony and R. C. Falcone, in "New Trends in Lipid Mediators Research" (U. Zor, Z. Naor, and A. Danon, eds.), Vol. 3, pp. 67-71. S. Karger, Basel, 1989.

[46]

ASSESSMENT OF LEUKOTRIENE D 4 RECEPTOR ANTAGONISTS

419

TABLE I INHIBITION OF 3H-LABELED LIGAND BINDING TO GPLM BY SELECTIVEAGONISTS AND LTD4 ANTAGONISTS Ki a(nM) vs.

Compound

[3H]ICI 198,615

[SIll LTD4

Agonists LTD4 LTE4 LTCa b YM 17690 Antagonists ICI 198,615 SKF 104,353 FPL 55,712 LY 171,883

0.7 2.8 66 5.9

± ±

0.1 0.6 12 0.7

0.3 4.6 2552 2871

± ± ± ±

0.1 0.8 555 378

8.7 - 1.4 24 ± 4 122 ± 34 198 --- 24 0.6 ± 0.1 21 ± 2 4129 ± 695 5822 ± 1147

Ki values are mean ± SEM of 3 to 7 experiments conducted in duplicate. b LTC4 was evaluated in the presence of 45 mM serine-borate to prevent its metabolism to LTD4.

110[.

'

I

•o

~8 0 + "',\~e

o

70

¢:

I

I

ICl

•

I ,,

0 0.01 0.;0

FPL 55,712

"~

,.

,,

~

j

198,515 \

10

"O'

iL

',

o 50t o< 30

I

--...

100

• ",,,

104,353

..

\

',, ,

. ",z~a "-A.A_. "4 110 10 100 11)00 10000100000 [Ligand]

nM

FIG. 3. Inhibition of [3H]ICI 198,615 (0.5 nM) binding to guinea pig lung membranes by structurally diverse antagonists. Nonspecific binding is defined by 100 nM ICI 198,615 or by 300 nM SKF 104,353. Results are mean of duplicate determinations from a typical experiment.

420

PHARMACOLOGY: ANTAGONIST/SYNTHESISINHIBITORS 50001

~

,

[46]

,

Gontrol

'7 \ . 1500. FPL 55,712 ~ 0 F

I

0

500

,

~

" 1 ~ 1~ 1000

\~"

1500 2000

Bound (fmol/mg)

FIG. 4. Scatchard analysis of [3H]ICI 198,615 (0.005-2.0 nM) binding to guinea pig lung membranes in the absence (control) and presence of I/~M FPL 55,712. Ki = 1.2/zM was calculated from the equation Kd(app) = Kd*(l + [I]/Ki), assuming competitive inhibition.

high-affinity (Kd = 0.6-1.0 nM), low-capacity (//max = 80-180 fmol/mg protein) binding sites for both ligands, which are antagonized by LTD4 antagonists, can also be demonstrated in human lung membranes. ~7 Summary Both [3H]LTD4 and [3H]ICI 198,615 bind selectively and with high affinity to LTD4 receptors on guinea pig and human lung membranes. Binding is inhibited by selective LTD4 antagonists. However there may be some advantages for preferring one over the other, which is largely due to the specific experimental design. For example, due to the apparent higher affinity of agonists in the [3H]LTD4 binding assay, one can use this ligand to measure competition by agonists, partial agonists, and to determine the stereoselectivity of LTD4 analogs. 14 The disadvantages are susceptibility to oxidation, double-bond isomerization under acidic condition, metabolism by membrane aminopeptidase, and requirement for optimization of "agonist binding conditions ''15 that may limit the use of this ligand in different tissues (i.e., ileum or brain). [3H]ICI 198,615 does not suffer from these disadvantages and allows the direct determination of potency for structurally diverse antagonists without the necessity to optimize the assay for agonist binding. 15An additional advantage is the ability to distinguish between agonists and antagonists at

[47]

RADIOLIGAND

BINDING

ASSAY FOR

LTDa R E C E P T O R

421

the receptor binding level, since only agonists inhibition curves (against [3H]ICI 198,615) are shifted to lower affinity by stable GTP analogs) 8 However, one has to bear in mind that although these binding assays are highly efficient, rapid, and possess high capacity to test antagonist potency and mechanism, they do not provide broad pharmacological information as do functional receptor assays that utilize viable tissues. Some of the more notable examples are the phosphodiesterase inhibitory properties of LY 171,883 (Ref. 7) and the thromboxane A2 inhibitory activity of FPL 55,712 (Ref. 19) that may enhance antibronchospastic properties in viable tissues or animal models, leading to an apparent overestimation of their potency. An additional important discrepancy is the ability of all LTD4 antagonists to inhibit LTC4 and LTD4 contractile activity on human lung equally well. z°'21 This is in contrast to functional3 and ligand binding ~7 experiments that demonstrate distinct binding sites for LTC4 and LTD4 (and ICI 198,615). Acknowledgments Mr. R. C. Falcone and Ms. C. A. Catanese are gratefully acknowledged for conducting these experiments and Dr. R. D. Krell for reviewing this manuscript. is C. A. Catanese, R. C. Falcone, and D. Aharony, J. Pharmacol. Exp. Ther. 251, 846 (1989). 19 A. F. Welton, W. C. Hope, L. D. Tobias, and J. G. Hamilton, Biochem. Pharmacol. 30, 1378 (1981). 2o C. K. Buckner, R. D. Krell, R. B. Laravuso, D. B. Coursin, P. R. Bernstein, andJ. A. Will, J. Pharmacol. Exp. Ther. 237, 558 (1986). 21 C. K. Buckner, R. Saban, W. L. Castleman, and J. A. Will, Ann. N. Y. Acad. Sci. 524, 181 (1988).

[47] S u l f i d o p e p t i d e L e u k o t r i e n e R e c e p t o r B i n d i n g Assays By SEYMOURMONG Introduction The sulfidopeptide leukotriene receptor binding assay has been used extensively in antagonist compound screening, ~ in the identification of C. D. Perchonock, I. Uzinkas, M. E. McCarthy, K. F. Erhard, J. G. Gleason, M, A. Wasserman, R. M. Muccitelli, J. F. Devan, S. S. Tucker, L. M. Vickery, T. Kirchner, B. M. Weichman, S. Mong, M. O. Scott, G. Chi-Rosso, H.-L. Wu, S. T. Crooke, and J. F. Newton, J. Med. Chem. 29, 1442 (1986).

METHODS IN ENZYMOLOGY, VOL. 187

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