Ž.European Journal of Pharmacology 325 1997 253±261
`Characterization of the binding and activity of a high affinity,
`pseudoirreversible morpholino tachykinin NK receptor antagonist1
`Margaret A. Cascieria,), Elzbieta Bera, Tung Ming Fonga, Jeffrey J. Haleb, Frank Tangc,
`Lin-Lin Shiaoa, Sander G. Millsb, Malcolm MacCossb, Sharon Sadowskia, Michael R.
`Tota a, Catherine D. Stradera,1
`a Departments of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, R80M-213, P.O. Box 2000, Rahway, NJ 07065, USA
`b Medicinal Chemical Research, Merck Research Laboratories, Rahway, NJ 07065, USA
`c Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065, USA
`Received 31 October 1996; revised 6 February 1997; accepted 11 February 1997
`Abstract
`Ž. Ž Ž Ž . . .Ž. Ž Ž . . Ž .2 S - 3,5-Bis trifluoromethyl benzyl -oxy -3S -phenyl-4- 3-oxo-1,2,4-triazol-5-yl methyl morpholine L-742,694 is a selective
`morpholino tachykinin NK receptor antagonist that inhibits the binding of125I-substance P to the human tachykinin NK receptor with a1 1
`K s37 pM. Increasing concentrations of L-742,694 added to cells 15 min prior to agonist progressively increase the apparent EC ofd 50
`Ž.substance P for inducing the synthesis of inositol phosphate in Chinese hamster ovary CHO cells expressing human tachykinin NK1
`receptor and decrease the maximal level of stimulation observed. In contrast, addition of substance P and L-742,694 to the cells at the
`same time results in an increase in the EC for substance P with no decrease in the maximal level of stimulation. The compound also50
`decreases the apparent number of binding sites for125I-substance P observed by Scatchard analysis. Analysis of the binding of
`w3 x 8 y1 y1H L-742,694 to the tachykinin NK receptor shows that it associates with the receptor withk s3.98=10 M min , and1 a
`dissociates withk s0.026 miny1 and t s27 min at 228C. The slow rate of dissociation of L-742,694 from the tachykinin NKd1 r2 1
`receptor and the observation that altering the order of addition of antagonist and substance P attenuates the effect of the antagonist on the
`maximal activation suggest that L-742,694 is a competitive antagonist that can behave as a pseudoirreversible antagonist under some
`experimental conditions. L-742,694 has reduced affinity for tachykinin NK receptors in which alanine has been substituted for Gln
`165,1
`His197 or His265 in transmembrane helices 4, 5 and 6, respectively. These three residues have previously been shown to be present in the
`binding site of tachykinin NK receptor antagonists of several structural classes. In addition, L-742,694 inhibits binding of the1
`Ž.Ž . wŽ. xw x Žw125 x .quinuclidine antagonist 2S,3S -cis-2- diphenyl methyl -N- 2-iodophenyl -methyl -1-azabicyclo 2.2.2 octane 3-amine I L-703,606
`with the same affinity as it inhibits binding of125I-substance P. These data indicate that L-742,694 binds to the same site within the
`transmembrane domain of the receptor as previously described competitive antagonists.q 1997 Elsevier Science B.V.
`Keywords: Tachykinin NK receptor antagonist; Antagonism, pseudo-irreversible1
`1. Introduction
`The endogenous ligands for the neurokinin receptors are
`peptides termed tachykinins that are widely distributed in
`the central and peripheral nervous systems, and whose
`C-terminal sequence is Phe-X-Gly-Leu-Met-NH . These
`2
`peptides interact with three related receptor subtypes, with
`) Ž. Ž.Corresponding author. Tel.: 1-908 594-4609; Fax: 1-908 594-3337;
`e-mail: peggy_cascieri@merck.com
`1 Current address: Schering Plough Research Institute, 2015 Galloping
`Hill Road, Kenilworth, NJ 07033, USA.
`substance P, neurokinin A and neurokinin B having high-
`Ž.est affinity for the tachykinin neurokinin-1 NK receptor,1
`the NK receptor and the NK receptor, respectively. All23
`three receptor subtypes are functionally coupled to G
`proteins and possess the seven hydrophobic transmem-
`brane domains that are characteristic of receptors of this
`class. Antagonists of the tachykinin NK receptor have
`1
`potential clinical utility in the treatment of chronic pain,
`Žmigraine, neurogenic inflammation and emesis Eglezos et
`al., 1991; Nagahisa et al., 1992; Laird et al., 1993; Bountra
`.et al., 1993; Tattersall et al., 1994 .
`The first non-peptidal tachykinin NK receptor antago-
`1
`Ž.Ž . wŽnist, 2 S,3S -cis-2- diphenylmethyl -N - 2-methoxy
`0014-2999r97r$17.00 Copyrightq 1997 Elsevier Science B.V. All rights reserved.
`Ž.PII S0014-2999 97 00122-2
`HELSINN EXHIBIT 2053
`Azurity Pharmaceuticals, Inc. v. Helsinn Healthcare S.A.
`IPR2025-00948
`Page 1 of 9
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`()M.A. Cascieri et al.rEuropean Journal of Pharmacology 325 1997 253±261254
`Fig. 1. Structure of L-742,694 and other structurally diverse tachykinin
`NK receptor antagonists.1
`. xw x Žphenyl -methyl -1-azabicyclo 2.2.2 octane-3-amine CP
`.Ž .96,345 , was reported by Snider et al. 1991 , and was a
`quinuclidine amine with subnanomolar affinity for the
`Žreceptor and a competitive mechanism of action See Fig.
`.1 for structures . Structure-activity studies of CP 96,345
`analogs combined with receptor mutagenesis experiments
`have shown that this and related compounds bind within
`the transmembrane domain of the receptor, and interact
`with Gln
`165, His197 and His265 of the human tachykinin
`NK receptor via a hydrogen bonding interaction with the1
`benzylic amine, an amino-aromatic interaction with the
`benzhydryl moiety and an aromatic interaction with the
`Žsubstituted benzyl moiety, respectively Fong et al., 1993,
`.1994a,b . These three mutant receptors have normal affin-
`ity for either substance P or other non-peptidyl ligands
`suggesting that the mutations do not produce gross alter-
`ations in the structure of the receptor. Subsequent studies
`have shown that several other structurally diverse competi-
`tive antagonists also interact within this binding site
`Ž.Cascieri et al., 1994, 1995a,b .
`Recent studies have shown that potent tachykinin NK
`1
`receptor antagonists, possessing dramatically improved oral
`activity, could be obtained after substitution of the ben-
`zylic amine in CP 96,345 and its phenyl piperidine analog
`CP 99,994 with an ether linkage, and after further modifi-
`Ž. Ž. Žcation of the aryl substituents. 2S -Phenyl-3S - 3,5-
`Ž. . Ž .bis trifluoromethyl benzyl-oxy piperidine L-733,060
`Ž. ŽFig. 1 inhibits binding with an ICs0.87 nM Harrison
`50
`.et al., 1995 . Substitution of the piperidine nitrogen with a
`Ž.methyl carboxamide moiety L-736,281, Fig. 1 did not
`alter affinity for the receptor, suggesting that a basic
`nitrogen in this position was not required for high affinity
`Ž.Harrison et al., 1995 . The cyclohexyl analog of L-733,060
`Ž.L-741,923, Fig. 1 has 2000-fold reduced affinity for the
`receptor, while the methyl carboxamide-substituted cyclo-
`Ž.hexyl analog L-741,344, Fig. 1 has the same affinity as
`Ž.L-736,281 Mills et al., 1995 . These data suggest that the
`ring nitrogen is normally involved in a hydrogen bonding
`interaction with the receptor, and that this interaction can
`be replaced with an interaction with the methyl carboxam-
`ide side chain with no loss in affinity.
`These data suggested that substitution at the ring nitro-
`gen might be productively utilized to introduce potentially
`potency-enhancing substituents onto the molecule. Such
`enhancements were achieved with the introduction of tria-
`zole or triazolinone substituents onto the phenylpiperidine
`Ž.or morpholine scaffolds L-741,671, L-742,694, Fig. 1
`Ž.Ladduwahetty et al., 1996; Hale et al., 1996 . These
`compounds have high affinity for the human tachykinin
`NK receptor with K s45 pM and 37 pM, respectively.
`1d
`Ž. ŽThe data in the present report indicate that 2S - 3,5-
`Ž. . . Ž . Ž Žbis trifluoromethyl benzyl -oxy -3S -phenyl-4- 3-oxo-1,
`.. Ž .2,4-triazol-5-yl methyl morpholine L-742,694 is a high
`affinity, selective and pseudoirreversible antagonist of the
`human tachykinin NK receptor.
`1
`2. Materials and methods
`[3 ]( )2.1. Synthesis of H L-742,694 45 Cirmmol
`The synthesis is described schematically in Fig. 2.
`ŽŽ . . Ž . Ž2- 3,5-Bis trifluoromethyl benzyloxy -3- S -3 , 5 -
`Ž.dibromophenyl-4-benzylmorpholine1 was prepared from
`Ž. Ž .S - 3,5-dibromo -phenylglycine with methods analogous
`Žto those described for the preparation of L-742,694 Hale
`.Ž .et al., 1996 and was obtained as a 2:1 mixture of 2-S
`Ž.and 2-R diastereomers. The mixture was combined with
`an equal amount of 10% palladium on carbon in iso-
`propanol and stirred under 0.3 atm of tritium gas for 2 h.
`The tritiated intermediate was further reduced with hydro-
`Ž. Ž.gen gas and the resulting 2-S and 2- R diastereomers
`were separated by high pressure liquid chromatography
`Ž. Ž .HPLC on a Zorbax Rx-C column, 50:50 to 85:15 vrv
`8
`MeCNrHO q0.1% trifluoroacetic acid gradient over 252
`.Ž . Žmin, 1.0 ml rm i n t oa f f o r d2 -S -3,5-bis trifluoro-
`.Ž . w 3 x Ž.methyl benzyloxy-3-S - 3,5- H phenylmorpholine 2 .
`w3 xThis material was converted to H L-742,694 using the
`Ž.methods previously described Hale et al., 1996 and was
`Žpurified by HPLC Zorbax Rx-C column, 20:80 to 65:358
`Ž.vrv MeCNrHO q0.1% trifluoroacetic acid gradient2
`.over 25 min, 1.0 mlrmin . The specific activity was
`calculated to be 45.47 Cirmmol. Non-radioactive L-
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`()M.A. Cascieri et al.rEuropean Journal of Pharmacology 325 1997 253±261 255
`w3 xFig. 2. Synthesis of H L-742,694.
`Ž742,694 was synthesized as previously described Hale et
`.al., 1996 .
`2.2. Cells and reagents
`Receptor assays and functional assays were performed
`Ž.using stable Chinese hamster ovary CHO cell lines ex-
`pressing 1=105 human tachykinin NK receptorsrcell1
`that were selected and maintained as described previously
`Ž.Cascieri et al., 1992 . Mutants of the tachykinin NK1
`receptor were prepared as described previously and were
`assayed after transient expression in SV-40 transformed
`Ž. ŽAfrican green monkey kidney COS cells Fong et al.,
`.1993, 1994a,b . The human tachykinin NK receptor ex-1
`Žpressed in baculovirus-infected Sf9 cells Mazina et al.,
`. w3 x1994 was utilized for binding experiments with H L-
`742,694.
`2.3. Receptor binding assays
`Ž
`4 .Ž .CHO cells 5=10 or membranes 1±2 mg express-
`ing the human tachykinin NK receptor were incubated1125 8 Žwith I-Tyr -substance P 0.1 nM, 2200 Cirmmol; New
`.England Nuclear at room temperature for 45 min and then
`filtered over GFrC filters that had been presoaked in 0.1%
`polyethylenimine using a Tomtec 96-well harvester
`Ž.Cascieri et al., 1992 . Experiments with mutant receptors
`were carried out under the same conditions after transient
`expression of the receptors in COS cells. Assays using
`w
`125 xI L-703,606 were performed in a similar fashion as
`Ž.described in detail previously Cascieri et al., 1992 . The
`free energy of binding and the change in the free energy of
`binding were calculated using the formulas DG s
`Ž. Ž .yRT lnK and DD G syRT ln K rK .
`dd ,w t d,m ut
`w3 xAlthough specific binding of H L-742,694 is observed
`using membranes prepared from CHO cells expressing the
`human tachykinin NK receptor, membranes prepared from
`1
`baculovirus-infected Sf9 cells expressing the human
`tachykinin NK receptor at higher density were utilized in
`1
`these experiments in order to increase the signal to noise
`ratio. Scatchard analysis was performed using various
`w
`3 x Ž.concentrations of H L-742,694 0.01±2 nM and 7mgo f
`membrane protein in 0.5 ml under the conditions described
`above. The data were analyzed using the LIGAND pro-
`Žgram as purchased from Biosoft Munson and Rodbard,
`.Ž .1980 . In order to determine the rate of dissociationk
`y1
`w3 x Ž.of H L-742,694 from the receptor, ligand 1.2 nM was
`Ž.incubated with receptor 7mg for 45 min at 228C, then
`dissociation was initiated by addition of 100-fold excess of
`w3 xunlabeled L-742,694. The amount of H L-742,694 re-
`maining bound to the receptor was measured at 228Ca t
`times from 0.5 to 120 min after the addition of unlabeled
`L-742,694. The dissociation was monophasic and the data
`were analyzed using the first-order rate equation,
`w
`3 xw 3 x .log H L-742,694)Rr H L-742,694)R =100 so
`.ykt r2.3 q2.y1
`Ž. w3 xThe rate of associationk of H L-742,694 with the1
`receptor was determined by measuring the kinetics of
`Žassociation at five ligand concentrations 0.3 nM, 1.2 nM,
`.3 nM, 7.1 nM and 15 nM at 228C. The data were analyzed
`using the equation describing a one-phase exponential
`w
`3 xw 3 xassociation, H L-742,694 Bound s HL - 7 4 2 , 6 9 4
`Ž yk obs t.Bound 1 ye , and the rate of association wasmax
`Ž. ww3 xcalculated using the equation,k s k yk r HL -1o b s y1
`x742,694 .
`2.4. Substance P-induced inositol phosphate production
`The assay was performed as described by Berridge et
`Ž.al. 1982 , using cells grown to confluence in 12-well
`wtissue culture dishes. Cells were prelabeled with myo- 2-
`3 xH inositol for 24 h, washed, and incubated with LiCl in
`the presence or absence of L-742,694 for 15 min at 378C.
`Substance P was added for 20 min, and inositol monophos-
`phate was isolated after extraction and ion exchange chro-
`Ž.matography Cascieri et al., 1992 .
`3. Results
`Ž. Ž Ž . . .Ž.2 S - 3,5-bis trifluoromethyl benzyl -oxy -3S -
`Ž.phenyl-morpholine L-742,311 , a morpholino tachykinin
`NK receptor antagonist with no substitution on the ring
`1 125 w 8 xnitrogen, inhibits I- Tyr substance P binding to the
`human tachykinin NK receptor with an ICs2.4"1.8
`15 0
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`()M.A. Cascieri et al.rEuropean Journal of Pharmacology 325 1997 253±261256
`125 w 8 xFig. 3. Inhibition of I- Tyr substance P binding to the human
`Ž.tachykinin NK receptor by L-742,694 closed circles and L-742,3111
`Ž. Ž .open circles . Ligand 0.1 nM and CHO cells expressing the human
`tachykinin NK receptor were incubated in the presence or absence of1
`competitor as described in Section 2. Data shown are % of maximal
`specific binding observed without antagonists, and are the average of
`ns5o rns3 experiments for L-742,694 and L-742,311, respectively.
`Ž.nM ns3, Fig. 3 . Introduction of the triazolinone substi-
`Žtution increases affinity 30-fold ICs0.08"0.02 nM,50
`.ns5 . TheK calculated from these data is 37 pM, withd
`a Hill coefficient of 0.86. L-742,694 inhibits the binding of
`ŽŽthe competitive quinuclidine benzylamine antagonist 2S,
`.Ž . wŽ. x3S -cis-2- diphenylmethyl -N- 2-iodophenyl -methyl -1-
`wx Žw125 x .azabicyclo 2.2.2 octane 3-amine I L-703,606
`Ž.Cascieri et al., 1992 to the tachykinin NK receptor with1
`Ž.an IC s0.1 nM data not shown . In contrast, L-742,69450
`has 80 000-fold and 1800-fold lower affinity for the human
`Ž.tachykinin NK receptor IC s7 mM and the human25 0
`Ž.tachykinin NK receptor IC s150 nM .35 0
`Ž.Preincubation 15 min at 378C of CHO cells expressing
`the human tachykinin NK receptor with increasing con-1
`centrations of L-742,694 increases the apparent EC for50
`substance P stimulation of inositol phosphate synthesis and
`Ž.decreases the maximal stimulation observed Fig. 4A .
`These data suggest that L-742,694 is a non-competitive or
`irreversible antagonist of the tachykinin NK receptor. In
`1
`contrast, the unsubstituted L-742,311 increases the appar-
`ent EC for substance P but does not change the maximal
`50
`Ž.stimulation observed under these conditions Fig. 4B .
`Schild analysis of the data in Fig. 4B gives aK s3n M
`b
`and a slope of 0.92, indicating that L-742,311 functions as
`a competitive antagonist.
`Incubation of CHO cells expressing the human
`tachykinin NK receptor with increasing concentrations of
`1125 w 8 xI- Tyr substance P results in saturable binding with a
`4 ŽK s0.1 nM and a B of 3=10 receptorsrcell Fig.dm ax
`.Ž .5 . L-742,694 30 pM or 100 pM decreases the apparent
`receptor number by 52% or 65%, respectively, without
`125 w 8 xaltering the apparent affinity for I- Tyr substance P
`Ž.Fig. 5 . These data are also consistent with L-742,694
`acting as a non-competitive or irreversible antagonist.
`125 w 8 xThe dissociation of I- Tyr substance P from the
`tachykinin NK receptor is biphasic, with dissociation1
`rates at 158C of 0.012"0.002 miny1 and 0.2"0.2 miny1
`for the high and low affinity states, respectively. In con-
`trast to the observations above, excess L-742,694 has no
`125 w 8 xaffect on the rate of dissociation of I- Tyr substance P
`w125 x Ž y1 .or I L-703,606 k s0.085 min at 158C from they1
`human tachykinin NK receptor, consistent with a compet-1
`Ž.itive mechanism data not shown .
`In order to assess if L-742,694 interacts with the same
`transmembrane domain binding site as other structurally
`diverse tachykinin NK receptor antagonists, the affinity
`1
`of L-742,694 and L-742,311 for receptors in which alanine
`was substituted for Gln
`165, His197 and His265 was deter-
`mined. Both compounds have reduced affinity for all three
`of these mutant receptors, indicating that they bind within
`Ž.the previously characterized binding site Table 1 . In
`Fig. 4. Stimulation of inositol phosphate synthesis in human CHO cells expressing the human tachykinin NK receptor by substance P in the presence of1
`Ž. Ž.increasing concentrations of L-742,692 A or L-742,311 B . Cells were incubated with LiCl and compound for 15 min before the addition of substance P.
`Ž. Ž . ŽThe reaction was terminated 30 min after addition of substance P. A Cells were incubated in the absence closed circles or presence of 1 nM open
`.Ž . Ž . Ž . Ž .circles , 3 nM closed diamonds or 30 nM open diamonds L-742,694. B Cells were incubated in the absence closed circles or presence of 100 nM
`Ž. Ž . Ž .open circles , 300 nM closed diamonds or 1mM open diamonds L-742,311. Inset: Schild analysis of the data for experiment shown. Data shown are
`representative of at least 2 experiments.
`Page 4 of 9
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`()M.A. Cascieri et al.rEuropean Journal of Pharmacology 325 1997 253±261 257
`125 w 8 xFig. 5. Scatchard analysis of the binding of I- Tyr substance P to the
`Ž.human tachykinin NK receptor in the absence closed circles or pres-1
`Ž. Ž .ence of 30 pM open circles or 100 pM closed squares L-742,694.
`Scatchard analysis was performed using freshly prepared ligand and CHO
`cells expressing the human tachykinin NK receptor as described in
`1
`Section 2. Data shown are representative of 3 experiments.
`ŽŽ . .addition, the loss in free energy of bindingDD G of
`L-742,311 and L-742,694 for each of these mutants in
`comparison with the wild-type receptor is comparable
`Ž.Table 1 . These data suggest that addition of the triazoli-
`none moiety in L-742,694 does not alter the way in which
`this compound interacts with the binding site in compari-
`son with the competitive antagonist L-742,311.
`Since L-742,694 has 30-fold higher affinity for the
`receptor than L-742,311, it is possible that L-742,694
`interacts with the receptor in an irreversible or pseudoirre-
`versible manner to produce its effects on the functional
`response to substance P. Therefore, we sought to character-
`ize the interaction of L-742,694 with tachykinin NK
`1
`receptors that have reduced affinity for this compound.
`The receptor in which His
`197 is replaced with serine has
`Ž.20-fold reduced affinity for L-742,694 ICs1.6 nM .50125 w 8 xScatchard analysis of I- Tyr substance P binding to the
`H197S tachykinin NK receptor expressed in COS cell
`1 125 w 8 xmembranes shows that the apparentK for I- Tyr sub-d
`stance P is increased 10-fold by inclusion of 10 nM
`Table 1
`Interaction of L-742,311 and L-742,694 with tachykinin NK receptors1
`with mutations in the non-peptidyl antagonist binding site
`Receptor L-742,311 L-742,694
`Ž. Ž . Ž. Ž .IC n DD G IC n DD G50 50
`Ž. Ž . Ž. Ž .nM kcalrmol nM kcalrmol
`Ž . Ž. Ž.NK-1R WT 1.8"0.8 3 0.18"0.09 8
`Ž. Ž.Q165A 38"8 2 1.8 1.9"0.4 2 1.4
`Ž. Ž.H197A 9"1 2 1 2.4"0.2 2 1.5
`Ž. Ž.H265A 73"27 2 2.2 4.6"0.4 2 2
`Table 2
`125 w 8 xScatchard analysis of I- Tyr substance P binding to wild-type and
`H197S human tachykinin NK receptors in the presence and absence of
`1
`L-742,694
`Ž. wx Ž.Receptor K pM Receptor pMd
`aaControl qL-742,694 Control qL-742,694
`Ž.NK R WT 19"48 0 "10 7.2"0.7 3.2"0.31
`H197S 27"3 300"30 9.3"0.3 8.5"0.7
`awxL-742,694s0.5 nM for NK R, 10 nM for H197S.1
`L-742,694, while there is no change in the apparent num-
`Ž.ber of receptor binding sites Table 2 . In contrast, the
`apparent number of binding sites observed for the wild-type
`receptor under these conditions is reduced 56% by inclu-
`sion of 0.5 nM L-742,694.
`125 w 8 xL-742,694 inhibits the binding of I- Tyr substance P
`to the rat tachykinin NK receptor with 1000-fold lower1
`Ž.affinity than the human receptor ICs80 nM . Preincu-50
`Ž.bation 15 min at 378C of CHO cells expressing the rat
`tachykinin NK receptor with up to 10mM L-742,694
`1
`increases the apparent EC for substance P stimulation of50
`inositol phosphate synthesis without altering the maximal
`Ž.response Fig. 6 . Schild analysis of these data gives a
`K s790 nM and a slopes0.97, indicating that L-742,694
`b
`acts as a competitive antagonist at the rat tachykinin NK1
`receptor. Although the 10-fold difference between the
`affinity determined in receptor binding and theK calcu-
`b
`lated from the Schild analysis suggests that equilibrium
`between antagonist and agonist had not been achieved,
`these data still suggest that the interaction of L-742,694
`with tachykinin NK receptors for which it has lower
`1
`Fig. 6. Stimulation of inositol phosphate synthesis in CHO cells express-
`Žing the rat tachykinin NK receptor by substance P in the absence closed1
`.Ž .circles or presence open squares of 10mM L-742,694. Cells were
`incubated with LiCl and L-742,694 for 15 min before addition of
`substance P. The reaction was terminated after 30 min as described in
`Section 2. Inset: Schild analysis of the data from this experiment.
`Page 5 of 9
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`()M.A. Cascieri et al.rEuropean Journal of Pharmacology 325 1997 253±261258
`w3 xFig. 7. Scatchard analysis of the binding of H L-742,694 to the human
`tachykinin NK receptor. Ligand and receptor as expressed in bac-1
`ulovirus-infected Sf9 cells were incubated as described in Section 2.
`Experiment shown is representative of 2 replicate experiments.
`affinity than the human tachykinin NK receptor does not1
`produce the apparent irreversible antagonism demonstrated
`with the human receptor.
`In order to directly assess the kinetics of L-742,694
`w3 xbinding to the human tachykinin NK receptor, H L-1
`Ž.742,694 45 Cirmmol was synthesized. The human
`tachykinin NK receptor expressed in baculovirus-infected
`1
`Sf9 cells was utilized for these experiments in order to
`increase the signal to noise ratio in the ligand binding
`w
`3 xassay. The binding of H L-742,694 to the tachykinin
`NK receptor reached equilibrium at 30 min and was
`1
`stable for at least 180 min at 228C. Scatchard analysis of
`w3 xthe binding of increasing concentrations of H L-742,694
`gave a K s0.12"0.01 nM and a maximal number of
`d
`Žbinding sites of 4.2"0.3 pmolrmg protein ns2, Fig.
`.7 . These data are consistent with the maximal number of
`receptor binding sites in this preparation determined using
`w3 xFig. 8. Dissociation of H L-742,694 from the human tachykinin NK1
`Ž.receptor. Ligand 1.2 nM was incubated with receptor as expressed in
`baculovirus-infected Sf9 cells until equilibrium, and dissociation was
`initiated with addition of 100-fold excess of unlabeled ligand. Data shown
`are the average of 2 experiments.
`the quinuclidine tachykinin NK receptor antagonist,1
`w125 xw 3 x Ž.I L-703,606 or H substance P Mazina et al., 1994 .
`The binding is inhibited by unlabeled L-742,694, CP 96,345
`and the inactive enantiomer of CP 96,345 withK s0.12i
`nM, 1.4 nM and )100 nM, respectively. These data are
`consistent with binding to the tachykinin NK receptor.1
`w3 xIn order to measure the rate of dissociation of H L-
`742,694 from the tachykinin NK receptor, membranes1
`Ž.were incubated with radioactive ligand 1.2 nM until
`Ž.equilibrium was achieved 45 min , and then 100-fold
`excess of unlabelled L-742,694 was added to the incuba-
`w
`3 xtion. H L-742,694 dissociates from the receptor with
`k s0.026"0.002 min
`y1 and t s27"2 min at 228Cy11 r2
`Ž.Fig. 8 .
`Ž.The rate of associationk measured for 0.3 nM,obs
`w3 x1.17 nM, 3 nM, 7.1 nM and 15 nM H L-742,694 is 0.40
`min
`y1, 0.61 miny1, 0.82 miny1, 1.28 miny1 and 6.4
`w3 x Ž. w3 xFig. 9. Association of H L-742,694 with the human tachykinin NK receptor. A The amount of specific binding of H L-742,694 vs. time at 0.3 nM1
`Ž. Ž . Ž . Ž .Ž .closed circles , 1.2 nM open circles , 3 nM closed diamonds , 7.1 nM open diamonds and 15 nM closed squares ligand was determined. The
`Ž. Ž .observed rate of associationk at each concentration was calculated as described in Section 2. B Correlation betweenk and concentration ofobs obs
`w3 x Ž.H L-742,694. The rate of associationk was calculated as described in Section 2.1
`Page 6 of 9
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`()M.A. Cascieri et al.rEuropean Journal of Pharmacology 325 1997 253±261 259
`Ž.Fig. 10. Stimulation of inositol synthesis in CHO cells expressing the human tachykinin NK receptor by substance P in the absence closed circles or1
`Ž.Ž .presence of 1 nM open circles or 100 nM closed diamonds L-742,694. Cells were preincubated with LiCl, then substance P and L-742,694 were added
`simultaneously. The incubation was terminated after 30 min. Inset: Schild analysis of these data. Data shown are representative of 2 experiments.
`y1 Ž.min , respectively Fig. 9A . The relationship ofk toobs
`w3 xconcentration of H L-742,694 is linear, and the associa-
`tion constantk calculated from these data is 3.76=108
`1
`M y1 miny1 when the Y-intercept is constrained to the
`Ž.value fork determined above Fig. 9B . TheK calcu-y1d
`Ž.lated from these kinetic measurements i.e.,k rk is 69y11
`pM.
`If the reduction in maximal responsiveness to substance
`P were produced by a pseudoirreversible interaction of
`L-742,694 with the tachykinin NK receptor due to a slow
`1
`rate of dissociation, alteration of the order of addition of
`antagonist and agonist might be expected to alter the
`effects of L-742,694 on the maximal responsiveness. In
`contrast to the data observed with preincubation of antago-
`Ž.nist Fig. 4 , simultaneous addition of agonist and antago-
`nist does not reduce the maximal responsiveness to sub-
`stance P at 1 nM L-742,694, and the maximal responsive-
`ness in the presence of 100 nM L-742,694 is reduced by
`Ž.only 50% Fig. 10 . Schild analysis of these data gives a
`K s40 pM and a slope of 0.83.
`b
`4. Discussion
`L-742,694 is a potent, selective tachykinin NK recep-1
`tor antagonist with increased receptor affinity and im-
`proved pharmacokinetic properties compared to previously
`Ž.described compounds Hale et al., 1996 . TheK of
`d
`L-742,694 for the tachykinin NK receptor is 37 pM, 691
`pM or 120 pM when determined by inhibition of125 I-
`w 8 xTyr substance P binding, measurement of the kinetic
`w3 xconstants for H L-742,694 binding, or Scatchard analysis
`w3 xof H L-742,694 binding, respectively. Although these
`experiments have been performed using the receptor ex-
`pressed in CHO cells, COS cells and baculovirus- infected
`Sf9 cells, these comparisons are valid since
`125 I-substance
`w3 xP and H L-742,694 have similar affinity in all three
`systems, and unlabeled L-742,694 inhibits this binding
`with similar affinity.
`Characterization of the effects of L-742,694 on sub-
`stance P stimulation of inositol phosphate synthesis and on
`125 w 8 xI- Tyr substance P binding isotherms indicates that the
`compound is an insurmountable antagonist. Thus, the max-
`imal level of receptor activation and the apparent number
`of
`125 I-substance P binding sites observed are decreased by
`addition of L-742,694. These data suggest that L-742,694
`is either a non-competitive or irreversible antagonist. How-
`ever, the compound has no effect on the rate of dissocia-
`125 w 8 xw 125 xtion of I- Tyr substance P or I L-703,606 from the
`tachykinin NK receptor, consistent with competitive an-
`1
`tagonism.
`L-742,311, thedes-triazolinone analog of L-742,694, is
`a competitive antagonist under these same experimental
`paradigms, and the reduction in the affinity of both these
`compounds for tachykinin NK receptor mutants at Gln
`165,1
`His197 and His265 is similar. Since these residues have been
`demonstrated previously to be components of the binding
`site for several structural classes of competitive non-
`Žpeptidal antagonists of this receptor Fong et al., 1994a,b;
`.Cascieri et al., 1994, 1995b , these data indicate that
`L-742,694 binds to the same site as competitive tachykinin
`NK receptor antagonists, and that addition of the triazoli-
`1
`none moiety does not alter the nature of its interaction with
`these three residues.
`Ž.Schambye et al. 1994a,b conclude that competitive
`and insurmountable antagonists of the angiotensin II type 1
`receptor bind to different, but overlapping sites within the
`transmembrane domain. Thus, alterations within trans-
`membrane domains VI and VII result in more pronounced
`loss in affinity for competitive antagonists than for insur-
`Page 7 of 9
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`()M.A. Cascieri et al.rEuropean Journal of Pharmacology 325 1997 253±261260
`mountable, non-competitive antagonists. However, it is not
`clear from these experiments if the mutated amino acid
`residues interact directly with the angiotensin II antago-
`nists, or if the mutations result in alterations in the size and
`shape of the antagonist binding pocket. Thus, it has not
`been demonstrated that the quantitative differences in
`affinity of competitive and insurmountable antagonists for
`the angiotensin II type 1 receptor mutants are due to
`differences in the molecular interactions shared by these
`antagonists.
`Several additional lines of evidence suggest that the
`apparent non-competitive behavior of L-742,694 is due to
`pseudoirreversible antagonism resulting from a slow rate
`of dissociation from the human tachykinin NK receptor.
`1
`L-742,694 is a competitive antagonist at the rat tachykinin
`NK receptor and at the human H197S mutant tachykinin
`1
`NK receptor, which have 1000-fold and 20-fold reduced1
`affinity for the compound, respectively. In addition, alter-
`ing the order of addition of agonist and antagonist in the
`inositol phosphate assay attenuates the effect of the antago-
`nist on maximal substance P-mediated activation.
`Lastly, direct measurement of the rate of dissociation of
`w
`3 xH L-742,694 from the tachykinin NK receptor gives a1
`k s0.026 miny1 and a t s27 min at 228C. They11 r2
`binding is fully reversible, and, thus, inconsistent with a
`covalent interaction between antagonist and receptor. Dis-
`125 w 8 xsociation of both I- Tyr substance P and the competi-
`w125 xtive quinuclidine antagonist I L-703,606 is too rapid to
`w125 xbe accurately measured at 228C. However, I L-703,606
`Ždissociates with at s8 min at 158C Cascieri et al.,1r2
`. 125 w 8 x1992 . I- Tyr substance P dissociates with at s461r2
`min and 0.2 min for the G-protein coupled and uncoupled
`Ž.receptor states, respectively, at 158C Cascieri et al., 1992 .
`Thus, the dissociation of L-742,694 from the tachykinin
`NK receptor is significantly slower than that of either
`1
`substance P or L-703,606. These data are consistent with
`L-742,694 acting as a pseudoirreversible antagonist when
`added to the receptor 15 min prior to addition of substance
`P in the functional assay. The apparent lack of reversibility
`of L-742,694 results from its slow rate of dissociation from
`the receptor, and correlates with the extremely high affin-
`ity of this antagonist for the human tachykinin NK recep-
`1
`tor.
`In summary, L-742,694 is a novel, high affinity
`tachykinin NK receptor antagonist whose binding kinetics1
`produce prolonged receptor antagonism. Thus, it is the first
`pseudoirreversible antagonist of the tachykinin NK recep-
`1
`tor described to date. In addition, we have demonstrated
`for the first time that such pseudoirreversible antagonists
`and competitive antagonists share qualitatively and quanti-
`tatively similar molecular interactions with the tachykinin
`NK receptor. The addition of the triazolinone moiety to
`1
`the competitive antagonist L-742,311 to give L-742,694
`results in an additional molecular interaction with the
`receptor that confers higher affinity and a slower rate of
`dissociation.
`References
`Berridge, M.J., Downes, C.P., Hanley, M.R., 1982. Lithium amplifies
`agonist dependent phosphatidylinositol responses in brain and salivary
`glands. Biochem. J. 206, 587±596.
`Bountra, C., Bunce, K., Dale, T., Gardner, C., Jordan, C., Twissell, D.,
`Ward, P., 1993. Anti-emetic profile of a non-peptide neurokinin NK1
`antagonist, CP 99,994 in ferrets. Eur. J. Pharmacol. 249, R3±R4.
`Cascieri, M.A., Ber, E., Fong, T.M., Sadowski, S., Bansal, A., Swain, C.,
`Seward, E., Frances, B., Burns, D., Strader, C.D., 1992. Characteriza-
`tion of the binding of a potent, selective, radioiodinated antagonist to
`the human neurokinin-1 receptor. Mol. Pharmacol. 42, 458±463.
`Cascieri, M.A., Macleod, A.M., Underwood, D., Shiao, L.-L., Ber, E.,
`Sadowski, S., Yu, H., Merchant, K.J., Swain, C.J., Strader, C.D.,
`Fong, T.M., 1