Supplemental material to this article can be found at:
`http://jpet.aspetjournals.org/content/suppl/2017/05/30/jpet.117.241422.DC1
`
`1521-0103/362/2/287–295$25.00
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright ª 2017 by The Author(s)
`This is an open access article distributed under the CC BY Attribution 4.0 International license.
`
`https://doi.org/10.1124/jpet.117.241422
`J Pharmacol Exp Ther 362:287–295, August 2017
`
`In Vitro and In Silico Characterization of Lemborexant (E2006), a
`Novel Dual Orexin Receptor Antagonist s
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`Carsten Theodor Beuckmann, Michiyuki Suzuki, Takashi Ueno, Kazuya Nagaoka, Tohru Arai,
`and Hiroyuki Higashiyama
`Neurology Business Group, Discovery (C.T.B.), Drug Metabolism and Pharmacokinetics (T.U.), hhc Data Creation Center (K.N.),
`and Medicine Development Center (T.A.), Eisai Co., Ltd., Tsukuba, Ibaraki, Japan; and Global Regulatory Affairs (M.S.),
`Neurology Business Group (H.H.), Japan and Asia Clinical Development, Eisai Co., Ltd., Bunkyo, Tokyo, Japan
`Received March 16, 2017; accepted May 23, 2017
`
`ABSTRACT
`Orexin (hypocretin) neuropeptides have, among others, been
`implicated in arousal/sleep control, and antagonizing the orexin
`signaling pathway has been previously demonstrated to pro-
`mote sleep in animals and humans. This mechanism opens up a
`new therapeutic approach to curb excessive wakefulness in
`insomnia disorder rather than to promote sleep-related signal-
`ing. Here we describe the preclinical pharmacological in vitro
`and in silico characterization of lemborexant ((1R,2S)-2-{[(2,4-
`dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-
`fluoropyridin-2-yl)cyclopropanecarboxamide)), a dual orexin
`receptor antagonist (DORA), as a novel experimental therapeutic
`agent for the symptomatic treatment of insomnia disorder and
`
`compare its properties to two other DORAs, almorexant and
`suvorexant. Lemborexant binds to both orexin receptors
`and functionally inhibits them in a competitive manner with low
`nanomolar potency, without any species difference apparent
`among human, rat, and mouse receptors. Binding and dissoci-
`ation kinetics on both orexin receptors are rapid. Lemborexant is
`selective for both orexin receptors over 88 other receptors,
`transporters, and ion channels of important physiologic function.
`In silico modeling of lemborexant into the orexin receptors
`showed that it assumes the same type of conformation within
`the receptor-binding pocket as suvorexant, the p-stacked
`horseshoe-like conformation.
`
`Introduction
`Insomnia disorder is a major problem in our societies,
`causing substantial individual and social burden. The major-
`ity of sleep medications enforce sleep-promoting signaling
`pathways, although recent neuroimaging evidence suggests
`that insomnia should be seen as inappropriate wakefulness or
`arousal at habitual bedtime rather than an inability to sleep
`(Nofzinger, 2004; Nofzinger et al., 2004).
`
`This research was supported by Eisai Co., Ltd.
`Part of the data in this manuscript has been presented previously at the
`following meetings:
`Beuckmann CT, Suzuki M, Nakagawa M, Akasofu S, Ueno T, Arai T,
`Higashiyama H (2014) Preclinical Pharmacological Characterization Of
`E2006, A Novel Dual Orexin Receptor Antagonist For Insomnia Treatment.
`(The 39th Annual Meeting of the Japanese Society of Sleep Research; July 3-4,
`2014, Tokushima City, Tokushima, Japan); Beuckmann CT, Suzuki M,
`Nakagawa M, Akasofu S, Ueno T, Arai T, Higashiyama H (2016) Preclinical
`Pharmacological Characterization Of Lemborexant, A Novel Dual Orexin
`Receptor Antagonist For Insomnia Treatment. (The 4th Annual International
`Institute for Integrated Sleep Medicine Symposium, February 26, 2016,
`Tsukuba, Ibaraki, Japan).
`https://doi.org/10.1124/jpet.117.241422.
`s This article has supplemental material available at jpet.aspetjournals.org.
`
`Since the simultaneous discovery of the orexin (also known
`as hypocretin) neuropeptide signaling system by two research
`groups (De Lecea et al., 1998; Sakurai et al., 1998), it has
`become clear that this system is involved in many physiologic
`functions, among them sleep/wake control (Chemelli et al.,
`1999), feeding (Sakurai et al., 1998), energy homeostasis
`(Hara et al., 2001; Yamanaka et al., 2003), and reward seeking
`(Boutrel et al., 2005; Harris et al., 2005), to name the most
`prominent ones.
`The two neuropeptides, orexin-A (OXA) and orexin-B (OXB),
`are derived from the common precursor prepro-orexin and
`activate the postsynaptically localized orexin-1 receptor
`(OX1R) and orexin-2 receptor (OX2R). OXA has similar
`affinity for both OX1R and OX2R, whereas OXB has higher
`preference for OX2R (Sakurai et al., 1998). Orexin-expressing
`neurons in the central nervous system are confined to the
`hypothalamus (De Lecea et al., 1998; Sakurai et al., 1998)
`from which they project to numerous wake-controlling nuclei
`such as the noradrenergic locus coeruleus, the serotonergic dorsal
`raphe nucleus, the cholinergic laterodorsal/pedunclopontine teg-
`mental nuclei, and the histaminergic tuberomamillary nucleus
`
`ABBREVIATIONS: Bmax, maximum binding; CHO, Chinese hamster ovary; DORA, dual orexin receptor antagonist; E2006, (1R,2S)-2-{[(2,4-
`dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide); EMPA, N-ethyl-2-[(6-methoxy-3-pyridinyl)
`[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide; FDSS, Functional Drug Screening System; HEK293, human embryonic kidney 293;
`hOX1R, human orexin-1 receptor; hOX2R, human orexin-2 receptor; hOXB, human orexin-B; Ki, Inhibition constant; koff, dissociation rate constant; kon,
`association rate constant; MD, molecular dynamics; MM-GBSA, molecular mechanics generalized born surface area; mOX2R, mouse orexin-2
`receptor; mOXB, mouse orexin-B; MT1R, melatonin-1 receptor; MT2R, melatonin-2 receptor; OX1R, orexin-1 receptor; OX2R, orexin-2 receptor; OXA,
`orexin-A; OXB, orexin-B; OXR, orexin receptor; PDB, Protein Data Bank; PLAP, placental alkaline phosphatase; RBA, receptor binding assay; REM,
`rapid eye movement; 2-SORA, orexin-2 receptor-selective antagonist.
`
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`
`Beuckmann et al.
`
`(for review, see Sakurai, 2007), all of which contain neurons
`expressing OX1R, OX2R, or both. Regarding the role of both
`orexin receptors (OXRs) in sleep/wake regulation, it seems that
`OX1R is suppressing the onset of rapid eye movement (REM)
`sleep, whereas the OX2R is mostly responsible for suppressing
`non-REM sleep onset and is also involved in REM sleep control
`to a certain extent (Willie et al., 2003).
`The discovery that orexins are involved in sleep/wake
`regulation (Chemelli et al., 1999; Lin et al., 1999) triggered
`efforts by pharmaceutical companies to develop OXR antag-
`onists for treating insomnia, which is believed to be inappro-
`priately timed hyperarousal or wakefulness, rather than the
`inability of the brain to sleep (Nofzinger, 2004; Nofzinger
`et al., 2004). Rather than supporting sleep-promoting circuits,
`inhibiting the wake-promoting orexin signaling pathway
`offers a novel therapeutic approach to dampen the excessive
`wakefulness in insomnia. Preclinical and clinical evaluation of
`DORAs included almorexant (Fig. 1B), which was discontinued
`in clinical phase 3 (Brisbare-Roch et al., 2007); SB-649868
`(Bettica et al., 2012a,b) and filorexant (Winrow et al., 2012),
`which both completed clinical phase 2; and suvorexant (Fig.
`1C), which was approved in the United States and Japan for
`treatment of insomnia (Cox et al., 2010; Winrow et al., 2011;
`Herring et al., 2012). Recently, two OX2R-selective antagonists
`(2-SORAs), MK-1064 (Roecker et al., 2014; Gotter et al., 2016)
`and JNJ-42847922 (Bonaventure et al., 2015), have been
`introduced into clinical testing and achieved proof-of-activity
`in healthy subjects.
`We have previously disclosed the DORA lemborexant
`[E2006: (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-
`2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropane
`carboxamide)] (Yoshida et al., 2015) (Fig. 1A), which is cur-
`rently in phase 3 clinical development for treatment of insom-
`nia disorder. Its medicinal chemistry evolution and initial
`pharmacological evaluation have already been reported
`(Yoshida et al., 2014, 2015). Here we describe in more detail
`the preclinical pharmacological in vitro and the in silico
`characterization of lemborexant.
`
`Materials and Methods
`Chemical Compounds
`DORAs lemborexant, almorexant, and suvorexant have been syn-
`thesized in-house, and concentrations indicate free bases. [125I]OXA
`was purchased from PerkinElmer (Waltham, MA), [3H] N-ethyl-2-[(6-
`methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-
`acetamide (EMPA) was purchased from Sekisui Medical (Tokyo,
`Japan), and unlabeled EMPA and SB-334867 were purchased from
`Tocris Bioscience (Bristol, UK).
`
`Measurement of Affinity by Receptor Binding Assay
`The binding affinity was assayed by receptor binding assay (RBA)
`using a 96-well Flashplate (PerkinElmer). The membrane fraction
`was prepared from Chinese Hamster Ovary (CHO) cells expressing
`human OX1R (hOX1R) or human OX2R (hOX2R). Membrane
`suspension of hOX1R or hOX2R (5 mg protein/assay) was mixed
`with test antagonists [lemborexant (0.6–200 nmol/l), almorexant
`(0.2–200 nmol/l), or suvorexant (0.2–60 nmol/l)], as well as OXA
`(10 mmol/l; Peptide Institute, Osaka, Japan) solution or vehicle and
`[125I]OXA solution (0.2 nmol/l; PerkinElmer). The mixtures (final
`volume, 100 ml) were incubated for 30 minutes at room temperature
`on a 96-well Flashplate. All reaction mixtures were discarded,
`followed by two washing steps with 200 ml of 25 mmol/l HEPES
`
`buffer containing 525 mmol/l NaCl. The remaining radioactivity (in
`dpm) of each well was measured by TopCount (PerkinElmer), and
`inhibitory activity of the test antagonist was calculated using the
`following formula:
`Inhibition  % 5 100 2 100  ðT 2 NÞ=ðC 2 NÞ
`
`where T is reported in dpm in the presence of test antagonist (test), N
`is reported in dpm in the presence of 10 mmol/l OXA (nonspecific
`binding), and C is reported in dpm in the absence of compound
`(control).
`Values in experiments were determined in triplicate (lemborexant,
`almorexant) or quadruplicate (suvorexant). Experiments with
`lemborexant were conducted three times in an identical fashion, and
`IC50 values were calculated for each experiment before averaging for
`the final IC50 value and its S.E.M. The experiments for almorexant
`and suvorexant were conducted once, with each value expressed as the
`mean 6 S.E.M. for statistical analysis.
`In all experiments, the mean IC50 value and S.E.M. were calculated
`based on the sigmoidal curves of inhibitory activity (normalized
`response in percentage) versus the respective antagonist concentra-
`tion (using least-squares fit without constraints and with variable
`slope). Statistical analyses were performed using GraphPad Prism
`version 6.02 (GraphPad Software, La Jolla, CA).
`
`Cell-Based Calcium Mobilization Assay upon Functional
`OXR Activation
`Measurements of intracellular calcium mobilization upon func-
`tional activation of recombinantly expressed OX1Rs and OX2Rs of
`human, rat, and mouse origin in human embryonic kidney
`293 (HEK293) cells by the addition of OXA (at approximately EC50),
`and the antagonistic effect on this activation by test compounds
`was performed as described previously (Marlo et al., 2009; Yoshida
`et al., 2015) using the Functional Drug Screening System (FDSS)
`6000 (Hamamatsu Photonics, Hamamatsu, Japan). Experiments were
`conducted independently three times with quadruplicate values, and
`IC50 as well as inhibition constant (Ki) values were calculated (using a
`least-squares fit without constraints and with variable slope in four
`parameters) from each independent experiment before averaging for
`the final result. Analysis was performed using GraphPad Prism
`(version 6.07; GraphPad Software).
`
`Cell-Based Functional Reporter Enzyme Assay
`HEK293 cells were stably transfected with human or mouse OX1R
`or OX2R and with a reporter system (Chen et al., 1995; Durocher et al.,
`2000) where a reporter enzyme [placental alkaline phosphatase
`(PLAP)] (Goto et al.,1996) could be induced upon functional OXR
`activation through an intracellular Ca21-dependent reporter unit.
`Cells were seeded into 96-well plates at a density of 10,000/well and
`cultivated overnight in culture medium. Next day, 5 ml of lemborexant
`solutions were added to cultured cells in 96-well plates to a final
`culture medium volume of 115 ml (23-fold dilution), resulting in 1, 3,
`10, 30, 100, 300, and 1000 nmol/l end concentrations for the incubation
`of cells.
`After the addition of lemborexant and incubation for approximately
`2–3 hours at room temperature, orexin peptide agonists human/mouse
`OXA (Peptide Institute), human OXB (hOXB; Peptide Institute),
`mouse OXB (mOXB; Peptide Institute), or modified [Ala11, D-Leu15]-
`OXB (Tocris Bioscience) were diluted in Dulbecco’s modified Eagle’s
`medium (containing 0.1% bovine serum albumin and 3.45 mmol/l
`forskolin), and 10 ml was added to cell wells, resulting in a 115-ml final
`volume. Final concentrations of peptide agonists ranged from 0.01 to
`1000 nmol/l. After mixing by agitation of the plates, cells were
`incubated at 37°C for about 20 hours, with each respective concen-
`tration combination of lemborexant and peptide agonist having been
`applied to four cell wells. There are two amino acids different between
`hOXB and mOXB. For this reason, hOX2R was activated with hOXB,
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`In Vitro Characterization of DORA Lemborexant
`
`289
`
`incubated with membranes of hOX1R-expressing CHO cells (20 mg
`protein) at 22°C to measure the association with rate [association rate
`constant (kon)]. Nonspecific binding was measured with OX1R-
`selective antagonist SB-334867 (1 mmol/l; Tocris Bioscience) for each
`incubation time point.
`The dissociation was initiated by the addition of an excess of
`SB-334867 (1 mmol/l) after 90 minutes of incubation of [125I]OXA
`(0.2 nmol/l) with the hOX1R-containing CHO cell membranes, and the
`time course of signal decrease was measured. The experiment was
`performed once in triplicate (n 5 3). All reaction mixtures were filtered
`rapidly under vacuum through glass fiber filters (GF/B; PerkinElmer)
`presoaked with 0.3% polyethylenimine and rinsed several times with
`an ice-cold buffer (50 mmol/l Tris-HCl/150 mmol/l NaCl) using a
`96-sample cell harvester (UniFilter; PerkinElmer). After drying,
`radioactivity on filters was measured in a scintillation counter (Top-
`Count; PerkinElmer) as cpm using a scintillation cocktail (Microscint
`0; PerkinElmer). Data were analyzed using GraphPad Prism software,
`where the kon value of [125I]OXA was calculated by fitting to the
`following formula:
`
`Y 5 Ymax  ð1-expð 2 kob  XÞÞ
`where Y is binding (in cpm), X is incubation time, Ymax is the
`maximum binding (Bmax) observed, and kob is kon  radioligand
`concentration 1 koff (dissociation rate constant).
`The koff value of [125I]OXA was calculated by fitting to the following
`formula:
`
`1 NS
`
`
`Y 5ðY0 2 NSÞ  exp
`
`2 koff  X
`
`
`
`Fig. 1. Chemical structures of lemborexant, almorexant, and suvorexant.
`
`and mouse OX2R (mOX2R) was activated with mOXB. [Ala11,
`D-Leu15]-OXB has been described to be of higher selectivity for
`OX2R than natural OXB (Asahi et al., 2003).
`Next day, 5 ml of cell supernatant was transferred from each cell
`well to 384-well plates and mixed with 20 ml of detection buffer and
`25 ml of Lumi-Phos 530 reagent (Wako, Osaka, Japan). After
`incubation at room temperature under light protection for 2 hours,
`receptor activation was determined via the luminescence intensity
`measurement of secreted PLAP activity using a Fusion a-FP HT
`device (PerkinElmer). PLAP activity of every cell well was de-
`termined as a single data point, and values of four identical cell
`wells were averaged for analysis.
`To assess cell viability after the removal of 5 ml of cell supernatant
`for PLAP activity measurements, 10 ml of alamarBlue reagent
`(BioSource, Camarillo, CA) was added to the cell-containing 96-well
`plates, mixed by agitation of plates, and incubated for 2–3 hours at
`37°C, after which fluorescence intensity was measured using a Fusion
`a-FP HT device (PerkinElmer)
`(excitation wavelength, 535 nm;
`emission wavelength, 590 nm). The viability value of every cell well
`was determined as a single data point. Quadruplicate measurements
`of luminescence were averaged and plotted as the mean. Analysis was
`performed using nonlinear regression and the Gaddum/Schild EC50
`shift method using GraphPad Prism (version 5.02; GraphPad Soft-
`ware). Parameters calculated were Ki values and Schild slopes.
`
`Kinetic RBA on hOX1R
`Determination of Association Rate Constant and Dissocia-
`tion Rate Constant of [125I]OXA. The methodology described here
`is based on Dowling and Charlton (2006) and Motulsky and Mahan
`(1984). [125I]OXA (PerkinElmer) at 0.2 nmol/l final concentration was
`
`where Y is binding (in cpm), X is incubation time, Y0 is binding at time
`zero, and NS is binding (nonspecific) at infinite times.
`Determination of kon, koff, and Dissociation Half-Life of
`Lemborexant. The association kinetics of the radioligand [125I]OXA
`were measured as described above in the absence and presence of 7,
`14, and 28 nmol/l unlabeled lemborexant in the same experiment.
`Three independent experiments were performed, with values being
`determined in triplicate (n 5 3).
`The results were analyzed as follows. The harmonic mean of the koff
`values of the radioligand [125I]OXA obtained in the three dissociation
`experiments was calculated and then used as a fixed constant (K2) for
`the analysis of the association experiments. The three association
`experiments were first analyzed individually. The kon and Bmax values
`of [125I]OXA were determined individually for each of the three
`association experiments. The kon (K3) and koff (K4) values of the
`unlabeled lemborexant were calculated individually from the results
`of each of the three association experiments, using the corresponding
`individual [125I]OXA kon (K1) and Bmax values and the harmonic mean
`of the [125I]OXA koff values (K2) of the three dissociation experiments.
`Finally, the harmonic means of the kon and koff values, and the
`arithmetic means of the dissociation half-lives, respectively, were
`calculated from the values of the three individual experiments.
`Data were analyzed using GraphPad Prism software (version 6.07;
`GraphPad Software), where the kon and koff values of lemborexant
`were calculated by fitting to the following formula:
`Y 5 Q  ðK4  DIFF=ðKF  KSÞ 1ððK4-KFÞ=KFÞ
` expð-KF  XÞ-ððK4-KSÞ=KSÞ  expð-KS  XÞÞ
`where Y is specific binding (in cpm), X is time, KA 5 K1  L  1029 1
`K2, KB 5 K3  I  1029 1 K4, S 5 SQRT((KA 2 KB)2 1 4  K1  K3 
`L  I  10218), KF 5 0.5  (KA 1 KB 1 S), KS 5 0.5  (KA 1 KB 2 S),
`DIFF 5 KF 2 KS, Q 5 Bmax  K1  L  1029/DIFF, L is the
`concentration of [125I]OXA (in nmol/l), K1 5 kon [125I]OXA, K2 5 koff
`[125I]OXA K3 5 kon lemborexant, K4 = koff lemborexant, half-life
`equals the ln(2) value divided by koff, and I is the concentration of the
`inhibitor.
`The S.E.M. values of kon and koff were calculated using SAS software
`(version 8.03; SAS Institute, Inc., Cary, NC), whereas S.E.M. values
`
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`
`Beuckmann et al.
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`for half-lives were calculated using GraphPad Prism (version 6.07;
`GraphPad Software).
`
`Kinetic RBA on hOX2R
`Determination of koff of [3H]EMPA. The koff value of [3H]EMPA
`was determined by allowing [3H]EMPA to reach equilibrium with
`hOX2R expressed in CHO cell membranes. After equilibrium was
`reached at 2 hours, the reassociation of [3H]EMPA was prevented by
`adding an excessive amount of EMPA. Bound [3H]EMPA was then
`measured at multiple time points over 90 minutes.
`Membrane suspension of hOX2R (final 0.8 mg protein/assay) and
`[3H]EMPA (final 3 nmol/l) were mixed, and the mixture (180 ml) was
`incubated for 2 hours at room temperature on a 96-well nonbinding
`surface plate (Corning, Corning, NY). Then, 20 ml of EMPA solution
`(100 mmol/l) was added and incubated at room temperature for
`between 5 and 90 minutes. For the 0 minute value, assay buffer
`instead of EMPA solution was added. All reaction mixtures were
`filtrated with UniFilter-96 GF/C (PerkinElmer) and washed twice
`with assay buffer containing 500 mmol/l NaCl using MicroBeta
`FilterMate-96 Harvester (PerkinElmer). UniFilter-96 was dried, and
`50 ml of Micro Scint 20 (PerkinElmer) was added to each well. The
`radioactivity of each well was measured by TopCount (PerkinElmer).
`The koff value of [3H]EMPA was calculated by fitting to the following
`formula:
`
`
`Y 5 ðY0 2 NSÞ  exp
`
`2 koff  X
`
`
`
`1 NS
`
`where Y is binding (in cpm), X is incubation time, Y0 is binding at time
`zero, NS is binding (nonspecific) at infinite times, and the half-life
`equals the ln(2) value divided by koff.
`GraphPad Prism version 6.02 (GraphPad Software) was used for
`the calculation. Each data point was measured in triplicate, and the
`experiment was repeated three times.
`Determination of kon of [3H]EMPA and kon and koff of
`Lemborexant, Suvorexant, and Almorexant. [3H]EMPA was
`added simultaneously with several concentrations of test antagonist
`(lemborexant, suvorexant, or almorexant) to hOX2R-expressing CHO
`cell membranes. The degree of [3H]EMPA bound to receptor was
`assessed at multiple time points over 4–6 hours after the addition of
`[3H]EMPA and a test antagonist mixture (Dowling and Charlton,
`2006).
`Membrane suspension (final 0.8 mg protein/assay), [3H]EMPA (final
`3 nmol/l), and test antagonist (0, 1, 3, or 10 nmol/l) were mixed at room
`temperature for 0.5–240 minutes (lemborexant and suvorexant) or
`0.5–360 minutes (almorexant). For the determination of nonspecific
`binding, [3H]EMPA (final 3 nmol/l), test antagonist (final 20 mmol/l),
`and membrane suspension (final 0.8 mg protein/assay) were mixed and
`incubated for 4 hours (lemborexant and suvorexant) or 6 hours
`(almorexant) at room temperature. All reaction mixtures were
`filtrated with UniFilter-96 GF/C and washed twice with the assay
`buffer containing 500 mmol/l NaCl using MicroBeta FilterMate-96
`Harvester. UniFilter-96 was dried, and 50 ml of Micro Scint 20 was
`added to each well. The radioactivity (in cpm) of each well was
`measured by TopCount (PerkinElmer).
`The kon and koff values of lemborexant were calculated as described
`previously (Mould et al., 2014), using the kon and koff values of [3H]
`EMPA (calculated with Phoenix WinNonlin version 6.3; Certara,
`Princeton, NJ).
`Each value is expressed as the mean and S.E.M. The mean value
`and S.E.M. were calculated based on the kon or koff values from three
`independent experiments in triplicate (EMPA and lemborexant) or
`triplicate measurements of one experiment (almorexant and
`suvorexant).
`
`Off-Target Panel Binding Assay
`A panel binding/functional assay was conducted on 80 receptors,
`transporters, and ion channels of important physiologic function
`
`(High-Throughput Profile; CEREP, Celle l’Evescault, France), as well
`as eight additional drug dependence liability–related and sleep/wake
`regulation–related targets (CEREP), as listed in Supplemental
`Table 1. Lemborexant was tested in two concentrations (1 and
`10 mmol/l), and values were determined in duplicate. Binding was
`calculated as the percentage of inhibition of the binding of a
`radioactively labeled ligand specific for each target. Significant
`binding was defined as more than 50% inhibition.
`
`Off-Target Functional Assay on Human Melatonin
`1 Receptor and Human Melatonin 2 Receptor
`Human melatonin 1 receptor (MT1R) was stably expressed in
`HEK293 cells containing Gqi5 chimeric G-protein (MT1R 1
`Gqi5/HEK293), which converts Gi-protein signaling from MT1R
`into intracellular calcium mobilization (Coward et al., 1998). Cells
`containing Gqi5 but no MT1R were used as control (Gqi5/HEK293).
`The same procedure was followed for cells expressing human melato-
`nin 2 receptor (MT2R). Cell-based calcium mobilization functional
`assay was carried out as described above for OXRs. Each data point
`was measured in quadruplicate, and each experiment was performed
`three times.
`
`Population Patch-Clamp Study on Human GABAA Receptor
`Functional GABAA receptor was stably expressed in HEK293 cells,
`which had been cotransfected via electroporation (microporator de-
`vice) with three separate expression plasmids containing respective
`human subunits a1, b3, and g2. Cells expressing human GABAA
`receptor (a1, b3, g2) were harvested via trypsinization and resus-
`pended in Dulbecco’s phosphate-buffered saline at a density of 2  106
`cells/ml. Chloride ion current through the GABAA receptor was
`measured by population patch clamping of cells (Hollands et al.,
`2009) in the presence of the positive allosteric modulator GABA on an
`IonWorks Quattro Instrument (Molecular Devices, Sunnyvale, CA).
`Forty microliters of cell suspension was placed into the wells of
`PatchPlate PPC (Molecular Devices). After allowing cells to seal to the
`substrate and achieving a stable patch-clamp configuration, a voltage
`ramp (300 ms, 280 to 160 mV) was applied, and the resulting currents
`were sampled at 2.5 kHz. After this initial signal measurement
`without GABA, 20 ml of compound solution containing 0.9 mmol/l
`GABA and 3-fold concentrated compounds lemborexant or phenobar-
`bital were added, and the same voltage ramp was applied again. Final
`concentrations for lemborexant or phenobarbital were 0.2, 1, and
`5 mmol/l, or 50 and 100 mmol/l, respectively.
`The influence of test compounds was measured as changes in the
`chloride ion current, with all electrophysiological measurements being
`conducted at room temperature. Phenobarbital at 50 and 100 mmol/l
`served as the positive control (reference compound). For data analysis,
`differences in currents at 0 mV voltage before and after GABA and
`compound addition were used. Each data point was measured in
`quadruplicate, and the experiment was performed once.
`
`Computational Method for Complex Modeling and Energy
`Calculation of Lemborexant in hOX1R and hOX2R
`Lemborexant was docked into the X-ray crystal structures of
`hOX1R [Protein Data Bank (PDB) identifier 4ZJ8; Yin et al., 2016]
`and hOX2R (PDB identifier 4S0V; Yin et al., 2015) after the
`elimination of fusion protein and protein modeling using the Homol-
`ogy Modeling tool in MOE 2014.09 (Chemical Computing Group,
`Montreal, Canada) and Protein Preparation Wizard in Maestro
`(version 10.7; Schrödinger, New York, NY) with default settings.
`Ligand docking simulations were conducted using Glide XP (version
`7.2; Schrödinger) (Friesner et al., 2004; Halgren et al., 2004) after
`conformational search by MacroModel (version 11.3; Schrödinger).
`The binding poses were chosen by the clustering of ligand conforma-
`tion and WaterMap/Molecular Mechanics Generalized Born Surface
`Area (MM-GBSA) scoring using WaterMap (version 2.8; Schrödinger).
`
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`In Vitro Characterization of DORA Lemborexant
`
`291
`
`murine OXRs or any influence on cell viability up to 1 mmol/l
`(data not shown). The activation curves of human and murine
`OX1R by OXA and OX2R by three different peptide agonists
`and the dextral shift caused by titration with lemborexant are
`shown in Supplemental Fig. 3.
`Data extracted from the curves in Supplemental Fig. 3 are
`listed in Table 2. Also in this assay, no species difference
`between human and murine OXRs could be found, and
`lemborexant showed higher affinity for OX2R than for
`OX1R. Schild slopes very close to a value of 1 indicate simple,
`competitive binding, and the fact that the compound shows
`comparable behavior against three different peptide agonists
`on the OX2Rs strongly suggests an orthosteric binding mode
`to the peptide binding pocket within the receptor. For the
`OX1R, only one peptide, OXA, was available as agonist;
`therefore, such a comparison could not be made.
`Association and Dissociation Kinetics on Human
`OXRs. Association rates to and dissociation rates from the
`receptors were determined on the hOX1R for lemborexant and
`on the hOX2R for lemborexant, almorexant, and suvorexant
`via a kinetic RBA. For this purpose, surrogate radiolabeled
`tracers, DORA [125I]OXA for hOX1R and 2-SORA [3H]EMPA
`for hOX2R, were used. Dissociation characteristics of the
`labeled tracer molecules [125I]OXA (Supplemental Fig. 4A)
`and [3H]EMPA (Supplemental Fig. 5A) were determined by
`exposing the tracer-receptor complex to excess concentrations
`of OX1R-selective antagonist SB-334867 or unlabeled EMPA,
`respectively. Subsequently, the influence of increasing con-
`centrations of lemborexant on the association kinetics of [125I]
`OXA to the hOX1R was assessed (Supplemental Fig. 4B). In a
`similar fashion, the effect of increasing concentrations of
`lemborexant, almorexant, and suvorexant on the association
`kinetics of [3H]EMPA to the hOX2R was determined (Supple-
`mental Fig. 5, B–D, respectively).
`Table 3 summarizes the kinetic parameters for labeled
`tracers and unlabeled DORAs on both human OXRs, as
`derived from data depicted in Supplemental Figs. 4 and 5.
`Lemborexant showed faster association to and dissociation
`from the hOX2R compared with suvorexant and almorexant,
`the kinetic profiles of which were consistent with what had
`previously been described (Gotter et al., 2013; Mould et al.,
`2014). Although the association speed of lemborexant to
`the hOX1R was comparable to that to the hOX2R, disso-
`ciation speed from the hOX1R was faster than from the
`hOX2R.
`
`After ligand-protein complex modeling, molecular dynamics (MD)
`simulations were performed using Desmond (version 4.7 Schrödinger)
`(Abel et al., 2008). Each lemborexant-human OXR complex model was
`then embedded in a 1-palmitoyl-2-oleoylphosphatidylcholine lipid
`bilayer and solvated using a TIP3P box water model with 0.15 M NaCl.
`Binding free energy of representative complex structures from MD
`simulation trajectory were calculated by MM-GBSA technology
`(Huang et al., 2006; Lyne et al., 2006) using Prime MM-GBSA (version
`3.0; Schrödinger)
`
`Results
`Binding Affinities and Antagonistic Activities of
`Lemborexant. The affinities for hOX2R and hOX1R were
`determined via RBA by the ability to inhibit binding of [125I]
`OXA to cell membranes prepared from either recombinant
`hOX1R- or hOX2R-expressing cells. Inhibition curves of ra-
`diolabeled tracer in the presence of lemborexant, almorexant,
`and suvorexant are depicted in Supplemental Fig. 1.
`In addition, we evaluated the antagonistic function of OXR
`antagonists on recombinantly expressed hOX1Rs and
`hOX2Rs, of which activation by OXA triggers an intracellular
`calcium signal increase. To investigate whether species dif-
`ferences exist, antagonists were evaluated on OXRs of human,
`rat, and mouse origin. Antagonist inhibition curves for
`lemborexant, almorexant, and suvorexant as determined via
`direct calcium mobilization upon receptor activation by OXA
`are depicted in Supplemental Fig. 2.
`Concentrations necessary for IC50 (via RBA) and Ki (via cell-
`based direct calcium imaging) values for the three DORAs
`were derived from data shown in Supplemental Figs.
`1 and 2 and are listed in Table 1. Although lemborexant
`and almorexant would be categorized as a DORAs in the RBA
`as well as even more in the functional assay, both compounds
`have higher affinity for the OX2R than for the OX1R. In contrast,
`suvorexant showed a slightly higher preference for the OX1R in
`our assay system. Furthermore, we could not detect any sub-
`stantial difference in OXR affinities of the three DORAs among
`the three species evaluated.
`Binding Mode and Site. To determine binding mode and
`site, another cell-based functional assay was conducted that
`measured the activity of reporter enzyme PLAP. This enzyme
`was expressed and released into cell medium in relation to the
`intracellular Ca21 increase upon OXR activation and was
`therefore a direct measure of OXR activation. In this assay,
`lemborexant did not show any agonistic activity on human or
`
`TABLE 1
`IC50 and Ki values of lemborexant, almorexant, and suvorexant when competing against OXA on human,
`rat, and mouse OX1R and OX2R in RBA and a cell-based FDSS
`Data represent the mean 6 S.E.M.
`
`RBA
`
`Suvorexant
`Lemborexant Almorexant
`IC50 in 1029 mol/l
`6.1 6 1.4
`8.6 6 6.5
`2.6 6 0.4
`4.6 6 1.6
`N.D.
`N.D.
`N.D.
`N.D.
`N.D.
`N.D.
`N.D.
`N.D.
`
`8.8 6 2.5
`12.0 6 2.8
`N.D.
`N.D.
`N.D.
`N.D.
`
`hOX1R
`hOX2R
`rOX1R
`rOX2R
`mOX1R
`mOX2R
`
`FDSS Ca2+ Imaging Assay
`
`Ki
`
`Lemborexant Almorexant