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Pharmacophore Model To Discover OX1 and OX2 Orexin Receptor Ligands Ainoleena Turku,†,∥ Alexandre Borrel,†,‡,§ Teppo O. Leino,† Lasse Karhu,† Jyrki P. Kukkonen,∥ and Henri Xhaard*,† †

Faculty of Pharmacy, Division of Pharmaceutical Chemistry and Technology, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland ∥ Faculty of Veterinary Medicine, Department of Veterinary Biosciences, University of Helsinki, P.O. Box 66, FIN-00014 Helsinki, Finland ‡ INSERM, §Université Paris Diderot, Sorbonne Paris Cité, UMRS-973, MTi, 35 Rue Héléne Brion, 75205 Paris Cedex 13, case courier 7113, Paris, France S Supporting Information *

ABSTRACT: Small molecule agonists and antagonists of the orexinergic system have key implications for research and therapeutic purposes. We report a pharmacophore model trained on ∼200 antagonists and prospectively validated by screening a collection of ∼137,000 compounds. The resulting hit list, 395 compounds, was tested for OX1 and OX2 receptor activity using calcium mobilization assay in recombinant cell lines. Validation was conducted using both calcium mobilization and [125I]orexin‑A competition binding. Compounds 4−7 have weak agonist activity and Ki’s in the 1−30 μM range; compounds 8−14 are antagonists with Ki’s in the 0.1−10 μM range for OX2 and 1−50 μM for the OX1 receptor. Docking simulations were used to devise a working hypothesis where two subpockets are important for activation, one between TM5 and TM6 lined by Phe5.42, Tyr5.47, and Tyr6.48 and another above the orthosteric pocket lined by Asp2.65 and Tyr7.32.

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INTRODUCTION The functional orexin/hypocretin system is composed of the peptides orexin-A and orexin-B (33- and 28-amino acids, respectively) and their G protein-coupled receptors OX1R and OX2R.1 Centrally, the orexin system regulates the sleep− wakefulness cycle as well as feeding and metabolic rate, stress response, and reward/addiction.2 Orexin responses are also detected in several peripheral tissues, such as the digestive track, pancreas, gonads, and adrenal glands, but the physiological role of these is unclear. Moreover, orexin receptor activation induces apoptosis in some cell populations; for instance, colon cancer cells express OX1Rs that induce apoptosis upon activation, unlike normal colon epithelium.3 Several genetic and pharmacological studies provide evidence for the principle of blocking orexin signaling in the development of sleep-promoting pharmacotherapies;4 the antagonist 1 (suvorexant/MK-4305; Figure 1)5 was recently approved by United States Food and Drug Administration for the treatment of primary insomnia. Conversely, orexin receptor activators, © 2016 American Chemical Society

either receptor agonists or potentiators, would have applications as wakefulness- and alertness-promoting agents6 as well as in other therapeutic areas, such as cancer.3 It is generally assumed that such compounds would have a strong therapeutic benefit for patients suffering from idiopathic (type I) narcolepsy as compared to the current therapeutics, and also in other diseases.7−9 Small molecular weight orexin receptor agonists are, furthermore, much needed research tools to study the orexin system. While development of nonpeptide dual and selective orexin receptor antagonists has been both popular and successful, discovery of nonpeptide agonists or potentiators has been reported to be extremely challenging.4 At the start of this study, only native and modified orexins had been reported to be activators of the orexin system.2 As peptides, native or modified orexins have short physiological half-lives, poor blood−brainReceived: March 3, 2016 Published: August 22, 2016 8263

DOI: 10.1021/acs.jmedchem.6b00333 J. Med. Chem. 2016, 59, 8263−8275

Journal of Medicinal Chemistry

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medicinal chemistry program that involved synthesis of more than 1000 analogues. The scale of the Nagahara study illustrates well the need to develop computational tools for the prescreening of compound libraries in order to discover new orexin receptor agonists. Here, we report the development of a pharmacophore-based screening approach tailored to discover ligands of OX1R and OX2R. The pharmacophore model is validated through the prospective testing of 395 compounds, leading to ligands with partial agonist activity and chemically novel antagonists.

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RESULTS AND DISCUSSION Pharmacophore Modeling. The goal of the procedure was to build a pharmacophore model that integrated: (1) the features of compound 2validated beforehand for its in vitro agonist activity at OX1R and OX2R (see the Experimental Section); (2) features present on the major classes of OX1Rspecific and dual orexin receptor antagonists; (3) information about the binding site, used to select the most interesting features and to define exclusion volumes. Such a model was hypothesized to be able to uncover both novel agonists and antagonists of OX1R and OX2R. All pharmacophore modeling was conducted using OX1R and the test set composed of OX1R-specific ligands. Nonetheless, molecular modeling, corroborated by the crystal structures of both receptor subtypes17,18 released after the pharmacological screening took place, suggests OX1R and OX2R to be nearly identical at their binding cavities; Ser103/Thr1112.61 (in superscript, the amino acid position using the Ballesteros−Weinstein convention19) and Ala127/Thr1353.33 are the only amino acids closer than 4 Å to the cocrystallized 1 that differ.17,18 Thus, the obtained hit list was tested on both OX1R and OX2R subtypes. Pharmacophore hypotheses were explored from different starting points: subsets of chemically diverse antagonists superimposed and/or docked, docked poses of the orexin-A peptide,20 and docked poses of 2 together with SAR data presented in the patent to guide the selection of the pharmacophoric features. The type of features tested was driven by the receptor−ligand interactions suggested by the predicted binding poses. At that stage, we used a neurotensin receptor 1 (NTSR1)-based OX1R homology model20 for all docking studies, since the orexin receptor crystal structures

Figure 1. Chemical structures of compounds 1, 2, and 3.

barrier permeation, and insignificant intestinal or topical absorption, which preclude their in vivo use.10−12 In 2010, Lee et al. reported screening of thousands of peptoids to identify orexin receptor activators, which led to an allosteric orexin receptor potentiator.13 In addition, the patent literature reports two series of non-peptide orexin receptor agonists.14,15 Compound 2 (Yan7874; Figure 1) is reportedly the most active among its congeners; 2 is reported to induce a stronger signal on OX2R as compared to OX1R, but only limited information regarding its potency and selectivity is provided in the patent.14 While this manuscript was under preparation, Nagahara et al. reported compound 3 (Figure 1), the first full orexin receptor agonist of small molecular size.16 This compound was discovered by the high-throughput screening of a chemical library containing 250,000 drug-like compounds followed by a

Figure 2. Best-performing pharmacophore model and comparison with binding modes deduced from the OX2R structure. (A) The best four-feature pharmacophore model described in the text: HBD, purple; R1 and R2, orange; and H, cyan. For clarity, exclusion volumes used for screening are not shown. (B) Mapping of the pharmacophore model on 1 bound to the OX2R crystal structure. The water molecule 4025 in the crystal is represented by a red sphere. 8264



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Calcium-Based Pharmacological Screening. The bestperforming pharmacophore model was used for virtual screening of an academic compound collection of about 137,000 compounds. The size of the hit list to be experimentally tested was set to be ∼400. Since the pharmacophore modeling provided a hit list of ∼800 compounds, its size was reduced by first filtering out compounds with toxic liabilities, too low molecular weight ( 10 000 nM, 41 molecules). The best performing pharmacophore hypothesis was built from the binding pose of 2. It discriminates the test set antagonists from the decoys with 59% sensitivity (96/162 true positives) and 90% specificity (37/41 true negatives), while the area under the receiving operator curve (AUC) is 0.85. This pharmacophore model consists of four features (Figure 2A): one hydrogen bond donor (HBD), two aromatic features (R1 and R2), and one hydrophobic feature (H). In space, these features form a triangle with side lengths of 5.6 Å (HBD−R2), 7.6 Å (R2−H), and 9.4 Å (H−HBD). The HBD is placed on 2’s hydroxyl group, and was defined based on the SAR data reported in the patent.14 The corresponding hydroxyl group cannot be changed to a carbonyl or methoxy group without losing the receptor activation, which indicates that either binding or the ability to activate the receptor has been altered. The two aromatic features R1 and R2 are located centrally on 2, and the hydrophobic feature H locates on 2’s tolyl group. This spatial arrangement of the H and R1−R2 features allows the test set antagonists to be placed in conformations that conform to the U-shape observed in the binding mode of 1 in the X-ray protein crystals.17,18 Exclusion volumes were added to the final pharmacophore model based on a distance threshold of 4 Å from 2. Since the pharmacophore model was built from 2 positioned inside the OX1R binding cavity, it allowed us to make inferences about the relationships between the pharmacophoric features and interacting amino acids at the OX1R (and/or OX2R) binding sites. In the docking mode of 2, HBD is at hydrogen bonding distance to Asn3186.55. Compound 2’s fused aromatic rings R1 and R2 are able to make T-shaped π−π stacking interactions with His2165.39 and Phe2195.42. The H feature lies in a vicinity of the β-carbon of Ser3156.52. When the three-dimensional NTSR1-based OX1R model containing docked 2 is superimposed as a whole body on the OX2R crystal structure bound to 1 (Figure 2B), the benzoxazole and phenyl rings of 1 map on top of the aromatic features R1 and R2, with H being in the vicinity of the 1,2,3-triazole ring. The binding mode of 1 to OX2R includes a water molecule (water 4025) that bridges the carbonyl groups of 1 and Asn3246.55. This water molecule is a critical feature of the binding mode of 1 to OX2R, and very interestingly, it aligns with the HBD feature. Caution should be kept, since these inferences are limited by the inaccuracies of the docking mode; multiple docking solutions of 2 are acceptable even in the X-ray structure of OX2R,17 which became available after the pharmacological screening had been conducted. The docking modes of 2 to the OX2R crystal structure entail several alternate binding possibilities caused by the flexibility of 2’s dichlorophenyl moiety and flipping of ligand around its short axis. Using a different docking solution logically leads to a different positioning of the pharmacophore in the binding site (Supporting Information Figure 1). 8265



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Figure 3. continued shown as gray squares. (B) Same as (A), but the measurement is after 30 min. (C) Correlation of the antagonist activities between OX1 and OX2 receptor-expressing cells. The activities of compounds 8−14 are shown as plain circles, and those of the rest of the 23 compounds selected for antagonist validation are shown as gray squares. Compound 2 is shown in all panels (star).

responses and availability for purchase. Following characterization and validation, two agonists were kept aside for medicinal chemistry optimization and are not presented in this manuscript. As potential agonists, we selected 18 compounds that showed a response of at least twice the background response on both receptor subtypes (Figure 3A), as well as five compounds that showed more than 15% response after 30 min stimulation (possibly slow-acting compounds; Figure 3B). In addition, one potential agonist with activity in only some replicates was retested. Since the observed calcium response is not necessarily mediated through orexin receptors, these 26 potential agonists were subsequently tested at 10 μM concentration in the Ca2+ assay in the absence and in the presence of a high-affinity orexin antagonist (Supporting Information Figure 3A). At the hit validation stage, we used 15 (TCS-1102; Supporting Information Figure 4) for both subtypes, which has been reported to have a Ki of 3 nM for OX1R and 0.2 nM for OX2R.25 In addition, the binding of the compounds was assessed at 10 μM concentration at both orexin receptors in a competition binding assay against [125I]-orexin-A (Supporting Information Figure 3B and Supporting Information Table 2). As a result, we present four compounds (referred to as compounds 4−7) that were validated as agonist hits, since they induced an intrinsic calcium response that could be at least partially blocked by 15 as well as displacement of [125I]-orexinA. Their best orexin receptor-specific responses at 10 μM ranged from 2.1 ± 1% (compound 6, OX1R) to 3.5 ± 1.7% (compound 7, OX2R), as expressed as a percentage of the full response to orexin-A. Two of these compounds, 4 and 5, furthermore showed over 70% inhibition of [125I]-orexin-A binding to either receptor subtype (Supporting Information Figure 3A−B and Supporting Information Table 2). Compounds 6 and 7 inhibited [125I]-orexin-A binding to either orexin receptor subtype by approximately 20−40%. Interestingly, the study also revealed an additional four compounds with a potential agonist effect that we did not select for characterization. Their structures are shown in Supporting Information Table 4; one is structurally very similar to compound 13, that is found to be an antagonist in this study. Another compound appears to be a weak potentiator that leads to increased binding of [125I]-orexin-A at the OX1R but not the OX2R subtype (Supporting Information Table 5). One of the compounds tested as a potential agonist showed over 50% inhibition of [125I]-orexin-A binding to OX1R, but no orexin receptor specific activation, and thus we considered it as an antagonist hit. As potential antagonists, we selected 21 compounds that showed ≥20% inhibition of the response to 0.3 nM orexin-A on either receptor subtype (Figure 3C). Two potential antagonists with high inhibition in some but not all of the replicates were also retested. Validation was conducted as above, i.e. by measuring the ability of 10 μM of the compound to displace

Figure 3. Pharmacological testing data for 393 virtual hits. (A) Correlation of the agonist activity (ability to elevate Ca2+) between OX1 and OX2 receptor-expressing cells measured within 2 min. The activities of compounds 4−7 are shown as plain circles, and those of the rest of the 24 compounds presented for agonist validation are 8266



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Figure 4. Characterization of compounds 4−7. (A) Orexin receptor-specific Ca2+ responses induced by the compounds, i.e. fraction of the calcium signal that can be blocked by an orexin receptor-specific antagonist. (B) Calcium response elicited by the compounds, i.e. after subtracting the background response. In panels A and B, values are given as percentage of the specific response to 10 nM orexin-A, and are averages of three independent experiments performed in quadruplicate. (C) pKi values for compounds 4−6. For 7, the pKi could not be determined from the binding data (see text). The data are averages of at least three independent experiments performed in quadruplicate.

[125I]-orexin-A (Supporting Information Figure 3C and Supporting Information Table 3). As a result, 21 out of 23 putative antagonist hits produced over 50% inhibition of [125I]orexin-A binding to either receptor subtype. Of these, we selected the seven most potent for further characterization (compounds 8−14). Characterization of the Agonist Hits. The four agonist hits (4−7) were scrutinized for calcium responses using a new set of experiments: responses to 1 and 10 μM of each compound were measured in CHO-hOX1 and -hOX2 cells in the absence and in the presence of the antagonists 16 (for OX1R SB-334867, Supporting Information Figure 4) and 15 (for OX2R) at 10 μM concentration (Figure 4A). We expect that the used antagonists are able to fully displace the tested compounds, since 16 has a Ki of 14−21 nM for OX1R and 15 has a Ki of 0.2 nM for OX2R, which are at least 50-fold lower than the Ki’s of 4−7 (see below). Furthermore, the response to 1 nM orexin-A, which already produces maximal or nearly maximal receptor activation (the EC50 of orexin-A is 0.2−0.4 nM at the receptor subtypes23), was fully blocked by these antagonists (data not shown). In the characterization phase, calcium responses are expressed as a percentage of the specific orexin-A response ± SEM (i.e., orexin-A induced response minus background response). For OX1R, at 10 μM compound concentration, all hits have a specific orexin receptor-mediated component in their calcium responses: 6.3 ± 1.2% for compound 4, 7.3 ± 0.6% for compound 5, and 5.4 ± 0.4% for compound 6; compound 7 is weaker, with an OX1R-specific calcium release around 2.7 ± 0.2%. In general, all our agonist hit compounds, except 7, show stronger activation of OX1R than of OX2R, with their OX2Rmediated calcium responses being 1.0−3.7% of the orexin-A induced response. We also tested control CHO cells (i.e., not expressing orexin receptors); in these cells, compounds 4−7 (but not orexin-A) produced calcium responses of similar magnitude as the non-orexin receptor-mediated responses in the CHO-OX1/OX2 cells (data not shown). No significant responses were obtained at 1 μM compound concentration. The specific responses measured for 4−7 are thus weak; however, in terms of magnitude, they are clearly larger than, for example, the background responses, which range approximately

from 2.5 to 3.5% of the orexin-A specific response. When considering the background responses measured within each individual experiment, at OX1R the specific response at 10 μM concentration for compound 4 is 3.3-fold the background response, that for compound 5 is 3.4-fold, that for compound 6 is 2.8-fold, and that for compound 7 is 1.9-fold. At OX2R the compounds’ specific responses are lower than those at OX1R, i.e. 1.4−2.2-fold the individual background responses. Comparing statistically the specific responses with the background responses using a t test shows that compounds 5 and 6 at OX1R (p values at 4.6e−7 and 1.1e−4) and, to a lesser extent, compound 4 at OX1R (p value of 9.0e−2) are significantly larger (other specific responses are not). It is important to note this refers to a comparison of the magnitude of the responses taken independently; the compound’s specific responses are, by definition, exempt of a background component. Higher background responses and lower specific activities were observed in the validation phase (see previous section), which can be easily explained by the larger number of replicas in the characterization phase that lead to more robust data points. The specific response of compound 5 at OX1R is at a similar level to the full calcium-induced response of this compound (Figure 4B), which indicates that most of the response is mediated through OX1R. This is also, to a lesser extent, true for compounds 6 and 7. Compound 4 appears, on the other hand, unspecific or very active at a secondary target, which is not surprising, since it is the nonselective β-adrenoceptor blocker carvedilol (chemical structures are presented below). The binding affinities (IC50) were determined based on the displacement of [125I]-orexin-A (Figure 4C, Table 1). The calculation of the pKi values from the IC50 values is not directly possible, owing to difficulties in determination of the Kd for [125I]-orexin-A, and thus, we used the functional Ki of a standard compound, 16, for normalization (see Experimental Section). The most potent binders with 1−3 μM Ki’s are 4, which binds equally well to both receptor subtypes, and 5, which prefers OX2R by about 10-fold (Figure 4C; Table 1). Compound 6 has a Ki of about 8 μM at OX1R and 18 μM at OX2R. We could not determine the binding affinity for 8267



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Table 1. IC50 and Ki Measured for Compounds 4−7 in Competition with [125I]-Orexin-A CHO-OX1

Compound

IC50 (binding) [μM]

4 5 6 7

2.3 ± 1.9 53.7 ± 23.2 13.3 ± 11.1 n.d.

an orexin receptor agonist.16 Compound 3 is built around a sulfonamide core, and alkylation of the secondary amine leads to compounds without agonist activity.16 Compounds 2, 6, and 7 thus demonstrate novel molecular frameworks that can lead to orexin receptor activation: 1-(2-imino-3-methyl-benzimidazol-1-yl)-2-propanol for 2 and the 1,3,5-triazine for 6 and 7 (Figure 6A). Compounds 4 and 5 do not bear clear scaffolding chemical units and are flexible in their central region. Compound 4 is carvedilol, a compound well-known as a βadrenoceptor blocker but whose action at orexin receptors was not previously identified. In β-adrenoceptors the central region of 4 (protonated amine, hydroxyl) is necessary for interaction via a salt bridge to the conserved aspartic acid 3.32; interaction with adrenoceptors, furthermore, requires one aromatic group near TM5.26,27 Although the agonist effect of compounds 4−7 is weak, they represent good starting points to develop chemical probes to study orexin receptor activation. When considering development of clinical candidates, the triazines 6 and 7 (as well as 9) have a high ratio of nitrogen to carbon atoms and, as such, are likely to be difficult to optimize as well as to have metabolic liabilities. Triazines have been reported to be ligands of several GPCRs, for example, the A2A adenosine, D3 dopamine, and CB2 cannabinoid receptors.28−30 Among the antagonists, compound 9 interestingly showed absorbance at wavelengths 350−530 nm and, thus, has potential for development as a chemical probe for binding or energy transfer studies. To date, there is no such probe available for the orexin receptors. Overall, compounds 4−14 share a framework often found in orexin antagonists, i.e. a central region containing several polar centers, capable of hydrogen bonding, surrounded by aromatic functional groups (Figure 6). The flexible regions of 2−12 and 14 should allow them to adopt conformations that reach the intramolecularly stabilized U-shape observed for 1; however, 13 is too rigid to do so. Compound 8 has a sulfonamide functionality, which is found in several OX2R-selective ligands such as the agonist 3 and the antagonist EMPA31 (see also Supporting Information Table 6). The structures of 10 and 11 include a larger substructure composed of a triazole ring, a sulfur atom, and an amide bond. Amide bonds are found, for example, in 5, 10−12, and 14. In order to represent the chemical novelty of these compounds, we described them using six intuitively under-

CHO-OX2

Ki (binding) [μM]


Ki (binding) [μM]

1.3 ± 1.1 30.7 ± 13.3 7.6 ± 6.4 n.d.

0.9 ± 0.2 1.7 ± 1.0 11.8 ± 7.7 n.d.

1.4 ± 0.3 2.5 ± 1.5 17.8 ± 11.6 n.d.

compound 7, since it somehow potentiated the binding of the radioligand, suggestive of co-operative binding (not shown). The specific responses induced by compounds 4−7 are thus, not surprisingly, much weaker than those of orexin-A, deeming them as partial agonists. It should, however, be noted that response saturation cannot be obtained due to compounds’ limited solubility to water. It should also be noted that the responses are obtained in an overexpressed system with a high receptor density and may not be relevant to normal physiology without further compound optimization. Nonetheless, the data provided can be with high confidence interpreted as a demonstration of a calcium elevation mechanism mediated by the orexin receptors. Characterization of the Antagonists. The Ki values of the antagonists 8−14 selected for characterization were determined in two ways: binding assay against [125I]-orexin-A, and calcium assay to assess their functional inhibition of orexinA (Figure 5 and Table 2). For OX1R, the functionally determined inhibitory constants are always higher than those determined in binding (by 2- to 9-fold). For OX2R, the results are consistent between the two methods, except for compound 9, for which the functionally determined Ki is about 20-fold higher than the Ki determined in the binding assay. The reasons for this discrepancy are not known. Compounds 9, 10, and 11 have the highest affinity for OX1R with binding-assaydetermined Ki’s of about 1 μM. For OX2R, compounds 8 and 10 are the most potent antagonists with submicromolar affinities (0.1−0.3 μM), identified by both Ki determination methods. Compounds 8 and 10 are thus at least 50-fold (8) and 20-fold (10) selective toward OX2R. Chemical Diversity of the Hits. To date, only a single compound, 3, has been reported in the literature to function as

Figure 5. Characterization of the seven antagonist compounds 8−14. (A) pKi values for OX1R and OX2R measured in the binding assay. (B) pKi values for OX1R and OX2R measured in the functional assay. The data are averages of at least three independent experiments performed in quadruplicate. 8268



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Table 2. IC50 and Ki Measured for Compounds 8−14 in Competition with [125I]-Orexin-A (binding), and the Ki Determined from Their Functional Inhibition of Orexin-A (functional) CHO-OX1 Compound 8 9 10 11 12 13 14

IC50 (binding) [μM] 11.1 1.9 2.2 1.4 4.8 20.9 11.7

± ± ± ± ± ± ±

2.2 1.7 0.2 0.4 2.8 7.7 4.2

Ki (binding) [μM] 6.3 1.1 1.2 0.8 2.7 12.0 6.7

± ± ± ± ± ± ±

1.2 1.0 0.1 0.2 1.6 4.4 2.4

CHO-OX2 Ki (functional) [μM]


± ± ± ± ± ± ±

0.07 ± 0.04 0.3 ± 0.1 0.13 ± 0.06 0.7 ± 0.4 0.8 ± 0.6 0.7 ± 0.3 0.5 ± 0.1

46.7 16.2 6.0 7.0 10.3 21.6 13.1

40.3 10.5 3.6 3.6 4.6 14.7 3.6

Ki (binding) [μM] 0.1 0.4 0.2 1.1 1.3 1.0 0.8

± ± ± ± ± ± ±

0.06 0.2 0.09 0.5 0.8 0.5 0.2

Ki (functional) [μM] 0.1 11.0 0.3 1.0 1.7 1.2 0.9

± ± ± ± ± ± ±

0.05 6.0 0.2 0.8 1.2 0.9 0.4

Figure 6. Chemical structures of compounds 4−14. (A) Partial agonist hits (4−7). (B) Antagonist hits (8−14).

the most unique. Among the antagonists, compound 8 is highly similar to an analogue of EMPA31 (Supporting Information Table 6). As could be expected from the chemical structures, 11 and 14 are part of a chemical space distinct from the other reported antagonists. Compound 11 is the largest, and it includes two amide bonds, reminiscent of a peptide mimetic compound. Docking Simulations of Compounds 2−8 and 10. In order to get theoretical insights into the mechanisms beyond the selectivity of compounds 8 and 10 and the activation by compounds 2−7, docking simulations were conducted (Figure 8). While the predicted binding modes of 2−7 lead us to devise a working hypothesis concerning their mode of action, it is important to keep in mind that 4−7 have only a weak agonist effect that could be difficult to study in modeled three-

standable molecular descriptors: molecular weight, topological surface, logP, molecular refractivity, unsaturation index, and hydrophilic index. We then mapped the variability of these descriptors on two dimensions using Principal Component Analysis (PCA), either with the screened database of 137,000 compounds (two dimensions explained ∼81% of the data variability) or with the positive test set used for the pharmacophore modeling (two dimensions explained ∼75% of the data variability) (Figure 7). The structures of the closest neighbors from the OX1R test set and the OX2R antagonists used in this study are provided as Supporting Information Table 6. Overall, the orexin antagonists used as training and test sets occupy a defined region of the chemical space within the studied library (Figure 7A). The agonist structures have global similarities to existing antagonists while 3 and 7 appear to be 8269



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Figure 8. (A) Compound 1 in the crystal structure of OX2R. (B−D) The top scoring binding poses of 8 in OX2R (B), 3 in OX2R (C), and 4 in OX1R (D) selected using Glide gscore. Color code: receptor (gray), antagonist (orange) and agonist (green) carbon atoms, and water molecules (red spheres). Orange surface, location of the crystallized 1; green dashes, possible hydrogen bonds. For clarity, only a subset of the amino acids and binding site waters is shown. Viewed from the extracellular side of the receptor.

vast knowledge gathered about the activation of other GPCR subfamilies, a picture is however emerging. Amino acids at positions 3.32 (Gln126/134 in OX1R/OX2R) and 6.48 (Tyr311/317 in OX1R/OX2R) are likely to play a key role in the activation process as they do in other GPCR subfamilies. Four salt bridges are lining the human OX2R binding crevice and are likely to be disrupted during the activation process: Asp211xl2.51− Arg3286.59; Glu212xl2.52 − His2245.39; Asp1152.65 − His3507.39 and Glu1182.68 − Arg3397.28 (see reference34 for the nomenclature used for loop amino acids). Three equivalent salt bridges are found in OX1R: Asp203xl2.51− Arg3226.59; Glu204xl2.52 − His2165.39; Asp1072.65 − His3447.39 but not the Glu1102.68 − Arg3337.28. As a result of docking simulations, compounds 2−7 were found to interact with Gln126/1343.32, Ala127/Thr1353.33, and Gln179/1874.60. Compound 3, a full agonist, was found to occupy a unique region of the binding pocket in comparison to the weak agonists 4−6 (but not 7 in OX1R) (Figure 8C−D; Supporting Information Figure 5). The 3-methyl-substituted aryl group (substituent of the B-ring, see ref erence16) of 3 was found 7/16 times in OX1R and 14/17 times in OX2R docked to a subpocket lined by amino acids Ser103/Thr1112.61, Asp107/ 1152.65, Tyr337/3437.32, and Phe340/3467.35, “above” the orthosteric pocket and especially “above” His344/3507.39. Analogues of 3 with different substituents in their B-ring have significantly different functional potencies (see Table 2 of ref 16). Interestingly, the majority of the poses of 2 (10/12) and one pose of 7 (out of 13) were also found docked in this subpocket. In OX1R the majority of the poses of 7 (13/14) occupy the subpocket with a dibromophenyl ring, while 5/12 poses of 2 and 4/15 poses of 5 dock to that subpocket entirely. A second subpocket, occupied by functional groups from compounds 2−7 but not 5, was also identified (compound 5 being shorter along its long axis in its extended conformation). This pocket, located between TM5 and TM6, is bordered by aromatic amino acids Phe219/2275.42, Tyr224/2325.47, and Tyr311/3176.48, and it offers two hydrogen bonding sites: the side chains of Thr223/2315.46 and Ser315/3216.52. All these

Figure 7. Chemical space covered by the compounds using Principal Component Analysis derived from six interpretable molecular descriptors (PC, principal components). Two independent PCAs were calculated: one (A) for the full library and the active ligands, and another one (B and C) for only the active ligands. (A) Projection of the full library (137,000 compounds, gray), the hit list selected by the pharmacophore model (395 compounds, black), the OX1R antagonist positive test set (162 compounds, green), the characterized agonists (four compounds, cyan), and the characterized antagonist hits (seven compounds, orange). Compounds 2 and 3 are also shown (dark blue). (B) Projection of the descriptors. (C) PCA that includes only the subset of active ligands.

dimensional complexes. The docking study of compounds 2−7 is more extensively described in Supporting Information S20− S26 (Supporting Information Figures 5−8). Docking simulations of compounds 8 and 10 were carried out on both the OX1R and the OX2R crystal structures. The best binding pose obtained for 8 clearly resembled the pose of 1 in OX2R, but not in OX1R (Figure 8A−B, Supporting Information Figure 9). This could be connected with the Ala127/Thr1353.33 amino acid that was located near 8’s sulfonamide group in OX2R and thus could be a determinant of selectivity. For 10, the docking simulations could not be interpreted with respect to selectivity. The activation mechanism of the orexin receptors by the orexin peptides or by compound 3 is, for the most part, unknown. Based on the three-dimensional structures of the human orexin receptors,17,18 on the docking simulations of orexin peptides supported by site-directed mutagenesis studies,20,32 on the docking simulations of 3,16,33 and on the 8270



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comparison of the chemical structure and predicted binding modes of the full agonist 3, docking simulations were used to suggest a working hypothesis where two subpockets are important for orexin receptor activation. This could help rationalizing the design of orexin receptor agonists, especially provided that, for GPCRs, including peptide-binding receptors, switching from antagonist to agonists may require only a small but well-placed chemical change.38 This study also identified seven compounds 8−14 as dual or OX2R-selective antagonists with Ki’s in the 0.1−10 μM (OX2R) or 1−50 μM range (OX1R). These expand our knowledge about the chemical space accessible by orexin ligands and offer new possibilities for the development of antagonists. Furthermore, compound 9 shows absorbance and thus has the potential to be developed as a chemical probe. Altogether, we hope that the presented structures, pharmacophore model, and screening data will be useful for a better understanding of orexin receptor activation, to conduct virtual screens, and for building predictive models of orexin receptor activity.

amino acids have been shown by alanine mutations to play a role in ligand binding or receptor activation or both in OX1R and/or OX2R receptor subtypes.35,36 Compounds 2−7 are at π−π stacking interaction distances of the phenyl group of Phe219/2275.42 and, with the exception of 2 (OX1R) and 5 (both receptor subtypes), of Tyr311/3176.48. Interactions with 6.48 (usually a tryptophan) are central to GPCR activation; an important role for amino acids 5.42 and 5.46, that are, for example, serines in adrenoceptors and dopamine receptors, is also well recognized.26,37 In OX2R, Compounds 3, 4, and 7 are at a suitable position to hydrogen bond to Ser321 6.52 (Supporting Information Figure 7C−D and G−H). In agreement with this manuscript, docking studies by Nagahara and co-workers suggest that the subpocket is occupied by the dimethylcarbamoyl group of 3 (substituent of the A-ring, see ref 16), but this is not the case in the alternative binding mode suggested by Heifetz et al.16,33 Analogues of 3 show a large variation in functional potencies and efficacies depending on the substituents used (see Table 1 of ref 16). The binding mode of 1 places its triazole group closest to the subpocket. In summary, the partial orexin receptor agonists 2−7 can occupy only one of these subpockets at a time. It is possible that 2 occupies a different pocket than 4−7, but further studies are needed to clarify the issue. In comparison, docking studies of the full agonist 3 suggest simultaneous occupancy of these subpockets, while maintaining interactions in the central region of the binding pocket, which may be a prerequisite for full efficacy. Occupancy of different regions of the binding site by 3 is corroborated by its structure−activity relationships, where independent modifications of the sulfonamide core, the A-ring, or the B-ring can significantly affect functional efficacy.

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EXPERIMENTAL SECTION

Computational Studies. Modeling Receptor−Ligand Complexes. Two sets of 3D structures are used in this study. At an early stage, we used a NTSR1-based homology model of OX1R presented by Karhu et al. (2015).20 Compounds were docked to the OX1R model employing the Glide software and the induced fit protocol of Schrödinger Maestro 2013v221 with default parameters. The centroid of the coordinates of the receptor’s amino acid residues that were reported important for receptor activation was used to define the binding site.35,36 While this study was ongoing, the crystal structures of OX1R and OX2R were published.17,18 Subsequent docking studies used the Glide software and the induced fit protocol of Schrödinger Maestro 2015v1,39 with the default parameters; the binding site was selected according to the location of 1 in the crystal. Docking to the crystal structures was tested in both the presence and absence of the binding site water molecules (waters 4021 and 4025). Pharmacophore Modeling. As a starting point for this study, we verified the ability of 2 to act as a specific OX1R and OX2R agonist. At 10 μM, 2 induced about 35% and 45% of the full calcium response to orexin-A in CHO-hOX1 and -hOX2 cells, respectively (see also Figure 3A and 3B for the activity at 5 μM). This signal could be blocked completely by the orexin receptor subtype-selective antagonists 16 for OX1R and 15 for OX2R. Furthermore, in the binding assay, 2 displaced [125I]-orexin-A with an IC50 value of 6.3 μM from human OX1R and 2.2 μM from OX2R, confirming that 2 indeed activates human orexin receptors. An ensemble of pharmacophore hypotheses was explored using Discovery Studio 3.5.40 Both manual and automated methods were tested for the construction of pharmacophore hypotheses. Both shared-feature and structure-based pharmacophore models with various features were tested for their ability to discriminate a test set of 162 known dual and OX1R selective antagonists from 41 decoys (i.e., compounds with no or weak binding to OX1R). The classification performance of the models was defined by calculating the sensitivity (eq 1) and the specificity (eq 2) of retrieving the test set compounds. Receiver operator curves were generated, and the area under the curve calculated by Schrödinger Maestro 2013v2 software.

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CONCLUSIONS In this manuscript, we present a pharmacophore model prospectively validated for its ability to recover not only antagonists but also partial agonists of the orexin receptors. The hit rates associated with the pharmacophore are 1.5% to 2.5% for fishing partial agonists (ten actives out of 395 virtual hits, i.e. compounds 4−7, two compounds not presented here, and the four compounds shown in Supporting Information Table 4) and from 1.7% to 5.6% for antagonists (22 out of 395 antagonists have passed the validation phase). Although these numbers are defined using different success criteria, they could be combined to give an overall hit rate for the pharmacophorebased screening from 3.3 to 8.1% to find orexin receptor ligands. This illustrates the power of such an integrated approach. These hit rates are most likely higher than those obtained in high-throughput screening campaigns, although no precise data is publicly available. We present four previously unreported compounds, 4−7, that show weak OX1R and OX2R agonist activity as well as binding to either or both of these receptors with Ki’s in the 1− 30 μM range. Compounds 5−7 show a reasonable specificity for the orexin receptors. Although they are likely only partial agonists with effects demonstrated in cells overexpressing the orexin receptors and thus unlikely to have effects at the physiological level, they represent an interesting starting point for optimization, since they already have Ki’s in the low micromolar range. The Ki’s reported for the optimized compound 3 are only slightly better, 0.14 μM at OX1R and 0.77 μM at OX2R (using [125I]-orexin-A binding assay) with an Emax of 98%.16 Taking advantage of the structural data available for cocrystallized 1, and using it as a starting point for

sensitivity =

TP TP + FN

(1)

specificity =

TN TN + FP

(2)

True positive (TP) refers to the number of active compounds that are correctly classified, and false negative (FN) is the number of active compounds that are incorrectly classified. True negative (TN) is the 8271



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number inactive compounds that are correctly classified, and false positive (FP) is the number of inactive compounds that are incorrectly classified. The pharmacophore model (in dsv file format), the collected antagonists (i.e. the test and training set, in sdf format), and full docking results are available upon request. Virtual Screening and Hit Selection. For virtual screening, the library from the Finnish Drug Discovery and Chemical Biology consortium was used (http://ddcb.fi/en/; v2011, about 137 000 compounds). The screening was performed with default parameters using Discovery Studio 3.5, leading to a hit list of 800 compounds. The postfiltering of the hits was done in Schrödinger Maestro 2013v2, and it included molecular weight (>400 g/mol passing) and reactivity filters (REOS), as well as visual examination. The absence of possible PAINS containing structures was checked by FAF-Drugs2.41 The final hit list consisted of 395 compounds. Chemical Space. The chemical space of the compound library and of the subset of active ligands were represented using PCAs built using an orthogonal linear transformation from molecular properties of the ligands considered. Six molecular ligand descriptors from the PyDPI42 python package were used: molecular weight, molar refractivity, LogP value based on the Crippen method, topological surface area, unsaturation index and hydrophilic index. All plots and statistical analyses were made using R software (version 3.2.2).43 Material. Human orexin-A was purchased from NeoMPS (Strasbourg, France), probenecid (p-(dipropylsulfamoyl) benzoic acid) from Sigma-Aldrich (St. Luis, MO), 16 [N-(2-methyl-6benzoxazolyl)-N′-1,5-naphthyridin-4-yl urea] and 15 [N-biphenyl-2yl-1-([(1-methyl-1H-benzimidazol-2-yl)sulfanyl]acetyl)-L-prolinamide] from Tocris Bioscience (Bristol, UK), dyngo 4a (3-hydroxy-N′-[(2,4,5trihydroxyphenyl)methylidene]naphthalene-2-carbohydrazide) from Abcam (Cambridge, UK), and [125I]-orexin-A from PerkinElmer (PerkinElmer Life and Analytical Sciences, Waltham, MA). The 395 compounds tested in the pharmocological screen were obtained cherry-picked by acoustic dispensing from the Finnish Institute of Molecular Medicine chemical library (Helsinki, Finland). Compound 2 was obtained from ChemBridge Co. (San Diego, CA), and the 47 compounds for the validation from Ambinter (c/o Greenpharma, Orleans, France). In addition to the vendor specification, the purity and identity of the final hits was independently verified by mass spectroscopy and [1H]NMR. LC-MS analyses for purity were performed using a Waters UPLC I-class with a photodiode-array detector (PDA) and Synapt G2Si mass spectrometer (QTof). Signal separation was carried out by use of Acquity UPLC BEH C18 column (1.7 μm, 3.0 mm × 50 mm) or Luna C18 (5 μm, 4.6 mm × 50 mm), eluent: A MQ-H2O 0,1% HCOOH and B 0,1% HCOOH in ACN (gradient: start 95:5 (A:B) → 9 min 20/80 → 9.9 min 95:5 → 11 min 95:5), and flow = 0.6 mL/min. Each compound was found to be more than 99% pure (for 4−14 see Supporting Information Figures 12 and 13). DMSO (Sigma-Aldrich) served as a solvent for all the studied compounds, and corresponding concentration of DMSO was used in the vehicle control in all experiments. Pharmacological Screening and Characterization. Cell Culture. CHO-hOX1 and -hOX2 cells, expressing human OX1 and OX2 receptors, respectively,44,45 and control CHO cells not expressing these, were cultured as previously described. 46 For the Ca2+ measurement, the cells were seeded on polyethylenimine-coated22 black, clear bottom 96-well half area polystyrene plates (Corning 3887; Corning Inc., Corning, NY) at the density of 1.5 × 104 cells/well, and cultured overnight. In the binding assay, the procedure was similar except that white, clear bottom 96-well half area polystyrene plates (Cellstar #675098; Greiner Bio-One) were employed. Experimental Media. Hepes-buffered medium (HBM) (137 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 20 mM Hepes, 10 mM glucose, and 1 mM CaCl2, pH adjusted to 7.4 with NaOH) was used. For Ca2+ measurements, it was supplemented with 2 mM probenecid (to inhibit probe extrusion from the cytosol), and the ligands were diluted in this same buffer

containing 0.05% w/v stripped bovine serum albumin (to aid in keeping the lipophilic compounds in solution). For binding, 0.1% w/v stripped bovine serum albumin (to reduce, in addition, nonspecific binding of [125I]-orexin-A) and 30 μM dyngo 4a (to block receptor internalization) were used.22 Ca2+ Measurements. Cell culture medium was removed, and the cells were loaded with the loading solution composed of FLIPR Calcium 4 Assay Kit (Molecular Devices, Sunnyvale, CA) dissolved in and diluted with HBM + 1 mM probenecid, for 60 min at 37 °C. Then, the plate was placed in a FlexStation 3 fluorescence plate reader (Molecular Devices) and the intracellular Ca2+ levels were measured as fluorescence changes (excitation at 485 nm, emission at 525 nm) at 37 °C. The screening assay was designed to show both agonist and antagonist properties. In the first round, the baseline was collected for 30 s for each well (i.e., a single column of wells, as in the flex-mode of Flexstation). The test compounds (in HBM) were added (final concentration of 5 μM) and the response collected for another 120 s, after which the next column of wells was exposed to the measurement cycle. The delayed agonistic response was assessed 30 min after agonist addition for the first 30 s (see Supporting Information Table 1). In the second round, 0.3 nM orexin-A was added at 30 s in all wells, allowing the assessment of the ability of the compound to inhibit orexin-A response. For both tests, vehicle was included for each column of wells to serve as both negative and positive control for both agonism and antagonism. Each test compound was tested once in triplicate. The response to each compound is reported as a percentage of full receptor activation, which was determined by constructing concentration−response curves for orexin-A for each experiment set for the response within 120 s. Determining the specific agonist activity of 4−7 followed essentially the first round of screening described above; in validation, each compound was tested twice at 10 μM and, in characterization, thrice at 1 and 10 μM in quadruplicate. Compounds 8−14 were analyzed for functional inhibition as in the second round of the screening assay, three times at concentrations 10 nM − 100 μM in the 10-fold dilution series. The antagonists were added to the wells and incubated for 10 min at 37 °C prior to the measurement. [125I]-Orexin-A Competition Binding Assay. Binding of the test compounds was assessed in intact cells. As reported earlier, [125I]orexin-A cannot be used with cell homogenates or membrane preparations of CHO cells.22 Culture medium was exchanged for HBM + 30 μM dyngo 4a ± 10 μM 15 (to determine nonspecific binding), and after a 10 min incubation, 0.05 nM [125I]-orexin-A, mixed with each test compound, was added. Dilution series of orexinA, 16, and 15 were used as controls. After a 90 min incubation at room temperature, the medium was removed by water suction and the wells were allowed to dry at 37 °C. Scintillation cocktail (Ultima Gold; PerkinElmer) was added, and the plates were incubated overnight and counted in a Wallac Microbeta Trilux microplate liquid scintillation counter (PerkinElmer). Initially, under hit validation, each ligand was tested twice at 10 μM in quadruplicate, and the characterized ligands at least three times at concentrations 10 nM − 100 μM in a 10-fold dilution series. Data Analysis. All data are presented as mean ± SEM. Microsoft Excel was used for nonlinear curve fitting. For functional (Ca2+ elevation data) the Ki values for the seven antagonist hit compounds were calculated from the IC50 values using the Cheng−Prusoff equation. Binding was analyzed utilizing eq 3. binding = (binding no competitor − nonspecific binding) −

[competitor] × (binding no competitor − nonspecific binding) [competitor] + IC50 (3)

Due to the several apparent affinity sites and apparently cooperative binding of orexin-A,22 calculation of Ki according to the Cheng−Prusoff equation is not directly possible, and thus the binding 8272


Journal of Medicinal Chemistry affinities are given as IC50 values. However, approximate Ki values were calculated according to eq 4.

K i(compound) =

IC50(compound) × K i(SB − 334867) IC50(SB − 334867) (4)

ASSOCIATED CONTENT

S Supporting Information *

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REFERENCES

(1) Sakurai, T.; Amemiya, a; Ishii, M.; Matsuzaki, I.; Chemelli, R.; Tanaka, H.; Williams, S.; Richardson, J.; Kozlowski, G.; Wilson, S.; Arch, J.; Buckingham, R.; Haynes, A.; Carr, S.; Annan, R.; McNulty, D.; Liu, W.-S.; Terret, J.; Elshourbagy, N.; Bergsma, D.; Yanagisawa, M. Orexins and Orexin Receptors: A Family of Hypo- Thalamic Neuropeptides and G Protein-Coupled Receptors That Regulate Feeding Behavior. Cell 1998, 92, 573−585. (2) Kukkonen, J. P. Physiology of the Orexinergic/hypocretinergic System: A Revisit in 2012. AJP Cell Physiol. 2013, 304, 2−32. (3) Laburthe, M.; Voisin, T. The Orexin Receptor OX1R in Colon Cancer: A Promising Therapeutic Target and a New Paradigm in G Protein-Coupled Receptor Signalling through ITIMs. Br. J. Pharmacol. 2012, 165, 1678−1687. (4) Gotter, A. L.; Roecker, A. J.; Hargreaves, R.; Coleman, P. J.; Winrow, C. J.; Renger, J. J. Orexin Receptors as Therapeutic Drug Targets. Prog. Brain Res. 2012, 198, 163−196. (5) Cox, C. D.; Breslin, M. J.; Whitman, D. B.; Schreier, J. D.; McGaughey, G. B.; Bogusky, M. J.; Roecker, A. J.; Mercer, S. P.; Bednar, R. a.; Lemaire, W.; Bruno, J. G.; Reiss, D. R.; Harrell, C. M.; Murphy, K. L.; Garson, S. L.; Doran, S. M.; Prueksaritanont, T.; Anderson, W. B.; Tang, C.; Roller, S.; Cabalu, T. D.; Cui, D.; Hartman, G. D.; Young, S. D.; Koblan, K. S.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Discovery of the Dual Orexin Receptor Antagonist [(7 R)-4-(5-Chloro-1,3- Benzoxazol-2-Yl)-7-Methyl-1,4-Diazepan-1-yl][5Methyl-2-(2 H −1,2,3-Triazol-2-Yl)phenyl]methanone (MK-4305) for the Treatment of Insomnia. J. Med. Chem. 2010, 53, 5320−5332. (6) Roecker, A. J.; Cox, C. D.; Coleman, P. J. Orexin Receptor Antagonists: New Therapeutic Agents for the Treatment of Insomnia. J. Med. Chem. 2016, 59, 504−530. (7) Chemelli, R. M.; Willie, J. T.; Sinton, C. M.; Elmquist, J. K.; Scammell, T.; Lee, C.; Richardson, J. a.; Clay Williams, S.; Xiong, Y.; Kisanuki, Y.; Fitch, T. E.; Nakazato, M.; Hammer, R. E.; Saper, C. B.; Yanagisawa, M. Narcolepsy in Orexin Knockout Mice: Molecular Genetics of Sleep Regulation. Cell 1999, 98, 437−451. (8) Lin, L.; Faraco, J.; Li, R.; Kadotani, H.; Rogers, W.; Lin, X.; Qiu, X.; De Jong, P. J.; Nishino, S.; Mignot, E. The Sleep Disorder Canine Narcolepsy Is Caused by a Mutation in the Hypocretin (Orexin) Receptor 2 Gene. Cell 1999, 98, 365−376. (9) Nishino, S.; Ripley, B.; Overeem, S.; Lammers, G. J.; Mignot, E. Hypocretin (Orexin) Deficiency in Human Narcolepsy. Lancet 2000, 355, 39−40. (10) Fujiki, N.; Yoshida, Y.; Ripley, B.; Mignot, E.; Nishino, S. Effects of IV and ICV Hypocretin-1 (Orexin A) in Hypocretin Receptor-2 Gene Mutated Narcoleptic Dogs and IV Hypocretin-1 Replacement Therapy in a Hypocretin-Ligand-Deficient Narcoleptic Dog. Sleep 2003, 26, 953−959. (11) Baier, P. C.; Hallschmid, M.; Seeck-Hirschner, M.; Weinhold, S. L.; Burkert, S.; Diessner, N.; Göder, R.; Aldenhoff, J. B.; Hinze-Selch, D. Effects of Intranasal Hypocretin-1 (Orexin A) on Sleep in Narcolepsy with Cataplexy. Sleep Med. 2011, 12, 941−946. (12) Weinhold, S. L.; Seeck-Hirschner, M.; Nowak, A.; Hallschmid, M.; Goder, R.; Baier, P. C. The Effect of Intranasal Orexin-A (Hypocretin-1) on Sleep, Wakefulness and Attention in Narcolepsy with Cataplexy. Behav. Brain Res. 2014, 262, 8−13. (13) Lee, J.; Reddy, M. M.; Kodadek, T. Discovery of an Orexin Receptor Positive Potentiator. Chem. Sci. 2010, 1, 48−54.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00333. Pharmacophore modeling (Figure 1). Pharmacological screening (Figure 2, Table 2). Hit validation (Figures 3 and 4, Tables 2−5). Chemical space using PCA, closest neighbors (Table 6). Docking study of compounds 2−7 (Figures 5−7). Docking study of compound 8 (Figure 8). Pose selection (Figure 9). Purity and identity (Figures 10−13) (PDF) Molecular formula strings (CSV) Representative docking poses of 2−8 in both receptor subtypes (in PDB file format) (ZIP)

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ABBREVIATIONS USED

AR, aromatic group (feature); CHO, Chinese hamster ovary (cells); H, hydrophobic group (feature); HBD, hydrogen bond donor (feature); HBM, Hepes-buffered medium; NTSR1, neurotensin receptor 1; OX1R and OX2R, OX1 and OX2 orexin receptor, respectively; PAINS, pan-assay interference compounds; PC, principal component; PCA, principal component analysis; probenecid, p-(dipropylsulfamoyl) benzoic acid; SAR, structure−activity relationships

The IC50 values were determined in the binding and the Ki values for 16, 21 nM for OX1R and 2.4 μM for OX2R, in the functional tests.47,48 The values are close to the ones determined in these cells earlier, i.e. 14 nM for OX1R and 2.0 μM for OX2R.49

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AUTHOR INFORMATION

Corresponding Author

*E-mail: henri.xhaard@helsinki.fi. Tel. +358-2941-59190. Author Contributions

J.P.K. and A.T. designed, conducted, and analyzed the pharmacological part of the study. H.X. designed and analyzed the computational part of the study together with A.T. (pharmacophore modeling, docking simulations), L.K. (receptor modeling), and A.B. (chemoinformatics). T.O.L verified the identity of the compounds. A.T., J.P.K., and H.X. wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Pirjo Puroranta and Santeri Suokas are acknowledged for technical assistance with the experiments and Kaj-Roger Hurme (Instrument Centre of Faculty of Agriculture and Forestry, University of Helsinki) for all the help with the liquid scintillation counting. Miikka Olin and Nina Sipari are acknowledged for the mass spectroscopy and analysis. Erik Wallén (Faculty of Pharmacy, University of Helsinki) is acknowledged for useful discussions about the project. The Drug Discovery and Chemical Biology (DDCB)Biocenter Finland consortium is acknowledged for providing physical access to compounds and computational infrastructure. The Center for Scientific Computing is acknowledged for organizing computational resources for the H.X. group. The COST action CM102-GLISTEN is thanked for organizing a pan-European research network. This study was supported by the University of Helsinki 3-year grant (H.X.), the Finnish Cultural Foundation (A.T.), the Magnus Ehrnrooth Foundation, and the Liv & Hälsa Foundation. 8273



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