Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships

Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

Very few opioid ligands show promiscuous high-affinity binding to all four opioid receptor subtypes, mu, kappa, delta and the nociceptin opioid receptors (MOP, KOP, DOP, NOP respectively). In fact, it is well documented in the literature that the most opioid ligands which have high affinity for the three classic opioid receptors, MOP, KOP and DOP, have little to no affinity for the NOP receptor [1][2][3] . Prior to the recent determination of the X-ray crystal structures of the four opioid receptors bound to their selective antagonist ligands, elegant structure-activity relationship (SAR) studies of opioid ligands, in conjunction with site-directed mutagenesis, provided seminal information on the similarities and differences in opioid receptor binding pockets and selectivity-enhancing pharmacophoric features of opioid ligands. Using these approaches receptor-selective opioid ligands were designed from universal opioid scaffolds; for example, kappa-selective antagonist norbinaltorphimine (norBNI) 4,5 and delta-selective antagonist naltrindole (NTI) 6 were designed from the non-selective opioid antagonist naltrexone (Fig. 1), and the kappa-selective antagonist, 5′-guanidinylnaltrindole (GNTI) was designed from the delta-selective antagonist NTI 7,8 . Binding modes of these antagonists in the opioid receptor homology-based models were derived by docking a universal opioid antagonist such as naltrexone as the 'common pharmacophore' or 'message' into the opioid binding pocket and refined based on the observed SAR of these ligands and the message-address concept 9 . The selectivity of the various naltrexone-derived antagonists was explained by the orientation and interaction of the 'address' elements of these ligands with different amino acid to obtain more information on the binding orientations of AT-076 in the opioid receptors, given the known differences in amino acid residues in the transmembrane domains and binding pockets of the four opioid receptors 19 .
Both the NOP and KOP receptors (but not DOP and MOP) contain anionic amino acid residues in their extracellular loop (EL) 2 (between transmembrane helices (TM) 4 and 5), which have been shown to function as the address domains for these receptors and interact with the cationic core residues of their respective endogenous peptide ligands nociceptin and dynorphin [20][21][22][23] . For kappa antagonists such as norBNI and 5′-GNTI, the selectivity-enhancing 'address' interaction occurs with the nonconserved residue Glu297 6.58 at the extracellular end of TM6 8,10-12 . To explore and confirm binding orientations of AT-076, we introduced various positively charged groups in the 7-hydroxy-Tic moiety by replacing the 7-hydroxy group with amine (3), guanidine (4) and N-methyl sulfonamide (5) groups; as well as substitution of the Tic-OH heterocycle with lysine (6) and arginine (7). For comparison with these charged analogs, the 7-OH was also replaced with a cyano group (2). Similarly, the 3-hydroxy group of the phenylpiperidine moiety was also replaced with a cyano (8), amine (10) and arginine groups (12). Additionally, for these compounds, the effect of removal of the 7-OH of the Tic moiety was also explored (9, 11 and 13, respectively).
As shown in the 2D diagram in Fig. 2, derived from the C-24 and SB-612111-bound NOP receptor crystal structures 18,24 , the benzofuran and 2,6-dichlorophenyl moieties at the 4-position of the piperidine are oriented towards the intracellular end of the TM binding pocket, surrounded by hydrophobic residues Met134 3.36 , Phe135 3.37 , Ile219 5.42 and Val283 6.55 . Docking of AT-076 in the NOP crystal structure also resulted in a similar orientation of the piperidine ring, where the 3-hydroxyphenyl ring at the 4-position of the piperidine ring was   Cyclohexyl analog 14 was prepared as shown in Fig. 7. (S)-3-methyl-1-(4-phenylpiperidin-1-yl)butan-2-amine IV-1, prepared according to previously reported methods 15 was hydrogenated to afford the corresponding cyclohexyl intermediate IV-2. Routine BOP-mediated amidation of IV-2 with carboxylic acid I-1, followed by HCl deprotection furnished 14.
In vitro pharmacological characterization. Compounds 2-16 were characterized in vitro for their binding affinities, intrinsic activity and antagonist potencies at the NOP, KOP, DOP and MOP receptors and compared to AT-076 (1) which was characterized in the same assays.  35 S]GTPγS binding to cell membranes in a six-point concentration curve up to 10 μM and compared to the standard agonists N/OFQ (NOP), DAMGO (MOP), U69,593 (KOP), and DPDPE (DOP), conducted as described in Methods 31,[34][35][36] . None of the analogs tested had any intrinsic activity in the GTPγS assay at the four receptor subtypes.
For compounds whose binding affinity K i was < 50 nM, the antagonist potencies (pA 2 ) were determined in the [ 35 S]GTPγS functional assay using Schild analysis, where the shift in EC 50 in the dose-response curve of the respective standard agonist is determined in the presence of at least 4 concentrations of the test antagonist. The pA 2 values obtained in these analyses are shown in Table 2.

Results
To explore binding orientations of 1 15 at the opioid receptors with an SAR analysis, we replaced the 7-hydroxy of the Tic moiety of 1 with positively charged aminergic substituents, as in analogs 3-7, which, we hypothesized, may interact with anionic residues in the EL2 loops of the NOP and KOP receptors 37 . The uncharged nitrile analog 2 was also synthesized to explore the importance of the 7-OH in the Tic moiety. To our surprise, these modifications decreased binding affinity at all four receptors compared to that of the lead AT-076 (Table 1). The drop in affinity at NOP was particularly pronounced, over three orders of magnitude for 3-7 (see Table 1). At the KOP, MOP and DOP receptors, the effect of these modifications was less pronounced. The polar but uncharged nitrile analog 2 showed only a 6-fold drop in KOP affinity, but charged substituents as in analogs 3 and 4 caused a > 100-fold decrease in binding affinity at KOP, MOP and DOP receptors. Replacing the entire Tic-OH moiety with lysine (6) or arginine (7) significantly decreased affinity for all four receptors.   Structural modifications at the opposite end of the molecule, i.e. replacing the 3-hydroxyl group of the phenylpiperidine with an amino group (10), interestingly increased NOP binding affinity 2-fold compared to the uncharged cyano precursor (8), giving a K i of 30.72 ± 14.5 nM. The arginine analog 12, has even higher NOP affinity (K i of 6.04 ± 1.32 nM), comparable to that of 1 at NOP. These modifications retained the high binding affinity at KOP and MOP but not the DOP receptors. Overall, the replacements of the 3-OH group were less detrimental to the affinity at NOP, and resulted in equi-potent binding affinity at KOP and MOP (analogs 8, 10 and 12). The importance of the 7-OH group in the Tic moiety was further confirmed with analogs 9, 11 and 13, because removal of this group significantly dropped affinity at all receptors, compared to 8, 10 and 12 respectively.
To further investigate the binding orientation of AT-076 at the NOP receptor, the 3-hydroxyphenyl group of AT-076 was replaced with hydrophobic moieties such as a cyclohexyl ring (14), indolinone (15) and indoline (16). Interestingly, these analogs show a significant decrease in binding affinity at the NOP receptor.
However, at the KOP receptor, analogs 14 and 16 have sub-nanomolar affinity, being about 2-4-fold higher affinity than AT-076 itself. There was a modest decrease in affinity at MOP and DOP for these compounds compared to AT-076. With this enhancement of binding affinity at the KOP receptor, compounds 14 and 16 are selective KOP ligands, showing greater binding selectivity for KOP over MOP (11-fold for 14, 20-fold for 16), DOP (49-fold for 14, 370-fold for 16) and NOP (333-fold for 14, >1000-fold for 16) receptors, compared to the KOP antagonist JDTic, which shows only a 4-fold binding selectivity over MOP, 25-fold over DOP and 39-fold over NOP as determined in our experiments ( Table 1).
Incorporation of a carbonyl group on 16 (to indolinone 15) reduces KOP affinity by 10-fold, and shows decreased affinity at MOP and DOP compared to 16.
Functional characterization of intrinsic (agonist) activity and antagonist potencies of the analogs was conducted using the [ 35 S]GTPγS binding assay. As expected, none of the analogs had any agonist activity at any of the opioid receptors. On the other hand, several analogs that had nanomolar binding affinities for any of the opioid receptors also showed significant antagonist potencies at that receptor, reported as pA 2 values shown in Table 2. Notably, compound 12, which has single digit nanomolar binding affinity at NOP, KOP and MOP (Table 1) also has high antagonist potency at these receptors (pA 2 values 8.3, 9.8 and 9.2 respectively, Table 2). The subnanomolar binding affinity and high selectivity for KOP observed with analogs 14 and 16 also translate to high antagonist potencies at KOP for these compounds (pA 2 −10.5 and −10.6, Table 2). Compounds 14 and 16 are therefore selective and potent KOP antagonists.

Discussion
The nanomolar binding affinity of AT-076 (1) at all four opioid receptors suggests that it has a chemotype that binds in the opioid binding pocket of all four opioid receptors, NOP, MOP, KOP and DOP 15 . 1 may possess a common opioid pharmacophore and can be used as a tool compound to probe ligand recognition features at all opioid receptors. Such information is useful for the design of multifunctional or selective opioid ligands, as needed, based on this scaffold. To aid such studies, we continued our SAR studies of 1 and investigated several chemical structure modifications, designed to inform the SAR at all four opioid receptors.
We previously reported the results of docking compound 1 in the NOP receptor crystal structure bound to antagonist C-24 (PDB No: 4EA3) 15 . 1 was found to bind in an extended conformation to NOP 15 , similar to the co-crystallized NOP antagonist C-24 18 . The piperidine nitrogen of 1 formed a salt bridge with the conserved Asp130 3.32 , a key interaction for ligands at all four opioid receptors. Similar to the benzofuran moiety of C-24, the 3-hydroxyphenyl moiety of 1 at the 4-position of the central piperidine ring, was oriented toward the intracellular end of the NOP binding site in a lipophilic pocket, comprised of Tyr 131 3.33 , Met 134 3.36 and Trp276 6.48 . The opposite end of the molecule, i.e. the Tic-OH moiety, of AT-076 in this docked pose was oriented towards the extracellular end of the binding cavity towards the EL2 loop, enriched with anionic residues such as Glu196, Glu197 and Glu199 in NOP 3,18,37 . However, the SAR of analogs 2-7 showed a large drop in NOP receptor binding affinity when the 7-OH group of the Tic-OH moiety was replaced with positively charged groups designed to interact with these anionic residues of the EL2 loop (Table 1 and Fig. 2). On the other hand, similar modifications of the 3-OH of the phenylpiperidine moiety retained NOP binding affinity similar to that of AT-076 (compound 12). SAR of analogs 2-7 suggests that AT-076 may not bind in the same orientation as the co-crystallized NOP antagonist C-24 as previously suggested by our docking results 15 . On the other hand, the high affinity of compounds 10 and 12 at NOP suggests that the positively charged moiet(ies) replacing the 3-OH of the phenylpiperidine instead, may likely contribute to the high affinity by interacting with the negatively charged residues near the extracellular end and EL2 loop of the NOP binding pocket. This SAR supports a reversed binding mode than previously proposed, such that the 3-hydroxyphenyl on the piperidine ring is oriented towards the extracellular end of the  Table 2. Antagonist potencies (pA 2 ) determined by Schild analysis in functional assays measuring inhibition of agonist-induced [ 35 S]GTPγS binding at the four opioid receptors ¶ . ¶ pA 2 values are given as mean ± SEM of at least two experiments performed in triplicate on two separate days. *ND = antagonist potency was not determined for compounds whose binding affinity was >50 nM. # N/C = compound showed a noncompetitive profile in Schild analysis. § pA 2 value from a single experiment done in triplicate.
NOP binding pocket, rather than towards the intracellular end, as previously found in the docked pose of 1 15 . Such an orientation would place the 7-hydroxy-4-(3-hydroxyphenyl)-1-piperidinyl]methyl}-2-methylpropyl)-1, 2,3,4-tetrahydro-3-isoquinolinecarboxamide in the hydrophobic pocket, lined with residues conserved among the four opioid subtypes such as Met134 3.36 . SAR showing poor NOP binding affinities for analogs 14 and 16, bearing hydrophobic replacements of the 3-hydroxyphenyl moiety, further suggests that these groups at the C-4 position of the central piperidine ring are likely oriented towards the polar extracellular end of the NOP binding pocket, supporting a flipped orientation of AT-076 analogs compared to co-crystallized antagonist C-24 at the NOP receptor.
Our SAR results suggest that NOP ligands of a chemotype different from the co-crystallized ligand C-24 may possibly bind in a different orientation than the co-crystallized ligand. Indeed, docking studies of other piperidine-based NOP antagonists J-113397 and its analog Trap-101 in the NOP crystal structure conducted by Miller et al. 24 showed that these antagonists favored the 'flipped' orientation, in which the piperidine C-4 heterocycle is oriented towards the extracellular end of the binding pocket, whereas the piperidine N-1 cyclooctyl substituent is buried in the intracellular hydrophobic pocket. SAR studies such as reported here are therefore useful for investigating possible binding orientations of ligand chemotypes different than the co-crystallized ligands in the opioid receptor crystal structures.
At the KOP receptor, the high KOP selectivity and antagonist potency of analogs 14 and 16 suggests that these compounds likely bind to the KOP receptor in an orientation similar to that of JDTic in the KOP receptor crystal structure 17 . The 3-hydroxyphenyl ring of JDTic is situated in a pocket comprised of Val118 2.63 , Cys131 3.25 , Val134 3.28 and Leu135 3.29 (See Fig. 9). These residues could likely provide strong hydrophobic interactions for the cyclohexyl and indoline group replacements in 14 and 16, respectively, which may explain their high binding affinity. Docking the selective KOP antagonists 14 and 16 in the KOP crystal structure 4DJH 17 confirmed this binding orientation similar to that of JDTic, in which the cyclohexyl and indoline groups of in 14 and 16 occupy the same pocket as the 3-hydroxyphenyl ring of JDTic, as shown in Fig. 9.

Conclusions
In summary, this SAR study of 1 reveals several interesting trends-(i) 1 represents an universal opioid antagonist chemotype that is not a morphinan scaffold. (ii) The 7-hydroxy-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide appears to be important pharmacophore for binding at all four opioid receptors since modifications in this moiety (2-7, 9, 11 and 13) causes significant loss of affinity at all four receptors. (iii) Substituents at the 4-position of the piperidinyl ring may be used to modulate affinity and selectivity, particularly for the KOP receptor. Such modifications resulted in the discovery of a selective KOP antagonist 16 from a pan antagonist lead compound 1. (iv) The SAR for 1 and its analogs at the NOP receptor highlights the limitations of docking using the X-ray crystal structures as a single tool for rational drug design. Rather, a combination of experimental SAR and docking allows for an accurate understanding of ligand recognition of structurally diverse ligands at the opioid receptors.

III-4b
(170 mg, 0.32 mmol). The solution was concentrated to dryness, then partitioned between CH 2 Cl 2 and satd. NaHCO 3 (aq). Solid NaCl was added. The layers were separated, and the aqueous solution was extracted 2X with CH 2 Cl 2 . The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated. The crude residue was purified via flash chromatography using Hexane/EtOAc/NH 4 OH(aq) 90/10/1 → 10/90/1 as the eluent to afford 120 mg of the title material in 87% yield, which was converted to the HCl salt by addition of 2 M HCl/ ether. 1     . The crude residue was diluted with CH 2 Cl 2 and satd. NaHCO 3 (aq). Solid NaCl was added. The layers were separated, and the aqueous solution was extracted 2X with CH 2 Cl 2 . The combined organic layers were washed with satd. NaCl(aq), dried over Na 2 SO 4 , filtered and concentrated. The aqueous layers were combined and extracted 2X with EtOAc. The combined organic layers were washed with satd. NaCl(aq), dried over Na 2 SO 4 , filtered and concentrated. The crude residue was purified via flash chromatography using Hexane/EtOAc/iPrOH/NH 4 OH 50/50/0/0 → 42.5/42.5/15/1 to afford 146 mg of the title material in 78% yield, which was converted to the HCl salt by addition of 2 M HCl/ether. 1   m/z 563.5 (M + H) + . The title material was prepared according to General Procedure 4 Method C from the Boc-protected intermediate (45 mg, 0.08 mmol). The crude residue was triturated with dioxane to afford 34 mg of the title material as a 2 HCl salt in 79% yield. 1  In vitro pharmacological Characterization. Cells. Human NOP, mu, delta, and kappa opioid receptors were individually expressed in Chinese hamster ovary cells stably transfected with the human receptor cDNA, as we have described previously 2,38 . Kappa-CN cells were used for KOP radioligand binding assays, while Kappa-FLG19 cells were used in KOP [ 35 S]GTPγS functional assays. The cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum, in the presence of 0.4 mg/ml G418 and 0.1% penicillin/ streptomycin, in 100-mm plastic culture dishes.

(R)-N-((S)-1-(4-(3-(((S)-2-amino-5-guanidinopentanamido)methyl)phenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (13).
Membrane preparation. The cell lines are grown to full confluency, then harvested for membrane preparation. The membranes are prepared in 50 mM Tris buffer (pH 7.4). Cells are scraped and centrifuged at 500 × g for 12 mins. The cell pellet is homogenized in 50 mM Tris with a Fisher Scientific PowerGen 125 rotor-stator type homogenizer, centrifuged at 20,000 × g for 25 mins, washed and recentrifuged once more and aliquoted at a concentration of 3 mg/mL protein per vial and stored in a −80 °C freezer till further use. . Nonspecific binding was determined by using 1.0 µM of the unlabeled nociceptin for NOP, 10 µM unlabeled DAMGO for MOP, 10 µM unlabeled DPDPE for DOP, and 10 µM unlabeled U69,593 for KOP. Assays were initiated by addition of membrane homogenates and samples were incubated for 60 min at 25 °C in a total volume of 1.0 mL. In NOP receptor experiments, 1 mg/mL BSA is added to the assay buffer. The amount of protein in the binding assay was 15 μg. The incubation was terminated by rapid filtration through 0.5% PEI-soaked glassfiber filter mats (GF/C Filtermat A, Perkin-Elmer) on a Tomtec Mach III cell harvester and washed 5 times with 0.5 mL of ice-cold 50 nM Tris-HCl, pH 7.4 buffer. The filters were dried overnight and soaked with scintillation cocktail before counting on a Wallac Beta plate 1205 liquid scintillation counter. Radioactivity was determined as counts per minutes (cpm). Full characterization of compounds includes analysis of the data for IC 50 values and Hill coefficients using GraphPad Prism. (ISI, San Diego, CA). K i values were determined by the method of Cheng and Prusoff 39 .
[ 35 S]GTPγS Functional assay. Functional assay is conducted in Buffer A, containing 20 mM HEPES, 10 mM MgCl 2 100 mM NaCl at pH 7.4. Membrane prepared as described above was incubated with [ 35 S]GTPγS (150,000 dpm/well), GDP (10 μM), and the test compound, in a total volume of 1 mL, for 120 minutes at 25 °C. Samples were filtered over Filtermat A and counted as described for the binding assays. A dose response curve with a prototypical full agonist at the respective receptor is conducted in each experiment to identify full and partial agonist compounds.
Determination of Antagonist potency. High affinity compounds (K i value < 50 nM) that demonstrate no agonist activity were evaluated for their antagonist potency by Schild analysis 40 , using an agonist full dose response curve in the presence of at least three concentrations of the test antagonist. pA 2 values and Schild slopes are determined using a statistical program designed for these experiments. If the Schild slope was found to be significantly different from -1.00, the antagonist activity was deemed non-competitive; in such cases, the pA 2 value is not reported. Equilibrium dissociation constants (K e values) were calculated as follows: where "a" is the nanomolar concentration of the antagonist and "DR" is the ratio of the agonist EC 50 in the presence of a given concentration of antagonist.
Molecular Docking. Compounds were sketched and minimized using MMFF94 force field and charges in Data availability. The authors declare that all data supporting the findings of this study are available within the article.