Discovery of a novel selective kappa-opioid receptor agonist using crystal structure-based virtual screening

Article abstract of DOI:10.1021/ci400019t

Kappa-opioid (KOP) receptor agonists exhibit analgesic effects without activating reward pathways. In the search for nonaddictive opioid therapeutics and novel chemical tools to study physiological functions regulated by the KOP receptor, we screened in silico its recently released inactive crystal structure. A selective novel KOP receptor agonist emerged as a notable result and is proposed as a new chemotype for the study of the KOP receptor in the etiology of drug addiction, depression, and/or pain.

Full text of DOI:10.1021/ci400019t

Letter
pubs.acs.org/jcim
Discovery of a Novel Selective Kappa-Opioid Receptor Agonist Using
Crystal Structure-Based Virtual Screening
Ana Negri,†,∥ Marie-Laure Rives,‡,⊥,∥ Michael J. Caspers,○ Thomas E. Prisinzano,○
Jonathan A. Javitch,‡,§,⊥ and Marta Filizola*,†
†Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United
States
§
‡Departments of Psychiatry and Pharmacology, Columbia University, College of Physicians and Surgeons, New York, New York
10032, United States
○Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045, United States
⊥Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, 10032, United States
S
* Supporting Information
endogenous opioid system, a safe, nonaddictive, and effective
opioid drug is yet to be discovered.3
Notable members of the superfamily of G protein-coupled
receptors (GPCRs), mu-, delta-, and kappa-opioid (or MOP,
DOP, and KOP) receptor subtypes4−7 are natural targets for
the majority of opioid ligands. The most clinically used opioid
drugs act as agonists at the MOP receptor3 and exert addiction
liability through activity at this receptor.8,9 Thus, it has been
proposed that high-affinity selective ligands of the DOP and
KOP receptors would provide more effective routes to
discovering nonaddictive analgesics.10,11 In particular, KOP
receptor agonists have been shown to be unable to activate the
reward pathway12 while still acting as effective pain suppressors
on the central nervous system (CNS) and/or the periphery,11
ABSTRACT: Kappa-opioid (KOP) receptor agonists
most likely through the Gi/o protein-mediated inhibition of
exhibit analgesic effects without activating reward path-
cAMP production,13 the blockade of calcium channels,14 and/
ways. In the search for nonaddictive opioid therapeutics
or the activation of the inward rectifier potassium channels.15
and novel chemical tools to study physiological functions
Unfortunately, the KOP receptor agonists developed to date
regulated by the KOP receptor, we screened in silico its
are not ideal drugs as they exert side effects such as dysphoria.16
recently released inactive crystal structure. A selective
However, KOP receptor-mediated dysphoric effects have
novel KOP receptor agonist emerged as a notable result
recently been attributed to the activation of the p38 MAPK
and is proposed as a new chemotype for the study of the
pathway following arrestin recruitment to the activated KOP
KOP receptor in the etiology of drug addiction,
receptor.16−18 Therefore, KOP receptor-selective G-protein
depression, and/or pain.
biased agonists, which do not recruit arrestin, have been
proposed to be more effective analgesics, without the adverse
effects triggered by the arrestin pathway.18 We have recently
INTRODUCTION
■
reported on such a functionally selective G protein-biased KOP
receptor ligand:19 6′-guanidinonaltrindole (6′-GNTI).
Opioids remain the most widely prescribed and abused class of
medicines.1,2 Addiction is not the only limiting factor for the
Although this morphine-derivative ligand is a promising lead
compound for nonaddictive analgesics acting at the KOP
effective use of these compounds as powerful painkillers,
antitussives, antidepressants, or antipruritic agents. In addition
to social and legal issues associated with their use for
nonmedical, recreational purposes, several adverse effects
receptor with reduced liability for dysphoria, its effective use as
a drug is severely limited by its physicochemical properties and
its inability to cross the blood−brain barrier.
The lack of a detailed molecular-level understanding of the
(e.g., dysphoria, constipation, respiratory depression, nausea,
interactions between opioids and their receptors has hindered
vomiting, etc.)3 hinder their clinical usefulness and justify the
successful receptor-based drug design. By revealing how opioid
enormous effort put forth by numerous investigators over the
years to discover safer opioid therapeutics and/or nonaddictive
medications. Notwithstanding the continued development of
many compounds with opioid activity, ranging from useful
agents in the clinic to important chemical tools to study the
ligands bind to their receptors, recent high-resolution crystal
structures of all four opioid receptor subtypes, i.e., the MOP,20
DOP,21 KOP,22 and nociceptin/orphanin FQ23 receptors, offer
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ci400019t | J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

Journal of Chemical Information and Modeling
Letter
an unprecedented opportunity to discover novel chemotypes
targeting these proteins that might eventually be developed into
more efficacious therapeutics.24,25
(Tc) to the nearest neighbors based on extended connectivity
fingerprint maximum distances 4 (ECFP4) and the protocol we
recently reported.33 Tc values range from 0 to 1, with the 0
value indicating maximally dissimilar compounds and 1
indicating maximally similar ones.34 As suggested in the
literature,35 molecules are considered reasonably similar if
their Tc value is above 0.40. Molecules for testing were
purchased from commercial vendors. Specifically, compounds
MCKK-1, MCKK-4, MCKK-8, MCKK-15, MCKK-18, and
MCKK-21−22 were obtained from ChemBridge, MCKK-2
from the National Cancer Institute of the National Institutes of
Health, MCKK-3 from Labotest, MCKK-5−7, MCKK-9−13,
MCKK-16−17, and MCKK-19 from Enamine, MCKK-14 from
Florida Heterocyclic Compounds, and MCKK-20 from
Molecular Diversity Preservation International.
Constructs for Expression Vectors and Transfection.
The cDNAs for human KOP (hKOP) receptor and the G
protein GαoB were obtained from the Missouri S&T cDNA
Resource Center. For arrestin recruitment experiments, full-
length Renilla luciferase 8 (RLuc8, provided by S. Gambhir)
was fused in-frame to the C terminus of the hKOP receptor in
the pcDNA3.1 vector. The following human G protein
constructs were provided by Gales:36,37 GαoB with RLuc8
inserted at position 91 (GαoB-RLuc8); untagged Gβ1 (β1);
untagged Gγ2 (γ2). The human γ2 subunit was fused to full-
length mVenus at its N terminus (mVenus-γ2), and we used
the fusion construct human arrestin3−mVenus previously
described.38 All constructs were confirmed by sequencing
analysis. A total of 20 μg of plasmid cDNA (e.g., 0.2 μg of
hKOR-RLuc8, 15 μg of arrestin3-mVenus, and 4.8 μg of
pcDNA3.1) was transfected into HEK-293T cells using
polyethylenimine (Polysciences Inc.) in a 1:3 ratio in 10-cm
dishes. Cells were maintained in culture with DMEM
supplemented with 10% FBS. The transfected ratio among
receptor, Gα, β1, and γ2, or arrestin was optimized by testing
various ratios of plasmids encoding the different sensors.
Experiments were performed 48 h after transfection.
Membrane Preparations and Binding Assays. Two
days after transfection with human KOP receptor and GαoB,
HEK293T cells were lysed and membranes were prepared in
HEPES buffer (NaCl 140 mM, KCl 5.4 mM, HEPES 25 mM,
EDTA 1 mM, MgCl2 2 mM, BSA 0.006%, pH 7.4) using a
Polytron homogenizer. Membranes were incubated with 3H-
diprenorphine (0.3 nM) (PerkinElmer) at room temperature
for 1 h in a final volume of 1 mL, in the absence or presence of
various concentrations of each small-molecule selected from the
virtual screening. Membranes were then harvested using a
Brandel cell harvester through a Whatman FPD-24 934AH
glass-fiber filter and washed three times with ice-cold wash
Buffer (Tris-HCl 10 mM, NaCl 120 mM, pH 7.4). Nonspecific
binding was determined using 400 nM of NorBNI.
BRET-Based G Protein Activation, Arrestin Recruit-
ment, and cAMP Accumulation Assays. BRET was
performed as described.39 Briefly, two days after transfection,
cells were harvested, washed, and resuspended in a phosphate-
buffered saline (PBS) solution. Approximately 200 000 cells/
well were distributed in 96-well plates, and 5 μM coelenterazine
H (luciferase substrate) was added to each well. Five minutes
after the addition of coelenterazine H, ligands were added to
each well. After 2 min for G protein activation or 5 min for
arrestin recruitment, the BRET signal was determined by
quantifying and calculating the ratio of the light emitted by
mVenus, the energy acceptor (510−540 nm), over that emitted
In the search for these nonaddictive therapeutics targeting
opioid-receptors, we screened in silico over 4.5 million
commercially available, “lead-like” small molecules accessible
in ready-to-dock three-dimensional format in the ZINC
database,26 based on complementarity with the crystallographic
binding mode of JDTic into the KOP receptor crystal
structure.22 To the best of our knowledge, this is the first
virtual screening study at the KOP receptor using its recently
released crystal structure.22 The study led to the identification
of 4 novel small-molecule chemotypes, out of 22 tested
molecules, acting at the KOP receptor. The S-stereoisomer of
one of these compounds was further characterized as a novel
selective KOP receptor ligand with agonistic activity at this
receptor, and as such, it represents a promising candidate for
structure-based drug design.
METHODS
■
Small-Molecule Subset, Docking, Selection, and
Novelty. We used the lead-like subset version of the ZINC
database26 that was accessible online on February 2, 2012,
when the molecular docking study was performed. This subset
version contained ∼4.5 million commercially available small-
molecules selected using the filtering criteria specified on the
ZINC database Web site. Molecular docking at chain A of the
recently released inactive KOP receptor structure (PDB ID:
4DJH22), following removal of all nonprotein atoms, was
performed with DOCK3.6.27−30 The atom positions of the
JDTic crystallographic ligand within the KOP receptor binding
pocket were replaced by 45 spheres that had been labeled for
chemical matching based on the local protein environment.
Default parameters, i.e., a bin size of 0.2 Å, a bin size overlap of
0.1 Å, and a distance tolerance of 1.2 Å for both the binding site
matching spheres and each docked small-molecule from the
lead-like subset, were used for ligand conformational sampling.
Partial charges from the united atom AMBER force field31 were
used for all receptor residues with the exception of Asp138 in
transmembrane helix 3. The dipole moment of this residue was
increased by 0.4 per polar atom to favor identification of small
molecules that would form ionic interactions with this
residue.32 The KOP receptor was kept rigid while each small
molecule was docked into the binding pocket in an average of
3073 orientations relative to the receptor and an average of
2132 conformations for each orientation. A score correspond-
ing to the sum of the receptor−ligand electrostatic and van der
Waals interaction energies, corrected for ligand desolvation, was
assigned to each docked molecule and configuration within the
KOP receptor binding pocket. The specific energy estimates
were obtained as we recently described for an analogous
study.33 The best scoring conformation of each docked
molecule was further subjected to 100 steps of energy
minimization with the protein residues kept rigid. Twenty-
two compounds, termed here MCKK-1−22, and listed in
Supporting Information Table S1, were selected from visual
inspection of the 500 top-scoring docked compounds
(Supporting Information Table S2) based on criteria discussed
in Results and Discussion. Similarity between these molecules
and the 9934 opioid receptor ligands that are annotated in the
ChEMBL database [https://www.ebi.ac.uk/chembl/] (Sup-
porting Information Table S1) was quantified using an in-
house script in R language that calculates Tanimoto coefficients
B
dx.doi.org/10.1021/ci400019t | J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

Journal of Chemical Information and Modeling
Letter
Figure 1. MCKK-17-S is a selective hKOP receptor agonist. (A and B) hKOP receptor (A), DOP or MOP receptors (B) was coexpressed with
GαoB−RLuc8, β1, and mVenus−γ2 to assay G protein activation. (A) Only MCKK-17-R/S exhibited agonistic activity at the hKOP receptor, and
MCKK-17S was the more active stereoisomer. (B) MCKK-17S is selective for hKOP relative to MOP and DOP receptors. (C) hKOP receptor was
coexpressed with a BRET-based CAMYEL sensor to assay inhibition of forskolin-stimulated cAMP accumulation. (D) hKOP receptor, fused to
RLuc8, was coexpressed with arrestin3 (Arr3) fused to mVenus to assay arrestin recruitment to the activated receptor. Error bars indicate SE.
by RLuc8, the energy donor (485 nm). The drug-induced
BRET signal was normalized, taking the Emax of the
ethylketocyclazocine (EKC)-induced response as 100%. To
measure cAMP accumulation, we used a BRET-based cAMP in
a previously described YFP-Epac-RLuc (CAMYEL) assay.40
GαoB, β1, and γ2 were coexpressed to enhance the signal-to-
noise ratio, and the cells were treated for 5 min with 100 μM
forskolin prior to stimulation.40 The data were normalized and
represented as the percentage of forskolin-stimulated cAMP
accumulation with 0 defined as the maximal inhibition elicited
by EKC.
Chemical Synthesis of MCKK-17 Stereoisomers. All
reagents purchased from chemical suppliers were used without
further purification and reactions monitored using thin-layer
chromatography (TLC) on 0.25 mm Analtech GHLF silica gel
plates using EtOAc/n-hexanes and visualized at 254 nm.
Column chromatography was performed on silica gel (40−63
μm particle size, 230−400 mesh) from Sorbent Technologies
(Atlanta, GA). NMR spectra were recorded on either a Bruker
DRX-400 with a H/C/P/F QNP gradient probe or a Bruker
Avance AV-III 500 with a dual carbon/proton cryoprobe using
δ values in parts per million as standardized from
tetramethylsilane (TMS) and J (Hz) assignments for 1H
resonance coupling and 13C fluorine coupling. High resolution
mass spectrometry data were collected on a LCT Premier
(Waters Corp.) time-of-flight mass spectrometer. Analytical
HPLC was performed on an Agilent 1100 Series Capillary
HPLC system with diode array detection at 254.8 nm on a
CRIRALCEL OD-H column (4.6 × 150 mm), Daicel Chemical
Industries, Ltd., using isocratic elution in 97% hexanes and 3%
2-propanol at a flow rate of 1.25 mL/min. General procedures
for the synthesis of tert-butyl 2-(thiazol-2-ylcarbamoyl)-
pyrrolidine-1-carboxylate (2S and 2R), as well as 1-(2-(3-
fluorophenylamino)-2-oxoethyl)-N-(thiazol-2-yl)pyrrolidine-2-
carboxamide (MCKK-17S and MCKK-17R) are provided in
the Supporting Information.
RESULTS AND DISCUSSION
■
Structure-Based Identification of Novel Chemotypes
Targeting the KOP Receptor. We screened in silico
4,554,059 commercially available, lead-like compounds from
the ZINC database26 based on complementarity with the
crystallographic binding mode of JDTic into the KOP receptor
binding pocket. The 500 top-scoring docking hits (Supporting
Information Table S2; 0.01% of the docked library) were
visually inspected and prioritized based on features that an
automatic molecular docking screen does not take into account.
Specifically, molecules were selected based on the following
criteria: (a) chemotype diversity; (b) the presence of polar
interactions between the ligand and the Asp138 residue; (c)
interactions with KOP receptor residues in the binding pocket
that are different in DOP and MOP receptors; (d) limited
flexibility; (e) different binding modes from classical alkaloids
as revealed by DOP21 and MOP20 receptor crystal structures;
and (f) purchasability, i.e., molecules were readily available for
purchase. On the basis of these criteria, 22 small molecules
were purchased from the set of 500 highest-scored compounds.
These molecules, labeled MCKK-1−22 in Supporting In-
formation Table S1, corresponded to the DOCK scoring ranks
1, 80, 87, 97, 111, 127, 137, 210, 253, 269, 276, 346, 347, 360,
379, 402, 403, 404, 411, 427, 452, and 472, respectively. As
shown in Table S1, these compounds were found to be
significantly different from annotated opioid receptor ligands in
the ChEMBL database, as indicated by small ECFP4-based Tc
C
dx.doi.org/10.1021/ci400019t | J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

Journal of Chemical Information and Modeling
Letter
Table 1. Active Compounds and Their Corresponding Chemical Structure, DOCK Scoring Rank from the Virtual Screening
a
Experiment, and Binding Affinity Values (Ki)
a
Values are the means S.E.M. (n = 3).
values.35 These data confirm the chemotype novelty of all
selected agents.
4.0 μM (Figure 1A). Thus, we proceeded to the chemical
synthesis of the R- and S-stereoisomers of MCKK-17 using
commercially available N-(tert-butoxycarbonyl)-L-proline (1R)
or N-(tert-butoxycarbonyl)-D-proline (1S) (Supporting Infor-
mation Figure S2) to identify the active molecule. The
appropriate proline was coupled to 2-aminothiazole using
1,1′-carbonyldiimidazole (CDI) in CH2Cl2 under anhydrous
conditions41,42 to afford the corresponding Boc-protected
thiazoles (2R and 2S). Removal of the Boc group under acidic
conditions followed by alkylation with 2-chloro-N-(3-
fluorophenyl)acetamide43,44 under basic conditions in DMF
overnight at 80 °C gave stereoisomers MCKK-17S and MCKK-
17R. The purity of MCKK-17S and MCKK-17R was
determined to be at least 99% by integration of the UV trace
from chiral HPLC (data not shown). MCKK-17S resulted in
the most active stereoisomer at the hKOP receptor. Indeed,
MCKK-17S displayed full agonism relative to EKC at the
hKOP receptor with an EC50 of 7.2 3.8 μM, whereas MCKK-
One of the Top-Scoring Docked Molecules: Selective
Agonist at the KOP Receptor. The primary experimental
testing of MCKK-1−22 consisted of performing a competitive
inhibition binding assay at the hKOP receptor. Membranes of
HEK293T cells transfected with the hKOP receptor and GαoB
were prepared and incubated with 3H-diprenorphine (0.3 nM)
in the absence or presence of 10 or 100 μM of each small-
molecule from the virtual screening. Four molecules, MCKK-4,
MCKK-5, MCKK-13, and MCKK-17 partially but significantly
inhibited 3H-diprenorphine binding (Supporting Information
Figure S1) at 100 μM, and their properties were therefore
further investigated.
To assess whether any of these molecules had agonistic
activity, we used a BRET-based G protein activation assay
where the hKOP receptor was coexpressed in HEK293T cells
with GαoB−RLuc8, β1, and mVenus−γ2, as discussed in
Methods. The drug-induced BRET signal is interpreted as a
dissociation of and/or conformational change within the Gαβγ
complex, and thus, as the activation of the coexpressed G
protein. Among the selected molecules, only the racemic
mixture MCKK-17R/S activated GαoB with a potency of 8.3
17R displayed a potency of only 120
9 μM. None of the
other molecules, MCKK-4, MCKK-5, and MCKK-13, signifi-
cantly activated GoB (Figure 1A), suggesting that, in contrast to
MCKK-17R/S, those molecules are antagonists at the hKOP
receptor.
D
dx.doi.org/10.1021/ci400019t | J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

Journal of Chemical Information and Modeling
Letter
To assess the degree of selectivity of MCKK-4, MCKK-5,
MCKK-13, MCKK-17R, or MCKK-17S for the hKOP over the
DOP and MOP receptors, we performed competitive inhibition
of 3H-diprenorphine (0.3 nM) binding in the absence or
presence of various concentrations of each molecule and
determined their Ki values for each receptor (Table 1). EKC
was used as a reference at all three receptors with recorded Ki
values of 20 6 nM at hKOP receptor, 250 31 nM at the
ASSOCIATED CONTENT
■
S
* Supporting Information
General procedures for the synthesis of MCKK-17S and
MCKK-17R. Table S1: List of the 22 tested compounds. Table
S2: Details of the 500 top-scoring docked compounds from
virtual screening at the KOP receptor. Figure S1: Plot of
competitive inhibition of 3H-diprenorphine binding at the KOP
receptor. Figure S2 Synthetic scheme used to obtain MCKK-
17R and MCKK-17S stereoisomers. Figure S3 shows the cAMP
accumulation inhibition curves at DOP and MOP receptors.
This information is available free of charge via the Internet at
http://pubs.acs.org.
DOP receptor, and 170
100 nM at the MOP receptor.
Consistent with the first experimental testing (Supporting
Information Figure S1), the aforementioned five molecules
displayed a relatively weak affinity (Ki values) at the hKOP
receptor, between 100 and 500 μM (Table 1). Among them,
only MCKK-17S exhibited selectivity for the hKOP receptor
with a measured affinity of 120 38 μM at this receptor, and
no detectable affinity at DOP and MOP receptors (>1000 μM).
To further assess the selectivity of MCKK-17S for the hKOP
receptor (Table 1), we also investigated whether the racemic
mixture MCKK-17R/S, as well as the two stereoisomers,
displayed agonistic activity at the DOP and MOP receptors
(Figure 1B). Neither the racemic nor the MCKK-17 stereo-
isomers displayed significant activity at the MOP receptor, and
they only weakly activated the DOP receptor (>1000 μM).
Notably, MCKK-17R, the less active steroisomer at the hKOP
receptor, was the most active enantiomer at the DOP receptor.
In contrast, MCKK-17S did not significantly activate the DOP
receptor, confirming its selectivity at the hKOP receptor. These
results were confirmed using a BRET-based cAMP accumu-
lation inhibition assay (Figure 1C) to monitor the agonistic
activity of the selected molecules at the hKOP receptor (Figure
1C), as well as at the DOP and MOP receptors (Supporting
Information Figure S3).
AUTHOR INFORMATION
■
Corresponding Author
*Mailing Address: Department of Structural and Chemical
Biology, Icahn School of Medicine at Mount Sinai, One
Gustave L. Levy Place, Box 1677, New York, NY 10029. Tel.:
212-659-8690. Fax: 212-849-2456. E-mail: marta.filizola@
mssm.edu.
Author Contributions
∥A.N. and M.-L.R. contributed equally to this work.
Notes
The authors declare the following competing financial
interest(s): Drs. Filizola, Javitch, and Prisinzano are in the
process of filing a provisional patent application through their
respective technology transfer offices.
ACKNOWLEDGMENTS
■
We thank Dr. Raymond C. Stevens for sharing with us the
atomic coordinates of the KOP receptor crystal structure as
well as Drs. Celine Gales and Sam Gambhir for providing
cDNA constructs and Dr. James Woods for providing EKC.
This work was supported, in whole or in part, by National
Institutes of Health grants DA026434 and DA034049 (to
M.F.), DA022413 and MH054137 (to J.A.J.), and DA018151
(to T.E.P.). M.F. is also grateful to CTSA grant UL1TR000067
awarded to the Icahn School of Medicine at Mount Sinai.
Computations were run on resources available through the
Scientific Computing Facility of Mount Sinai School of
Medicine.
Finally, we investigated whether MCKK-17 and the
corresponding stereoisomers could recruit arrestin. MCKK-
17R/S and MCKK-17S recruited arrestin3, with potencies of
160 38 and 120 47 μM, respectively, whereas MCKK-17R
only weakly recruited arrestin3 at the highest concentration
(potency >1000 μM) (Figure 1D), consistent with its weaker
potency at the hKOP receptor for G protein activation.
CONCLUSIONS
■
Although several nonpeptidic selective molecules targeting the
KOP receptor have been developed, we are still far from a
therapeutically effective KOP receptor drug. Our structure-
based virtual screening and compound selection criteria yielded
the discovery of a novel small-molecule chemotype that acts as
a selective, full agonist at the KOP receptor. To the best of our
knowledge, this is the first time that a virtual screening based on
an antagonist-bound GPCR crystal structure has identified an
agonist. We were pleased to note that the chemical scaffold of
the identified hit refers to a selective KOP receptor compound
that has very little similarity with all opioid receptor agonists or
antagonists annotated in ChEMBL, and that it has never been
reported to be an opioid receptor ligand. In summary, MCKK-
17S is a promising new lead compound for structure-based
ligand optimization aimed at discovering potent nonaddictive
analgesics. Although the parent compound MCKK-17S is not
biased toward G protein activation over arrestin, the chemical
scaffold is well suited to structure-guided modifications, raising
the prospects of maintaining selectivity while increasing
potency and building G protein bias.
REFERENCES
■
(1) Results from the 2009 National Survey on Drug Use and Health:
Summary of National Findings; Office of Applied Studies: Rockville,
MD, 2010.
(2) Annual report on the state of the drugs problem in Europe; Office of
the European Union: Lisbon, 2010.
(3) McCurdy, C. R.; Prisinzano, T. E., Opioid receptor ligands. In
Burger’s Medicinal Chemistry, Drug Discovery, and Development, Seventh
ed.; Abraham, D. J., Rotella, D. R., Eds.; John Wiley & Sons, Inc.: New
York, 2010; pp 569−735.
(4) Evans, C. J.; Keith, D. E., Jr.; Morrison, H.; Magendzo, K.;
Edwards, R. H. Cloning of a delta opioid receptor by functional
expression. Science 1992, 258, 1952−1955.
(5) Kieffer, B. L.; Befort, K.; Gaveriaux-Ruff, C.; Hirth, C. G. The
delta-opioid receptor: isolation of a cDNA by expression cloning and
pharmacological characterization. Proc. Natl. Acad. Sci. U S A 1992, 89,
12048−12052.
(6) Minami, M.; Toya, T.; Katao, Y.; Maekawa, K.; Nakamura, S.;
Onogi, T.; Kaneko, S.; Satoh, M. Cloning and expression of a cDNA
for the rat kappa-opioid receptor. FEBS Lett. 1993, 329, 291−295.
E
dx.doi.org/10.1021/ci400019t | J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

Journal of Chemical Information and Modeling
Letter
(7) Chen, Y.; Mestek, A.; Liu, J.; Hurley, J. A.; Yu, L. Molecular
cloning and functional expression of a mu-opioid receptor from rat
brain. Mol. Pharmacol. 1993, 44, 8−12.
(8) DeLander, G. E.; Portoghese, P. S.; Takemori, A. E. Role of spinal
mu opioid receptors in the development of morphine tolerance and
dependence. J. Pharmacol. Exp. Ther. 1984, 231, 91−96.
(9) Fields, H. L. Understanding how opioids contribute to reward
and analgesia. Reg. Anesth. Pain Med. 2007, 32, 242−246.
(10) Nagase, H.; Fujii, H. Opioids in preclinical and clinical trials.
Top. Curr. Chem. 2011, 299, 29−62.
(11) Vanderah, T. W. Delta and kappa opioid receptors as suitable
drug targets for pain. Clin. J. Pain 2010, 26 (Suppl 10), S10−15.
(12) Wang, Y. H.; Sun, J. F.; Tao, Y. M.; Chi, Z. Q.; Liu, J. G. The
role of kappa-opioid receptor activation in mediating antinociception
and addiction. Acta Pharmacol. Sin. 2010, 31, 1065−1070.
(13) Taussig, R.; Iniguez-Lluhi, J. A.; Gilman, A. G. Inhibition of
adenylyl cyclase by Gi alpha. Science 1993, 261, 218−221.
(14) Tallent, M.; Dichter, M. A.; Bell, G. I.; Reisine, T. The cloned
kappa opioid receptor couples to an N-type calcium current in
undifferentiated PC-12 cells. Neuroscience 1994, 63, 1033−1040.
(15) Henry, D. J.; Grandy, D. K.; Lester, H. A.; Davidson, N.;
Chavkin, C. Kappa-opioid receptors couple to inwardly rectifying
potassium channels when coexpressed by Xenopus oocytes. Mol.
Pharmacol. 1995, 47, 551−557.
(16) Land, B. B.; Bruchas, M. R.; Schattauer, S.; Giardino, W. J.; Aita,
M.; Messinger, D.; Hnasko, T. S.; Palmiter, R. D.; Chavkin, C.
Activation of the kappa opioid receptor in the dorsal raphe nucleus
mediates the aversive effects of stress and reinstates drug seeking. Proc.
Natl. Acad. Sci. U S A 2009, 106, 19168−19173.
(17) Bruchas, M. R.; Chavkin, C. Kinase cascades and ligand-directed
signaling at the kappa opioid receptor. Psychopharmacology (Berl)
2010, 210, 137−47.
(18) Chavkin, C. The therapeutic potential of kappa-opioids for
treatment of pain and addiction. Neuropsychopharmacology 2011, 36,
369−370.
(19) Rives, M. L.; Rossillo, M.; Liu-Chen, L. Y.; Javitch, J. A. 6′-
Guanidinonaltrindole (6′-GNTI) is a G protein-biased kappa-opioid
receptor agonist that inhibits arrestin recruitment. J. Biol. Chem. 2012,
287, 27050−27054.
(20) Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F. S.;
Mathiesen, J. M.; Sunahara, R. K.; Pardo, L.; Weis, W. I.; Kobilka, B.
K.; Granier, S. Crystal structure of the mu-opioid receptor bound to a
morphinan antagonist. Nature 2012, 485, 321−326.
(21) Granier, S.; Manglik, A.; Kruse, A. C.; Kobilka, T. S.; Thian, F.
S.; Weis, W. I.; Kobilka, B. K. Structure of the delta-opioid receptor
bound to naltrindole. Nature 2012, 485, 400−404.
(22) Wu, H.; Wacker, D.; Mileni, M.; Katritch, V.; Han, G. W.;
Vardy, E.; Liu, W.; Thompson, A. A.; Huang, X. P.; Carroll, F. I.;
Mascarella, S. W.; Westkaemper, R. B.; Mosier, P. D.; Roth, B. L.;
Cherezov, V.; Stevens, R. C. Structure of the human kappa-opioid
receptor in complex with JDTic. Nature 2012, 485, 327−332.
(23) Thompson, A. A.; Liu, W.; Chun, E.; Katritch, V.; Wu, H.;
Vardy, E.; Huang, X. P.; Trapella, C.; Guerrini, R.; Calo, G.; Roth, B.
L.; Cherezov, V.; Stevens, R. C. Structure of the nociceptin/orphanin
FQ receptor in complex with a peptide mimetic. Nature 2012, 485,
395−399.
(24) Filizola, M.; Devi, L. A. Structural biology: How opioid drugs
bind to receptors. Nature 2012, 485, 314−317.
(25) Filizola, M.; Devi, L. A. Grand opening of structure-guided
design for novel opioids. Trends Pharmacol. Sci. 2013, 34, 6−12.
(26) Irwin, J. J.; Shoichet, B. K. ZINC-a free database of
commercially available compounds for virtual screening. J. Chem. Inf.
Model. 2005, 45, 177−182.
(29) Kuntz, I. D.; Blaney, J. M.; Oatley, S. J.; Langridge, R.; Ferrin, T.
E. A geometric approach to macromolecule-ligand interactions. J. Mol.
Biol. 1982, 161, 269−288.
(30) Mysinger, M. M.; Shoichet, B. K. Rapid context-dependent
ligand desolvation in molecular docking. J. Chem. Inf. Model. 2010, 50,
1561−1573.
(31) Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.;
Alagona, G.; Profeta, S.; Weiner, P. A. A new force-field for molecular
mechanical simulation of nucleic-acids and proteins. J. Am. Chem. Soc.
1984, 106, 765−784.
(32) Carlsson, J.; Yoo, L.; Gao, Z. G.; Irwin, J. J.; Shoichet, B. K.;
Jacobson, K. A. Structure-based discovery of A2A adenosine receptor
ligands. J. Med. Chem. 2010, 53, 3748−3755.
(33) Negri, A.; Li, J.; Naini, S.; Coller, B. S.; Filizola, M. Structure-
based virtual screening of small-molecule antagonists of platelet
integrin alphaIIbbeta3 that do not prime the receptor to bind ligand. J.
Comput. Aid. Mol. Des. 2012, 26, 1005−1015.
(34) Rogers, D.; Brown, R. D.; Hahn, M. Using extended-
connectivity fingerprints with Laplacian-modified Bayesian analysis in
high-throughput screening follow-up. J. Biomol. Screen. 2005, 10, 682−
686.
(35) Wawer, M.; Bajorath, J. Similarity-potency trees: a method to
search for SAR information in compound data sets and derive SAR
rules. J. Chem. Inf. Model. 2010, 50, 1395−1409.
(36) Gales, C.; Van Durm, J. J.; Schaak, S.; Pontier, S.; Percherancier,
Y.; Audet, M.; Paris, H.; Bouvier, M. Probing the activation-promoted
structural rearrangements in preassembled receptor-G protein
complexes. Nat. Struct. Mol. Biol. 2006, 13, 778−786.
(37) Sauliere, A.; Bellot, M.; Paris, H.; Denis, C.; Finana, F.; Hansen,
J. T.; Altie, M. F.; Seguelas, M. H.; Pathak, A.; Hansen, J. L.; Senard, J.
M.; Gales, C. Deciphering biased-agonism complexity reveals a new
active AT1 receptor entity. Nat. Chem. Biol. 2012, 8, 622−630.
(38) Klewe, I. V.; Nielsen, S. M.; Tarpo, L.; Urizar, E.; Dipace, C.;
Javitch, J. A.; Gether, U.; Egebjerg, J.; Christensen, K. V. Recruitment
of beta-arrestin2 to the dopamine D2 receptor: insights into anti-
psychotic and anti-parkinsonian drug receptor signaling. Neuro-
pharmacology 2008, 54, 1215−1222.
(39) Guo, W.; Urizar, E.; Kralikova, M.; Mobarec, J. C.; Shi, L.;
Filizola, M.; Javitch, J. A. Dopamine D2 receptors form higher order
oligomers at physiological expression levels. Embo J. 2008, 27, 2293−
2304.
(40) Jiang, L. I.; Collins, J.; Davis, R.; Lin, K. M.; DeCamp, D.;
Roach, T.; Hsueh, R.; Rebres, R. A.; Ross, E. M.; Taussig, R.; Fraser, I.;
Sternweis, P. C. Use of a cAMP BRET sensor to characterize a novel
regulation of cAMP by the sphingosine 1-phosphate/G13 pathway. J.
Biol. Chem. 2007, 282, 10576−10584.
(41) Luo, Q. L.; Li, J. Y.; Chen, L. L.; Li, J.; Ye, Q. Z.; Nan, F. J.
Inhibitors of type I MetAPs containing pyridine-2-carboxylic acid
thiazol-2-ylamide. Part 2: SAR studies on the pyridine ring 3-
substituent. Bioorg. Med. Chem. Lett. 2005, 15, 639−644.
(42) Yu, C.; Qiu, J.; Zheng, F.; Zhong, W. Highly efficient
bifunctional organocatalysts for the asymmetric Michael addition of
ketones to nitroolefins. Tetrahedron Lett. 2011, 52, 3298−3302.
(43) Ma, L.; Xie, C.; Ma, Y.; Liu, J.; Xiang, M.; Ye, X.; Zheng, H.;
Chen, Z.; Xu, Q.; Chen, T.; Chen, J.; Yang, J.; Qiu, N.; Wang, G.;
Liang, X.; Peng, A.; Yang, S.; Wei, Y.; Chen, L. Synthesis and biological
evaluation of novel 5-benzylidenethiazolidine-2,4-dione derivatives for
the treatment of inflammatory diseases. J. Med. Chem. 2011, 54, 2060−
2068.
(44) Gu, S. J.; Lee, J. K.; Pae, A. N.; Chung, H. J.; Rhim, H.; Han, S.
Y.; Min, S. J.; Cho, Y. S. Synthesis and biological evaluation of 1,4-
diazepane derivatives as T-type calcium channel blockers. Bioorg. Med.
Chem. Lett. 2010, 20, 2705−2708.
(27) Lorber, D. M.; Shoichet, B. K. Flexible ligand docking using
conformational ensembles. Protein Sci. 1998, 7, 938−950.
(28) Lorber, D. M.; Shoichet, B. K. Hierarchical docking of databases
of multiple ligand conformations. Curr. Top. Med. Chem. 2005, 5,
739−749.
F
dx.doi.org/10.1021/ci400019t | J. Chem. Inf. Model. XXXX, XXX, XXX−XXX