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Title
In silico design of novel probes for the atypical opioid receptor MRGPRX2.
Permalink
https://escholarship.org/uc/item/7kk0c31w
Journal
Nature chemical biology, 13(5)
ISSN
1552-4450
Authors
Lansu, Katherine
Karpiak, Joel
Liu, Jing
et al.
Publication Date
2017-05-01
DOI
10.1038/nchembio.2334
Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California
In silico design of novel probes for the atypical opioid receptor
MRGPRX2
Katherine Lansu
†,1
, Joel Karpiak
†,2
, Jing Liu
5
, Xi-Ping Huang
1,6
, John D. McCorvy
1
, Wesley
K. Kroeze
1
, Tao Che
1
, Hiroshi Nagase
3
, Frank I. Carroll
4
, Jian Jin
5
, Brian K. Shoichet
2
, and
Bryan L. Roth
1,6,7
1
Department of Pharmacology, University of North Carolina, Chapel Hill NC
2
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA
3
University of Tsukuba, International Institute for Integrative Sleep Medicine, Tsukuba, Japan
4
Research Triangle Institute International, Center for Drug Discovery, Research Triangle Park, NC
5
Department of Pharmacological Sciences and Department of Oncological Sciences, Icahn
School of Medicine at Mount Sinai, New York, NY
6
National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP),
University of North Carolina, Chapel Hill NC
7
Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy,
University of North Carolina at Chapel Hill, Chapel Hill, NC
Abstract
The primate-exclusive MRGPRX2 G protein-coupled receptor (GPCR) has been suggested to
modulate pain and itch. Despite putative peptide and small molecule MRGPRX2 agonists,
selective nanomolar potency probes have not yet been reported. To identify a MRGPRX2 probe,
we first screened 5,695 small molecules and found many opioid compounds activated MRGPRX2,
including (−)- and (+)-morphine, hydrocodone, sinomenine, dextromethorphan and the
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†
These authors contributed equally.
Competing Financial Interests Statement:
The authors report no competing financial interests.
AUTHOR CONTRIBUTIONS
K.L. performed the
in vitro
pharmacology, molecular biology, and wrote the paper. J.K. designed and developed homology models,
carried out docking screens, analyzed results, and wrote the paper. J.L. synthesized the probe enantiomers. X.P.H. performed
GPCRome screening and assisted with
in vitro
pharmacology experiments. J.D.M. performed binding studies and
in vitro
pharmacology. W.K. assisted in the
in vitro
small molecule screening and helped with data and statistical analyses. T.C. performed
in
vitro
pharmacology experiments. N.H. synthesized (+)-TAN-67 and KNT-127. F.I.C. synthesized several compounds and advised SAR
studies. J.J. supervised chemical synthesis of probe enantiomers. B.L.R. and B.K.S. coordinated and supervised the project, and with
the other authors wrote the paper.
Code Availability
The DOCK3.6 program
29,34
is freely accessible to academic labs at http://dock.compbio.ucsf.edu/DOCK3.6/ or http://
blaster.docking.org/ for open access use.
Data Availability
The generated and analyzed raw data sets that support the findings of this study are available from the corresponding author upon
reasonable request.
HHS Public Access
Author manuscript
Nat Chem Biol
. Author manuscript; available in PMC 2017 September 13.
Published in final edited form as:
Nat Chem Biol
. 2017 May ; 13(5): 529–536. doi:10.1038/nchembio.2334.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
prodynorphin-derived peptides, dynorphin A, dynorphin B, and α- and β-neoendorphin. We used
these to select for mutagenesis-validated homology models and docked almost 4 million small
molecules. From this docking, we predicted ZINC-3573, which represents a potent MRGPRX2-
selective agonist, showing little activity against 315 other GPCRs and 97 representative kinases,
and an essentially inactive enantiomer. ZINC-3573 activates endogenous MRGPRX2 in a human
mast cell line inducing degranulation and calcium release. MRGPRX2 is a unique atypical opioid-
like receptor important for modulating mast cell degranulation, which can now be specifically
modulated with ZINC-3573.
G protein-coupled receptors (GPCRs) are seven transmembrane receptors that transduce
extracellular signals into biological responses via heterotrimeric G-proteins and β-arrestins
1
.
GPCRs are involved in nearly every known biological system and, unsurprisingly, GPCR-
targeting small molecules represent the largest target class for FDA-approved drugs
2
.
Despite their therapeutic utility, only 10% of GPCRs function as therapeutic targets for FDA
approved drugs
2
,
while ~120 of 394 non-olfactory GPCRs represent “orphan receptors” or
understudied GPCRs (“oGPCRs”
3
) without useful probes and, frequently, without validated
endogenous ligands
4,5
. The process of discovering specific and potent probes for oGPCRs
yields useful research tools and can also illuminate previously unrealized drug interactions,
potentially establishing new drug targets.
To identify oGPCR ligands, we have developed and tested new physical and computational
approaches for screening these receptors. The first physical method, dubbed “PRESTO-
Tango,” involves high-throughput, massively parallel screening of potential modulators
(including small molecules, bioactive peptides, and other reagents) for their ability to
stimulate β-arrestin recruitment at most non-olfactory receptors in the GPCRome
6
. The
second orthogonal and complementary approach relies on the principle that over-expressed
G proteins facilitate a partially active state for most GPCRs enabling the discovery of both
allosteric and orthosteric modulators for oGPCRs using engineered yeast
3
. Encouraged by
the success of other structure-based drug design methods in GPCRs
7–9
, our
in silico
approach leverages the physical screens to develop comparative structural models of the
receptors, and then computationally screens a much wider chemical space—typically several
million commercially available molecules—to find specific ligands for the oGPCRs.
We apply this strategy to the Mas-related G protein-coupled receptor X2 (MRGPRX2) -- a
primate-exclusive
10,11
class A orphan GPCR expressed in mast cells and small diameter
neurons in the dorsal root and trigeminal ganglia
12–16
. Several unrelated peptides are
reported to activate MRGPRX2 including Cortistatin-14
16
, Substance P
13
, and PAMP(9–
20)
14
, whether any proposed peptides are endogenous MRGPRX2 agonists is unknown.
MRGPRX2 remains an oGPCR in part because no convincing rodent ortholog has been
validated (see Discussion) and because MRGPRX2-selective nanomolar potency probes are
unavailable. Although several selective agonists are reported for MRGPRX2
17,18
, the
compounds are not easily obtained and have not been validated for specificity or potency.
The identification of demonstrably selective, potent MRGPRX2 agonist probes represents an
essential step toward illumination of its function
in vitro
and
in vivo.
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Nat Chem Biol
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Here, we describe how an integrated approach combining our PRESTO –Tango method and
our modeling and docking platforms led to the identification of MRGPRX2 agonists,
including exogenous and endogenous opioids and a selective MRGPRX2 probe ZINC-3573.
We confirmed ZINC-3573’s selectivity for MRGPRX2 via testing at 315 class A GPCRs
using PRESTO-Tango, binding assays performed by the National Institute of Mental Health
Psychoactive Drug Screening Program (NIMH-PDSP), and by testing the parent scaffold
using a commercial (DiscoverX) kinome screen. Using ZINC-3573, we show MRGPRX2
activation induces intracellular calcium release and degranulation in a human mast cell line.
We also demonstrate that MRGPRX2 represents a novel G
αq
-coupled opioid-like receptor
that could mediate some peripheral side effects of commonly prescribed opiate medications.
This discovery of the specific MRGPRX2 agonist ZINC-3573, matched with an inactive
enantiomer, provides the community with a pair of chemical probes by which the
in vivo
function of this fascinating target may be investigated with exquisite specificity and control.
RESULTS
Identification of MRGPRX2 agonists
We initially attempted to replicate prior reports of potential MRGPRX2 agonists to
determine if any might prove suitable as leads for probe development. Unfortunately, we
could not replicate the activities of most reported MRGPRX2 agonists when tested at the
highest concentrations possible for our assays (Fig 1, Supplementary Results,
Supplementary Table 1). Thus, of the many putative MRGPRX2-activating peptides and
peptide-like compounds, we could replicate activities only for substance P, cortistatin-14 and
PAMP (9–20) (Fig 1a,b). Mastoparan, octreotide, leuprolide, and kallidin were inactive or
only marginally so (e.g. G
αq
EC
50
= 11.5 μM for kallidin) at human MRGPRX2 (Fig 1c,
Supplementary Table 1).The putative MRGPRX2 agonist mastoparan
19
was active in cells
with and without MRGPRX2 expression suggesting non-specific activity (Fig 1d.,
Supplementary Table 1).
Of the more than one dozen non-peptide compounds reported to activate MRGPRX2, we
could only replicate four (TAN-67
20
, compound 48/80
19
, cetrorelix
19
and the proposed
selective agonist complanadine A
18
) and even these had low potencies or have other known
receptor targets (Fig 1c,d, Supplementary Table 1). Notably, we could not validate several
recently reported secretagogue agonists for MRGPRX2 including the THIQ motif-
containing octreotide, rocuronium, ciprofloxacin, atracurium, moxifloxacin, and
levofloxacin
19
even when tested up to 100 μM (Fig 1c,d, Supplementary Table 1). Likewise,
although the proposed nanomolar MRGPRX2-selective agonist complanadine A
18
was
active in our assays, we measured a G
αq
EC
50
of 18 μM and could not obtain a β-arrestin
EC
50
due to apparent cytotoxicity above 1 μM using PRESTO-Tango, a luciferase reporter-
based β-arrestin screening platform
6
(Fig 1c,d, see Supplementary Table 1 for a full list of
validated compounds).
As MRGPRX2 is expressed only in primates, finding a rodent analog would enable the use
of genetic techniques to probe for the receptor’s functional roles, with the caveat that
GPCRs may often be knocked down without incurring phenotypes that recapitulate their
roles in pharmacology. The suggested MRGPRX2 rodent ortholog MRGPRB2
19
only shares
Lansu et al. Page 3
Nat Chem Biol
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52% sequence identity with MRGPRX2 (Supplementary Fig 1a) and we could not
substantiate any proposed shared ligands except for Cortistatin-14, which had high
micromolar activity at MRGPRB2 (Fig 1e, f, Supplementary Fig 1b). Thus, although there
have been prior studies of MRGPRX2 and/or MRGPRB2’s pharmacology, many remain
insufficiently robust for facile replication; and no independent studies have emerged that
replicate these prior findings.
Accordingly, we chose an unbiased approach to identify reliable MRGPRX2 probes. We
recently proposed parallel screening strategies
3,6
using small libraries of drugs and drug-like
compounds as fruitful initial approaches to discover active compounds at oGPCRs
6
. Here we
screened 5,695 unique compounds for agonist activity at three members of the MRGPRX
family (MRGPRX1, MRGPRX2, and MRGPRX4) using the PRESTO-Tango platform (see
Online Methods). Our strategy was to screen against three MRGPRX family receptors in
parallel to find active compounds with selectivity within the family.
The screening revealed MRGPRX1 had the fewest number of actives (39), followed by
MRGPRX4 (54), and MRGPRX2 (81) (Fig 2a). We were most interested in compounds
showing selective MRGPRX2 agonism without apparent activity at other MRGPRX
receptors. Among the 67 compounds that activated MRGPRX2, and neither MRGPRX1 nor
MRGPRX4, were five opioid-related ligands ADL-5859, sinomenine, dextromethorphan,
dextromethorphan’s metabolite dextrorphan, and the previously reported MRGPRX2 ligand
TAN-67
20
, a delta opioid receptor agonist.
Confirmatory concentration response curves using the PRESTO-Tango platform indicated
the five opioid-like compounds had low micromolar potency (Fig 2b, c). To confirm the
MRGPRX2-Tango construct performed similarly to the unmodified wild-type (WT)
receptor, we tested previously reported MRGPRX2 agonists TAN-67
20
, cortistatin-14
16
,
substance P
13
, and compound 48/80
19
and found all activated the MRGPRX2-Tango
receptor at similar reported potencies for G
αq
assays at the WT receptor (Fig 2d).
To confirm MRGPRX2 G
αq
-mediated functional activity of agonists, we used a tetracycline-
inducible WT MRGPRX2 stable HEK-T cell line to test for intracellular calcium release.
Dextromethorphan, dextrorphan, sinomenine, and TAN-67 promoted intracellular calcium
release when MRGPRX2 expression was induced by tetracycline (1 μg/ml; Supplementary
Fig 2a) but not in the absence of tetracycline (e.g, MRGPRX2 is not expressed,
Supplementary Fig 2b).
Preference for dextro-enantiomers and N-methyl scaffolds
As several dextrorotatory opiate ligands activated MRGPRX2, we initially investigated
ligand stereochemistry, aware that classical opioid receptors prefer levorotary morphinans
and benzomorphans
21
. We assayed levorphanol and levallorphan, enantiomers of the
screening hits dextrorphan and dextromethorphan, respectively, for activity at MRGPRX2 in
β-arrestin recruitment and calcium mobilization assays. Levorphanol was approximately ten-
fold less potent at MRGPRX2 than dextrorphan and levallorphan was completely inactive up
to 100 μM (Fig 3a). Likewise the dextrorotary morphinan sinomenine had comparable
potency to dextrorphan and dextromethorphan at MRGPRX2 (Supplementary Table 2), more
Lansu et al. Page 4
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. Author manuscript; available in PMC 2017 September 13.
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