In silico design of novel probes for the atypical opioid receptor MRGPRX2

Article abstract of DOI:10.1038/nchembio.2334

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 MRGPRX

Full text of DOI:10.1038/nchembio.2334

article
puBLIsHed OnLIne: 13 MarCH 2017 | dOI: 10.1038/nCHeMBIO.2334
In silico ꢀꢁꢂigꢃ of ꢃovꢁl ꢄꢅobꢁꢂ foꢅ ꢆhꢁ ꢇꢆyꢄicꢇl
oꢄioiꢀ ꢅꢁcꢁꢄꢆoꢅ MrGprX2
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Kꢇꢆhꢁꢅiꢃꢁ Lꢇꢃꢂꢈ , Joꢁl Kꢇꢅꢄiꢇk , Jiꢃg Liꢈ , Xi-piꢃg Hꢈꢇꢃg , Johꢃ d McCoꢅvy , Wꢁꢂlꢁy K Kꢅoꢁzꢁ ,
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tꢇo Chꢁ , Hiꢅoꢂhi nꢇgꢇꢂꢁ , Fꢅꢇꢃk I Cꢇꢅꢅoll , Jiꢇꢃ Jiꢃ , Bꢅiꢇꢃ K shoichꢁꢆ & Bꢅyꢇꢃ L roꢆh
*
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 that many opioid compounds activated
MRGPRX2, including (−)- and (+)-morphine, hydrocodone, sinomenine, dextromethorphan, and the prodynorphin-derived
peptides dynorphin A, dynorphin B, and a- and b-neoendorphin. We used these to select for mutagenesis-validated homol-
ogy models and docked almost 4 million small molecules. From this docking, we predicted ZINC-3573—a potent MRGPRX2-
selective agonist, showing little activity against 315 other GPCRs and 97 representative kinases—along with 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.
protein-coupled receptors (GPCRs) are seven-transmembrane
We apply this strategy to the Mas-related G protein-coupled
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receptors that transduce extracellular signals into biologi- receptor X2 (MRGPRX2), a primate-exclusive
class A orphan
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G
cal responses via heterotrimeric G proteins and β-arrestins . GPCR expressed in mast cells and small-diameter neurons in the
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GPCRs are involved in nearly every known biological system, and, dorsal-root and trigeminal ganglia . Several unrelated peptides
unsurprisingly, GPCR-targeting small molecules represent the arereportedtoactivateMRGPRX2, includingcortistatin-14(ref. 16),
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largest target class of drugs approved by the US Food and Drug substance P , and PAMP(9–20) (ref. 14); whether any proposed pep-
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Administration (FDA) . Despite their therapeutic utility, only 10% tides are endogenous MRGPRX2 agonists is unknown. MRGPRX2
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of GPCRs function as therapeutic targets for FDA-approved drugs , remains an oGPCR in part because no convincing rodent ortholog
while ~120 of 394 nonolfactory GPCRs represent ‘orphan recep- has been validated (see Discussion) and because MRGPRX2-
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tors ’, or understudied GPCRs (“oGPCRs” ), for which there are no selective nanomolar potency probes are unavailable. Although
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useful probes and, frequently, no validated endogenous ligands . several selective agonists are reported for MRGPRX2 (refs. 17,18),
The process of discovering specific and potent probes for oGPCRs the compounds are not easily obtained and have not been validated
yields useful research tools and can illuminate previously unrealized for specificity or potency. The identification of demonstrably selec-
drug interactions, potentially establishing new drug targets.
tive, potent MRGPRX2 agonist probes represents an essential step
To identify oGPCR ligands, we have developed and tested new toward illumination of its function in vitro and in vivo.
physical and computational approaches for screening these recep- Here, we describe how an integrated approach combining
tors. The first physical method, dubbed “PRESTO-Tango ,” involves our PRESTO-Tango method and our modeling and docking plat-
high-throughput, massively parallel screening of potential modu- forms led to the identification of MRGPRX2 agonists, including
lators (including small molecules, bioactive peptides, and other exogenous and endogenous opioids and a selective MRGPRX2
reagents) for their ability to stimulate β-arrestin recruitment at most probe, ZINC-3573. We confirmed ZINC-3573’s selectivity for
6
nonolfactory receptors in the GPCRome . The second, an orthog- MRGPRX2 via testing of 315 class A GPCRs using PRESTO-Tango,
onal and complementary approach, relies on the principle that binding assays performed by the National Institute of Mental Health
overexpressed G proteins facilitate a partially active state for most Psychoactive Drug Screening Program (NIMH-PDSP), and testing
GPCRs, enabling the discovery of both allosteric and orthosteric of the parent scaffold using a commercial (DiscoveRX) kinome
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modulators for oGPCRs using engineered yeast . Encouraged by the screen. Using ZINC-3573, we showed that MRGPRX2 activation
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success of other structure-based drug-design methods in GPCRs , induces intracellular calcium release and degranulation in a human
we designed an in silico approach that leverages the physical screens mast cell line. We also demonstrated that MRGPRX2 represents a
to develop comparative structural models of the receptors and then novel Gα -coupled opioid-like receptor that could mediate some
q
computationally screens a much wider chemical space—typically peripheral side effects of commonly prescribed opiate medica-
several million commercially available molecules—to find specific tions. This discovery of the specific MRGPRX2 agonist ZINC-3573,
ligands for the oGPCRs.
matched with an inactive enantiomer, provides the community
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dꢃꢉarꢁmꢃꢀꢁ ꢅf pharmaꢄꢅꢇꢅgy, uꢀꢆꢊꢃrsꢆꢁy ꢅf nꢅrꢁh carꢅꢇꢆꢀa, chaꢉꢃꢇ Hꢆꢇꢇ, nꢅrꢁh carꢅꢇꢆꢀa, uSA. dꢃꢉarꢁmꢃꢀꢁ ꢅf pharmaꢄꢃꢂꢁꢆꢄaꢇ chꢃmꢆsꢁry, uꢀꢆꢊꢃrsꢆꢁy
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Sꢄhꢅꢅꢇ ꢅf Mꢃꢋꢆꢄꢆꢀꢃ aꢁ Mꢅꢂꢀꢁ Sꢆꢀaꢆ, nꢃw Yꢅrk, nꢃw Yꢅrk, uSA. naꢁꢆꢅꢀaꢇ iꢀsꢁꢆꢁꢂꢁꢃ ꢅf Mꢃꢀꢁaꢇ Hꢃaꢇꢁh psyꢄhꢅaꢄꢁꢆꢊꢃ drꢂg Sꢄrꢃꢃꢀꢆꢀg prꢅgram (niMH pdSp),
uꢀꢆꢊꢃrsꢆꢁy ꢅf nꢅrꢁh carꢅꢇꢆꢀa, chaꢉꢃꢇ Hꢆꢇꢇ, nꢅrꢁh carꢅꢇꢆꢀa, uSA. iꢀꢁꢃrꢀaꢁꢆꢅꢀaꢇ iꢀsꢁꢆꢁꢂꢁꢃ fꢅr iꢀꢁꢃgraꢁꢆꢊꢃ Sꢇꢃꢃꢉ Mꢃꢋꢆꢄꢆꢀꢃ, uꢀꢆꢊꢃrsꢆꢁy ꢅf tsꢂkꢂꢈa, tsꢂkꢂꢈa,
Jaꢉaꢀ. cꢃꢀꢁꢃr fꢅr drꢂg dꢆsꢄꢅꢊꢃry, Rꢃsꢃarꢄh trꢆaꢀgꢇꢃ iꢀsꢁꢆꢁꢂꢁꢃ iꢀꢁꢃrꢀaꢁꢆꢅꢀaꢇ, Rꢃsꢃarꢄh trꢆaꢀgꢇꢃ park, nꢅrꢁh carꢅꢇꢆꢀa, uSA. dꢆꢊꢆsꢆꢅꢀ ꢅf chꢃmꢆꢄaꢇ bꢆꢅꢇꢅgy
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aꢀꢋ Mꢃꢋꢆꢄꢆꢀaꢇ chꢃmꢆsꢁry, eshꢃꢇmaꢀ Sꢄhꢅꢅꢇ ꢅf pharmaꢄy, uꢀꢆꢊꢃrsꢆꢁy ꢅf nꢅrꢁh carꢅꢇꢆꢀa aꢁ chaꢉꢃꢇ Hꢆꢇꢇ, chaꢉꢃꢇ Hꢆꢇꢇ, nꢅrꢁh carꢅꢇꢆꢀa, uSA. thꢃsꢃ aꢂꢁhꢅrs
ꢄꢅꢀꢁrꢆꢈꢂꢁꢃꢋ ꢃqꢂaꢇꢇy ꢁꢅ ꢁhꢆs wꢅrk. *ꢃ-maꢆꢇ: ꢈryaꢀ_rꢅꢁh@mꢃꢋ.ꢂꢀꢄ.ꢃꢋꢂ
nature CHeMICaL BIOLOGY | AdvAnce online publicAtion | www.ꢀaꢁꢂrꢃ.ꢄꢅm/ꢀaꢁꢂrꢃꢄhꢃmꢆꢄaꢇꢈꢆꢅꢇꢅgy
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with a pair of chemical probes by which the in vivo function of this
fascinating target may be investigated with exquisite specificity
and control.
a 5
+X2
Compound 48/80
b 5
–X2
Compound 48/80
Substance P
Substance P
4
3
2
1
Cortistatin-14
4
3
2
1
Cortistatin-14
PAMP-12
TAN-67
DMSO
PAMP-12
TAN-67
DMSO
RꢁSꢀꢃTS
Identification of MRGPRX2 agonists
We initially attempted to replicate prior reports of potential
MRGPRX2 agonists to determine whether any might prove suitable
as leads for probe development. Unfortunately, we could not repli-
cate the activities of most reported MRGPRX2 agonists when tested
even 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
–
12 –9 –8 –7 –6 –5 –4
log [drug] (M)
–12 –9 –8 –7 –6 –5 –4
log [drug] (M)
c 5
+X2
d 5
–X2
Moxifloxacin
Levofloxacin
Ciprofloxacin
Olfloxacin
Moxifloxacin
Levofloxacin
Ciprofloxacin
Olfloxacin
4
3
2
1
4
3
2
1
TAN-67
TAN-67
DMSO
DMSO
Octreotide
Atracurium
Rocuronium
Complanadine A
Mastoparan
Octreotide
Atracurium
Rocuronium
Complanadine A
Mastoparan
kallidin were inactive or only marginally active (for example, Gα
q
effector concentration for half-maximal response (EC ) = 11.5 μM
50
–12 –9 –8 –7 –6 –5 –4
log [drug] (M)
–12 –9 –8 –7 –6 –5 –4
log [drug] (M)
for kallidin) at human MRGPRX2 (Fig. 1c; Supplementary Table 1).
19
The putative MRGPRX2 agonist mastoparan was active both in
cells with and without MRGPRX2 expression, suggesting nonspe-
cific activity (Fig. 1d; Supplementary Table 1).
e 4
f 4
MRGPRX2 cortistatin-14
MRGPRB2 cortistatin-14
MRGPRX2 substance P
MRGPRB2 substance P
MRGPRX2 TAN-67
MRGPRX2 cortistatin-14
MRGPRB2 cortistatin-14
MRGPRX2 substance P
MRGPRB2 substance P
MRGPRX2 TAN-67
3
2
1
3
2
1
Of the more than one dozen nonpeptide compounds reported
to activate MRGPRX2, we could only replicate four (TAN-67
MRGPRB2 TAN-67
MRGPRB2 TAN-67
1
9
(
ref. 20), compound 48/80 (ref. 19), cetrorelix , and the proposed
18
selective agonist complanadine A ), and even these had low poten-
cies 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-
–
12
–8
–6
–4
–12 –9
–8
–7
–6
–5
–4
log [drug] (M)
log [drug] (M)
containing octreotide, rocuronium, ciprofloxacin, atracurium, Figure 1 | Validation of MRGPRX2 and MRGPRB2 Agonists.
19
moxifloxacin, and levofloxacin , even when tested at up to 100 μM (a–d) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs ꢆꢀ ꢁhꢃ Fꢇꢂꢅrꢃsꢄꢃꢀꢁ ꢆmagꢆꢀg
(
Fig. 1c,d; Supplementary Table 1). Likewise, although the pro- ꢉꢇaꢁꢃ rꢃaꢋꢃr (FlipR) ꢆꢀꢁraꢄꢃꢇꢇꢂꢇar ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ assay (n = 3 ꢃxꢉꢃrꢆmꢃꢀꢁs
18
posed nanomolar MRGPRX2-selective agonist complanadine A
ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ wꢃꢇꢇs) ꢆꢀ MRGpRX2-ꢆꢀꢋꢂꢄꢆꢈꢇꢃ ꢄꢃꢇꢇs, ꢋꢃsꢆgꢀaꢁꢃꢋ +X2 ꢅr –X2 fꢅr
was active in our assays, we measured a Gα EC of 18 μM and ꢁhꢃ ꢉrꢃsꢃꢀꢄꢃ ꢅr aꢈsꢃꢀꢄꢃ ꢅf ꢁꢃꢁraꢄyꢄꢇꢆꢀꢃ-ꢆꢀꢋꢂꢄꢃꢋ rꢃꢄꢃꢉꢁꢅr ꢃxꢉrꢃssꢆꢅꢀ, fꢅr
q
50
could not obtain a β-arrestin EC because of apparent cytotoxic- ꢉrꢃꢊꢆꢅꢂsꢇy ꢉꢂꢈꢇꢆshꢃꢋ MRGpRX2 agꢅꢀꢆsꢁs. thꢃ y axꢆs shꢅws ꢁhꢃ fꢅꢇꢋ ꢄhaꢀgꢃ
50
ity above 1 μM using PRESTO-Tango, a luciferase-reporter-based ꢅf ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ ꢅꢊꢃr ꢁhꢃ ꢈasaꢇ rꢃꢄꢃꢉꢁꢅr sꢆgꢀaꢇꢆꢀg. (e) cꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–
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β-arrestinscreeningplatform (Fig.1c,d;seeSupplementaryTable1 rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs ꢆꢀ FlipR ꢆꢀꢁraꢄꢃꢇꢇꢂꢇar ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ assay fꢅr sꢁaꢈꢇꢃ ꢄꢃꢇꢇ
for a full list of validated compounds).
ꢇꢆꢀꢃs ꢃxꢉrꢃssꢆꢀg ꢃꢆꢁhꢃr hꢂmaꢀ MRGpRX2 (sꢅꢇꢆꢋ ꢇꢆꢀꢃs) ꢅr ꢁhꢃ ꢉrꢅꢉꢅsꢃꢋ
As MRGPRX2 is expressed only in primates, finding a rodent mꢅꢂsꢃ ꢅrꢁhꢅꢇꢅg MRGpRb2 (ꢋꢅꢁꢁꢃꢋ ꢇꢆꢀꢃs) wꢆꢁh X2-aꢄꢁꢆꢊaꢁꢆꢀg ꢉꢃꢉꢁꢆꢋꢃs
analog would enable the use of genetic techniques to probe for the ꢄꢅrꢁꢆsꢁaꢁꢆꢀ-14 aꢀꢋ sꢂꢈsꢁaꢀꢄꢃ p aꢀꢋ smaꢇꢇ-mꢅꢇꢃꢄꢂꢇꢃ agꢅꢀꢆsꢁ tAn-67.
receptor’s functional roles, with the caveat that GPCRs may often Y axꢆs ꢆs fꢅꢇꢋ-ꢄhaꢀgꢃ ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ ꢅꢊꢃr ꢈasaꢇ (n = 3 ꢆꢀ qꢂaꢋrꢂꢉꢇꢆꢄaꢁꢃ)
be knocked down without producing phenotypes that recapitu- (f) cꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs fꢅr ꢄꢅrꢁꢆsꢁaꢁꢆꢀ-14, sꢂꢈsꢁaꢀꢄꢃ p, aꢀꢋ
late their roles in pharmacology. The suggested MRGPRX2 rodent smaꢇꢇ-mꢅꢇꢃꢄꢂꢇꢃ agꢅꢀꢆsꢁ tAn-67 wꢆꢁh MRGpRX2-taꢀgꢅ (sꢅꢇꢆꢋ ꢇꢆꢀꢃs) aꢀꢋ
ortholog MRGPRB2 (ref. 19) shares only 52% sequence identity with MRGpRb2-taꢀgꢅ (ꢋꢅꢁꢁꢃꢋ ꢇꢆꢀꢃs) ꢆꢀ ꢁhꢃ pReSto-taꢀgꢅ arrꢃsꢁꢆꢀ rꢃꢄrꢂꢆꢁmꢃꢀꢁ
MRGPRX2 (Supplementary Fig. 1a), and we could not substantiate assay (n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ). thꢃ y axꢆs shꢅws ꢁhꢃ fꢅꢇꢋ ꢄhaꢀgꢃ rꢃsꢉꢅꢀsꢃ
any proposed shared ligands except for cortistatin-14, which had ꢅꢊꢃr ꢈasaꢇ ꢇꢂmꢆꢀꢃsꢄꢃꢀꢁ sꢆgꢀaꢇ. Rlu, rꢃꢇaꢁꢆꢊꢃ ꢇꢂmꢆꢀꢃsꢄꢃꢀꢁ ꢂꢀꢆꢁs.
high-micromolar activity at MRGPRB2 (Fig. 1e,f; Supplementary Aꢇꢇ ꢃrrꢅr ꢈars rꢃꢉrꢃsꢃꢀꢁ s.ꢃ.m.
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 five opioid-related ligands: ADL-5859, sinomenine, dextrometho-
emerged that replicate these prior findings.
rphan, dextromethorphan’s metabolite dextrorphan, and the previ-
Accordingly, we chose an unbiased approach to identify reli- ously reported MRGPRX2 ligand TAN-67 (ref. 20), a delta opioid
able MRGPRX2 probes. We recently proposed parallel screening receptor agonist.
3,6
strategies using small libraries of drugs and drug-like compounds
Confirmatory concentration–response curves using the PRESTO-
as fruitful initial approaches to discover active compounds at oGP- Tango platform indicated that the five opioid-like compounds had
6
CRs . Here we screened 5,695 unique compounds for agonist activity low-micromolar potency (Fig. 2b,c). To confirm the MRGPRX2-
at three members of the MRGPRX family (MRGPRX1, MRGPRX2, Tango construct performed similarly to the unmodified wild-type
and MRGPRX4) using the PRESTO-Tango platform (see Online (WT) receptor, we tested previously reported MRGPRX2 ago-
1
3
Methods). Our strategy was to screen against three MRGPRX fam- nists TAN-67 (ref. 20), cortistatin-14 (ref. 16), substance P , and
ily receptors in parallel to find active compounds with selectivity compound 48/80 (ref. 19), and found that they all activated the
within the family.
MRGPRX2-Tango receptor at similar potencies to those reported
The screening revealed MRGPRX1 had the smallest number from G assays at the WT receptor (Fig. 2d).
αq
of activating compounds (39), followed by MRGPRX4 (54), and
To confirm MRGPRX2 G -mediated functional activity of ago-
αq
MRGPRX2 (81) (Fig. 2a). We were most interested in compounds nists, we used a tetracycline-inducible WT MRGPRX2 stable HEK-T
showing selective MRGPRX2 agonism without apparent activ- cell line to test for intracellular calcium release. Dextromethorphan,
ity at other MRGPRX receptors. Among the 67 compounds that dextrorphan, sinomenine, and TAN-67 promoted intracellular
activated MRGPRX2 but neither MRGPRX1 nor MRGPRX4 were calcium release when MRGPRX2 expression was induced by
2
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a
b
replacement of the morphine’s 3-hydroxy with gluconuride to yield
morphine-3-β-gluconuride eliminated MRGPRX2 agonist activ-
ity (Fig. 3c,d; Supplementary Table 2; structures in Fig. 3e). In
the C6 position, gluconuride or acetyl modifications to morphine
and codeine all retained agonist activity, albeit with loss in potency
compared to (−)-morphine and (−)-codeine, respectively (Fig. 3c,d;
Supplementary Table 2). These preliminary opioid structure–
activity relationships (SAR) demonstrate that larger modifications
on C6 are more tolerated than those on C3.
MRGPRX1
MRGPRX2
TAN-67
ADL5859
Sinomenine
1
00
4
6
20
67
50
9
4
3
5
0
–
12 –8
–7
–6
–5
–4
We then explored the effect of substituents on the cationic nitrogen
in the morphinans on MRGPRX2 activation. N-methyl-substituted
scaffolds, as in codeine, morphine, and metazocine, conferred recep-
tor agonism while the N-unsubstituted-norcodeine was inactive up to
100μM (Fig.3b–d;SupplementaryTable2). Morphinansandbenzo-
morphans with substituents larger than N-methyl, such as the N-allyl
of levallorphan and N-allylnormetazocine, and the N-cyclopropyl of
naltrexone and cyclazocine, were inactive at MRGPRX2 (Fig. 3a,b;
Supplementary Table 2). The inactive morphinans and benzomor-
phans showed no antagonist activity against MRGPRX2, suggesting
a tight SAR around substitution of this position.
MRGPRX4
log drug (M)
c
d
Dextrorphan
Dextromethorphan
TAN-67
TAN-67
Cortistatin-14
Substance P
1
00
100
50
0
Compound 48/80
50
0
–
12
–8
–7
–6
–5
-4
–12 –8
–7
–6
–5
–4
log drug (M)
log [drug] (M)
As previously mentioned, TAN-67 is a δ-opioid receptor agonist
known to activate MRGPRX2 (ref. 20). The active enantiomer at
the δ-opioid receptor is (−)-TAN-67 as (+)-TAN-67 displays pro-
Figure 2 | PRꢁSTꢄ-Tango Screening of MRGPRX2 reveals new agonists.
a) vꢃꢀꢀ ꢋꢆagram ꢋꢃꢉꢆꢄꢁꢆꢀg ꢉꢅꢅꢇꢃꢋ pReSto-taꢀgꢅ sꢄrꢃꢃꢀꢆꢀg aꢄꢁꢆꢊꢃs
fꢅr MRGpRX1, MRGpRX2, aꢀꢋ MRGpRX4 fꢅr aꢇꢇ hꢆꢁs wꢆꢁh grꢃaꢁꢃr ꢁhaꢀ
wꢅ-fꢅꢇꢋ aꢄꢁꢆꢊaꢁꢆꢅꢀ ꢅꢊꢃr ꢈasaꢇ (kꢀꢅwꢀ faꢇsꢃ ꢉꢅsꢆꢁꢆꢊꢃs aꢀꢋ ꢋꢂꢉꢇꢆꢄaꢁꢃs
ꢃxꢄꢇꢂꢋꢃꢋ). (b,c) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs (n = 3 ꢆꢀ
rꢆꢉꢇꢆꢄaꢁꢃ wꢃꢇꢇs fꢅr aꢇꢇ, ꢃxꢄꢃꢉꢁ Adl5859, fꢅr whꢆꢄh n = 1) fꢅr ꢁhꢃ fꢆꢊꢃ
ꢄꢅmꢉꢅꢂꢀꢋs frꢅm ꢁhꢃ sꢄrꢃꢃꢀꢆꢀg shꢅw ꢇꢅw mꢆꢄrꢅmꢅꢇar aꢄꢁꢆꢊaꢁꢆꢅꢀ ꢅf
MRGpRX2. (d) cꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃ fꢅr ꢉrꢃꢊꢆꢅꢂsꢇy ꢉꢂꢈꢇꢆshꢃꢋ
MRGpRX2 ꢉꢃꢉꢁꢆꢋꢃ agꢅꢀꢆsꢁs aꢁ ꢁhꢃ MRGpRX2-taꢀgꢅ ꢄꢅꢀsꢁrꢂꢄꢁ. y axꢃs arꢃ
shꢅwꢀ as ꢉꢃrꢄꢃꢀꢁ ꢅf tAn-67 aꢄꢁꢆꢊꢆꢁy. Aꢇꢇ ꢃrrꢅr ꢈars rꢃꢉrꢃsꢃꢀꢁ s.ꢃ.m.
(
2
3
nociceptive activity in vivo through an unidentified receptor .
We tested (+)-TAN-67 for MRGPRX2 activity in PRESTO-Tango
and calcium mobilization assays and found that (+)-TAN-67 was
ꢁ
more potent than racemic (+/−)-TAN-67 (Gα EC = 290 nM vs.
ꢁ
q
50
7
40 nM, respectively) (Supplementary Fig. 4), demonstrating that
MRGPRX2 prefers dextro-enantiomers at most chemical scaffolds,
with some exceptions. Neither (−)- nor (+)-naloxone antagonized
(
+/−)-TAN-67 agonist activity (Supplementary Fig. 5a,b), and no
other canonical opioid antagonists, including naltrindole, naltrex-
one, and β-chlornaltrexamine, inhibited (+/−)-TAN-67 activity.
tetracycline (1 μg/ml; Supplementary Fig. 2a) but not in the absence (Supplementary Fig. 5c–d). These SARs suggests that MRGPRX2,
of tetracycline (i.e., MRGPRX2 is not expressed; Supplementary despite its distant sequence relationship to the four classic opioid
Fig. 2b).
receptors (transmembrane region sequence identity no greater than
6%), is an opioid-responding oGPCR with important differences
2
Preference for dextro-enantiomers and N-methyl scaffolds in ligand recognition patterns.
As several dextrorotatory opiate ligands activated MRGPRX2, we
initially investigated ligand stereochemistry, aware that classical Prodynorphin-derived peptides activate MRGPRX2
opioid receptors prefer levorotary morphinans and benzomor- A key question is whether MRGPRX2 is also activated by endoge-
2
1
phans . We assayed levorphanol and levallorphan, enantiomers of nous opioid ligands. Accordingly, we tested a panel of 20 endogenous
the screening hits dextrorphan and dextromethorphan, respectively, opioid peptides for activity in PRESTO-Tango and intracellular cal-
for activity at MRGPRX2 in β-arrestin recruitment and calcium cium assays. MRGPRX2 was preferentially activated by prodynor-
mobilization assays. Levorphanol was approximately ten-fold less phin-derived peptides (Fig. 4a,b) and only minimally activated, if
potent at MRGPRX2 than dextrorphan, and levallorphan was com- at all, by other opioid peptides (Fig. 4c,d). The full-length dynor-
pletely inactive up to 100 μM (Fig. 3a). Likewise, the dextrorotary phin A(1–17) and several truncated prodynorphin peptides acti-
morphinan sinomenine had comparable potency to dextrorphan vated MRGPRX2 (Fig. 4a). C-terminal amino acid truncation of
and dextromethorphan at MRGPRX2 (Supplementary Table 2), dynorphin peptides progressively reduced agonist potency until the
more potent than sinomenine’s purported activity at the opioid dynorphin A(1–7) and dynorphin A(1–6) fragments, which were
22
receptors (Supplementary Fig. 3). MRGPRX2 also preferred dex- completely inactive (Fig. 4a; Supplementary Fig. 6a).
trorotary benzomorphan compounds, as (−)-cis-normetazocine To determine whether the C-terminal portion of dynorphin A
was ten-fold less potent than (+)-cis-normetazocine (Fig. 3b; was sufficient for MRGPRX2 activation, we assayed dynorphin
Supplementary Table 2). A(13–17) and dynorphin A(6–17) and found that dynorphin
To determine whether other opioids activated MRGPRX2 with A(6–17) retained minimal activity (EC = 39.6 μM) and dynor-
50
similar stereochemical preferences, we tested a panel of 11 mor- phin A(13–17) was inactive (Fig. 4b), suggesting that both the
phine and codeine analogs for activity in PRESTO-Tango and cal- N-terminal YGGF motif and C-terminal cationic tail are important
cium mobilization assays, including the most common metabolites for MRGPRX2 activation (Supplementary Fig. 6a). Dynorphin
of morphine and codeine. Both (+)- and (−)-morphine enantiomers B(1–13) and α- and β-neoendorphin also activated MRGPRX2,
activated MRGPRX2 with comparable potency in PRESTO-Tango with dynorphin B and α-neoendorphin having almost identical
and calcium assays, as did (+)- and (−)-codeine and thebaine (Fig. μM potency (Fig. 4b). Intriguingly, EC values for the dynorphin
50
3
c,d; Supplementary Table 2; structures in Fig. 3e). For the metab- peptides were right-shifted in the PRESTO-Tango assay, indicating
olites, we observed differential effects of substitutions on C3 and C6 perhaps modest G protein bias, similar to observations made for
24
positions in the morphine scaffold, as replacement of morphine’s dynorphin peptides at the κ-opioid receptor .
3
-hydroxy with a 3-methoxy (for example, codeine and thebaine)
To determine whether more closely related receptors might
25
had little effect on affinity or efficacy at MRGPRX2, whereas share peptide ligands with MRGPRX2, we used the GPCRDB
nature CHeMICaL BIOLOGY | AdvAnce online publicAtion | www.ꢀaꢁꢂrꢃ.ꢄꢅm/ꢀaꢁꢂrꢃꢄhꢃmꢆꢄaꢇꢈꢆꢅꢇꢅgy
3

NATꢀRꢁ CꢂꢁMICAꢃ BIꢄꢃꢄGꢅ dOI: 10.1038/nCHeMBIO.2334
article
a
4
3
2
1
b
4
3
2
1
e
Dextromethorphan
Dextrorphan
Levorphanol
(+)-Metazocine
Active
Inactive
(+)-cis-Normetazocine
(–)-cis-Normetazocine
(+)-N-allylnormetazocine
N
N
Levallorphan
(+)-Cyclazocine
HO
O
OH
O
HO
OH
Morphine
β-Arr EC50: 6.4 µM
Gαq EC50: 7 µM
Nalorphine
β-Arr EC50 inactive
:
Gαq EC50
:
inactive
N
N
–
12
–8
–7
–6
–5
–4
–12
–8
–7
–6
–5
–4
log [drug] (M)
log [drug] (M)
c
d
HO
HO
4
3
2
1
4
3
2
1
(+)-Codeine
(–)-Codeine
(
(
+)-Morphine
–)-Morphine
Dextrorphan
Levorphanol
β-Arr EC50: 24 µM
Gα
β-Arr EC50: 4.3 µM
Gα
6
-Acetyl-codeine
q
EC50: 6 µM
q
EC50: 14 µM
Thebaine
Morphine-6G
Morphine-3G
Codeine-6G
Nor-codeine
N
N
O
HO
HO
O
O
O
OH
HO
O
OH
HO
OH
Morphine
β-Arr EC50: 6.4 µM
Gαq EC50: 7 µM
Morphine-3G
β-Arr EC50: 28 µM
inactive
–
12
–8
–7
–6
–5
–4
–12
–8
–7
–6
–5
–4
Gαq EC50
:
log [drug] (M)
log [drug] (M)
Figure 3 | MRGPRX2 is activated by many opioid scaffolds. (a–d) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs (n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ) fꢅr sꢁrꢂꢄꢁꢂraꢇꢇy rꢃꢇaꢁꢃꢋ
ꢄꢅmꢉꢅꢂꢀꢋs, mꢅrꢉhꢆꢀaꢀs (a), ꢈꢃꢀzꢅmꢅrꢉhaꢀs (b), mꢅrꢉhꢆꢀꢃ aꢀꢋ aꢀaꢇꢅgs (c), aꢀꢋ ꢄꢅꢋꢃꢆꢀꢃ aꢀꢋ aꢀaꢇꢅgs (d), ꢆꢀ aꢀ ꢆꢀꢁraꢄꢃꢇꢇꢂꢇar ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ assay ꢆꢀ
whꢆꢄh ꢁhꢃ y axꢆs ꢆs fꢅꢇꢋ-ꢄhaꢀgꢃ ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ ꢅꢊꢃr ꢈasꢃꢇꢆꢀꢃ. errꢅr ꢈars rꢃꢉrꢃsꢃꢀꢁ s.ꢃ.m. (e) Sꢂmmary ꢅf majꢅr fꢆꢀꢋꢆꢀgs ꢆꢀ sꢁrꢂꢄꢁꢂrꢃ–aꢄꢁꢆꢊꢆꢁy rꢃꢇaꢁꢆꢅꢀshꢆꢉs
fꢅr ꢅꢉꢆꢅꢆꢋ sꢄaffꢅꢇꢋs aꢁ MRGpRX2, ꢆꢀꢄꢇꢂꢋꢆꢀg sꢆzꢃ ꢅf N-sꢂꢈsꢁꢆꢁꢂꢃꢀꢁ, sꢁꢃrꢃꢅꢄhꢃmꢆsꢁry ꢅf ꢄhꢆraꢇ ꢄꢃꢀꢁꢃrs, aꢀꢋ ꢈꢂꢇk ꢅꢀ ꢁhꢃ 3-ꢉꢅsꢆꢁꢆꢅꢀ ꢅf ꢁhꢃ mꢅrꢉhꢆꢀaꢀ.
to identify class A receptors with ≥30% sequence similarity to which shares only 23.3% sequence identity in the transmembrane
MRGPRX2 in the class A binding pocket (Supplementary Fig. 6b). regions with MRGPRX2. We calculated an initial MRGPRX2 struc-
28
We identified 14 receptors, 11 of which had established probes ture using Modeller and expanded on this using elastic network
(
10 ligands total). Three receptors were oGPCRs with no known modeling to increase the amount of sampled backbone conforma-
29
ligands. We tested the ten established ligands for activity and tions . From the initial model, we calculated 360 further structures
found that only one, somatostatin-14, activated MRGPRX2 using the elastic network modeling program (ENM); half of these
(
Supplementary Fig. 5b,d; EC = 380 nM). This result is consistent
50
with previous reports that somatostatin-14, somatostatin-28, and
16,20
cortistatin-14 are MRGPRX2 agonists . Interestingly, the canoni-
cal opioid receptors and somatostatin receptors are closely related
in binding-site-sequence space but not in small-molecule chemical
a
b 4
Dynorphin A(1–17)
Dynorphin A(1–13)
Dynorphin A(1–9)
Dynorphin A(1–8)
Dynorphin A(1–7)
Dynorphin A(1–6)
Dynorphin A(2-8)
Dynorphin A(6-17)
Dynorphin A(13-17)
Dynorphin B(1-13)
α-neoendorphin
4
3
2
1
3
2
1
26
space . Our data, along with previously proposed peptide ligands,
suggest that although MRGPRX2 can be activated by many peptide
ligands, this cannot be predicted from sequence similarity alone.
MRGPRX2 is an opioid-like oGPCR that responds to endog-
enous pro-dynorphin-derived opioid peptides and binds to many
well-known synthetic opioid agonists, but differs from classic opi-
oid receptors in its unique preference for dextromorphinans and
dextrobenzomorphans as well as its inability to be antagonized by
potent and classic opioid receptor antagonists. Although none of
these compounds are suitable as a selective probe or tool, the chemi-
cal matter identified was useful to launch an in silico campaign to
identify novel chemotypes active at MRPGRX2.
β-neoendorphin
–12
–8
–7
–6
–5
–4
–12
–8
–7
–6
–5
–4
log [drug] (M)
log [drug] (M)
c 4
d 4
Leu-enkephalin
Met-enkephalin
Dermorphin
BAM-12
β-endorphin
Endomorphin-2
Endomorphin-1
Nociceptin
3
2
1
3
2
1
BAM-22
BAM(8–22)
Structure-based docking predicts MRGPRX2-selective ligands
We next turned to our computational approach, using a 1,000-fold
larger compound library than that used in the physical screen—the
–
12
–8
–7
–6
–5
–4
–12
–8
–7
–6
–5
–4
log [drug] (M)
log [drug] (M)
over 3.7-million commercially available ‘lead-like’ molecules then Figure 4 | MRGPRX2 is preferentially activated by prodynorphin-derived
27
in the ZINC database . Our strategy was to calculate many 3D peptides. (a,b) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs (ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ wꢃꢇꢇs;
MRGPRX2 models, select those few that recapitulated the ligand n = 3 fꢅr aꢇꢇ ꢃxꢄꢃꢉꢁ dyꢀ A fragmꢃꢀꢁs ꢈꢃꢇꢅw 7 amꢆꢀꢅ aꢄꢆꢋs ꢆꢀ ꢇꢃꢀgꢁh aꢀꢋ
recognition patterns we observed experimentally, and use these α-ꢀꢃꢅꢃꢀꢋꢅrꢉhꢆꢀ fꢅr whꢆꢄh n = 2 ; sꢃꢃ Supplementary Table 2) fꢅr ꢉrꢅ-
to create a template of a final model to screen the full, larger com- ꢋyꢀꢅrꢉhꢆꢀ-ꢋꢃrꢆꢊꢃꢋ ꢉꢃꢉꢁꢆꢋꢃs ꢆꢀ MRGpRX2 tꢃꢁ-oꢀ ꢄꢃꢇꢇs, ꢆꢀ whꢆꢄh ꢁhꢃ y axꢆs
pound library for novel molecules unrelated to opioid ligands but ꢆs fꢅꢇꢋ-ꢄhaꢀgꢃ ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ ꢅꢊꢃr ꢈasꢃꢇꢆꢀꢃ. errꢅr ꢈars rꢃꢉrꢃsꢃꢀꢁ s.ꢃ.m.
selective for MRGPRX2.
(c,d) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs (ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ wꢃꢇꢇs; n = 3 fꢅr
Thegeneralapproachformodelinganddockinghasbeenrecently aꢇꢇ ꢃxꢄꢃꢉꢁ ꢀꢅꢄꢆꢄꢃꢉꢁꢆꢀ aꢀꢋ bAM(8–22) fꢅr whꢆꢄh n = 2 aꢀꢋ β-ꢃꢀꢋꢅrꢉhꢆꢀ
3
described , and we summarize it here (see Online Methods for more aꢀꢋ bAM-22 fꢅr whꢆꢄh n =1 ; sꢃꢃ Supplementary Table 2) ꢋꢃꢉꢆꢄꢁꢆꢀg
details). We first sought GPCRs of known structure with MRGPRX2 ꢀꢅꢀꢉrꢅꢋyꢀꢅrꢉhꢆꢀ-ꢋꢃrꢆꢊꢃꢋ ꢉꢃꢉꢁꢆꢋꢃs wꢆꢁh mꢆꢀꢆmaꢇ aꢄꢁꢆꢊꢆꢁy ꢄꢅmꢉarꢃꢋ ꢁꢅ
sequence similarity; intriguingly, this led to the κ-opioid receptor, ꢋyꢀꢅrꢉhꢆꢀs ꢆꢀ A aꢀꢋ b. errꢅr ꢈars rꢃꢉrꢃsꢃꢀꢁ s.ꢃ.m.
4
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NATꢀRꢁ CꢂꢁMICAꢃ BIꢄꢃꢄGꢅ dOI: 10.1038/nCHeMBIO.2334
article
Asp184 is necessary for opioid activation
WT
4
N
O
E164Q
D184N
3
2
Sample
binding sites
1
Glu164
Align and model MRGPRX2
with DOCK
on the κ-opioid receptor
–10
–8
–6
–4
Asp184
(
25% identity)
log [DXM] (M)
4
WT
D184N
E164Q
N
N
N
3
2
1
N
N
N
N
N
N
N
+
N
N
Medicinal
chemistry
N
N
N
0
–
12
–8
–7
–6
–5
DOCK ~3.7
million molecules
ZINC-9232 is a
specific 3,000-nM agonist
(R)-ZINC-3573
760 nM
(S)-ZINC-3573
>100,000 nM
log [(R)-ZINC-3573] (M)
Figure 5 | In silico MRGPRX2 homology modeling predicts a selective agonist. Wꢅrkfꢇꢅw ꢋꢃꢉꢆꢄꢁꢆꢀg MRGpRX2 hꢅmꢅꢇꢅgy mꢅꢋꢃꢇ ꢄꢅꢀsꢁrꢂꢄꢁꢆꢅꢀ (ꢁꢅꢉ ꢇꢃfꢁ)
fꢅꢇꢇꢅwꢃꢋ ꢈy ꢆꢋꢃꢀꢁꢆfꢆꢄaꢁꢆꢅꢀ ꢅf a ꢉꢂꢁaꢁꢆꢊꢃ ꢈꢆꢀꢋꢆꢀg sꢆꢁꢃ (ꢁꢅꢉ mꢆꢋꢋꢇꢃ) ꢁhaꢁ was ꢄꢅꢀfꢆrmꢃꢋ ꢈy ꢁꢃsꢁꢆꢀg ꢁhꢃ mꢂꢁaꢁꢆꢅꢀs e164Q aꢀꢋ d184n (ꢁꢅꢉ rꢆghꢁ, aꢊꢃragꢃ ꢋꢅsꢃ
rꢃsꢉꢅꢀsꢃ, n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ, shꢅwꢀ wꢆꢁh ꢋꢃxꢁrꢅmꢃꢁhꢅrꢉhaꢀ (dXM). thꢃꢀ, ~3.7 mꢆꢇꢇꢆꢅꢀ mꢅꢇꢃꢄꢂꢇꢃs wꢃrꢃ ꢋꢅꢄkꢃꢋ ꢁꢅ ꢉrꢃꢋꢆꢄꢁ ꢁhꢃ agꢅꢀꢆsꢁ Zinc-9232 (ꢈꢅꢁꢁꢅm
ꢇꢃfꢁ). Fꢂrꢁhꢃr ꢆꢁꢃraꢁꢆꢅꢀ aꢀꢋ ꢁꢃsꢁꢆꢀg rꢃꢊꢃaꢇꢃꢋ ꢁhꢃ ꢁꢅꢅꢇ aꢀꢋ sꢃꢇꢃꢄꢁꢆꢊꢃ ꢄꢅmꢉꢅꢂꢀꢋs (S)- aꢀꢋ (R)-Zinc-3573 (ꢈꢅꢁꢁꢅm mꢆꢋꢋꢇꢃ aꢀꢋ rꢆghꢁ). dꢅꢄkꢆꢀg ꢉꢅsꢃ ꢅf
sꢃꢇꢃꢄꢁꢆꢊꢃ ꢄꢅmꢉꢅꢂꢀꢋ ꢆs sꢂꢉꢉꢅrꢁꢃꢋ wꢆꢁh mꢂꢁaꢁꢆꢅꢀ ꢃxꢉꢃrꢆmꢃꢀꢁs ꢆꢀ pReSto-taꢀgꢅ (ꢈꢅꢁꢁꢅm rꢆghꢁ, aꢊꢃragꢃ ꢋꢅsꢃ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃ, aꢊꢃragꢃ ꢅf n = 4 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ).
errꢅr ꢈars ꢅꢀ graꢉhs shꢅwꢀ ꢆs rꢃꢉrꢃsꢃꢀꢁaꢁꢆꢊꢃ ꢅf s.ꢃ.m.
structures had an extracellular disulfide bond imposed and half related opioids in the PRESTO-Tango assay (Fig. 5; Supplementary
did not. Each of these was expanded three-fold with Modeller (i.e., Fig. 7a). We then tested the importance of the length of the acidic
using the ENM models as templates for Modeller). Overall, 1,080 residue via E164D and D184E mutations and observed that E164D
models were calculated.
increased the efficacy and reduced the potency of both dextrorphan
We docked the 5,695 unique compounds from the physical and morphine (Supplementary Fig. 7a,c) whereas D184E ablated
screening against all 1,080 models (i.e., over 6 million compound– activity of all opioids tested (Fig. 5; Supplementary Fig. 7a–d).
12
receptor docking calculations, and over 10 individual configura- The substitutions support the importance of the proposed ionic
tions of the library molecules in the receptor). We looked for mod- interaction and the putative binding site predicted by the in silico
4.60
5.38
els that best enriched the discovered opioid agonists over the vast model, suggesting both Glu164 and Asp184 are necessary for
number of inactive decoy molecules. The top-enriching models MRGPRX2 opioid activation.
were inspected visually for binding poses that captured sensible
As an aside, dynorphin A(1–13) activity was lost with the D184N
ion-pairing interactions with the conserved aminergic group of the but not the E164Q mutation, suggesting that only one of these resi-
opioid agonists. This prioritized a model that had a background- dues is required for the opioid–peptide interaction (Supplementary
corrected area under the receiver operating characteristic curve Fig. 7d). We modeled the putative MRGPRX2–dynorphin binding
30,31
(
logAUC), for enrichment
of 8.42—which happened to be the site by overlaying the MRGPRX2 homology model with the previ-
35
top-enriching model (this is not always the case, as sometimes the ouslypublishedκ-opioidreceptordockedwithdynorphinA(1–13) .
32
top-enriching model does not have sensible geometries , though The N-terminal Tyr1 of dynorphin A(1–13) is accommodated
usually the model chosen is among the best enriching models). within a negatively charged aromatic pocket, whereas Arg7 and
5.38
Residues within 6 Å of the dextromethorphan pose were mini- Phe4 appear to interact with Asp184 (Supplementary Fig. 8a–c).
5
.38
mized around the docked ligand with protein local optimization The predicted orientation of dynorphin A(1–13) at Asp184 and
33
4.60
program (PLOP) and the 5,695 molecules redocked. Two rounds Glu164 shows a clear preference for charged interactions between
5.38
of docking and PLOP-based refinement led to the final modeled dynorphin A and Asp184 , an outcome supported by the muta-
receptor conformation, which predicts the opioid agonists making genesis results (Supplementary Fig. 8d).
4.60
5.38
ionic interactions with Glu164 and Asp184 (Fig. 5, showing
With experimental support for the MRGPRX2 comparative
dextromethorphan); superscripts indicate Ballesteros–Weinstein model, we proceeded to virtually screen the ZINC library for new
34
residue numbering based our MGPRX2 alignment with κ-opioid ligands. Over 3.7 million commercially available molecules were
receptor (Supplementary Fig. 6c). The exact rotamer state of these docked, each in an average of 926 orientations and 558 conforma-
residues allowed some flexibility; i.e., several different rotamers of tions; overall, 1.9 trillion docked complexes were sampled and scored
the Asp and Glu led to productive placement of the known agonists (for details, see Online Methods). We selected, purchased, and
(Supplementary Fig. 7e).
assayed 20 compounds from the 0.13% of top-ranked compounds
To test the predicted ion pair between the receptor and the for activity at MRGPRX2. As there is little scoring difference among
4.60
5.38
ligands, we made a series of mutations at Glu164 and Asp184
.
the top-ranked molecules, we chose molecules following our usual
36
E164Q and D184N substitutions retained steric properties of the strategy and prioritized those with diverse, nonopioid chemo-
WT residues but removed their negative charges, and both resulted types. We deprioritized molecules with apparently high internal
in a loss of activity for dextromethorphan, (−)-morphine, and energies, which the docking scoring function does not well account
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5

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36
for , and prioritized those that made ionic interactions with the
a
b
5.38
4.60
Asp184 and Glu164 pair, similarly to the modeled opioids.
Of the 20 ZINC compounds tested, one, ZINC-72469232 (1, here
on referred to as ZINC-9232), had substantial activity below
ZINC-9232
ZINC-3573
6
4
2
0
0
0
0
ZINC-3573 (S)
ZINC-3573 (R)
3
2
1
1
0 μM in PRESTO-Tango and calcium release assays (Fig. 5).
To improve potency, 22 analogs of ZINC-9232 topologically simi-
lar to the lead were obtained and tested. Of these 22, seven were
active, with the most potent being ZINC-72453573 (2, from here on
referred to as ZINC-3573), having submicromolar potency (EC =
–20
–12
–8 –7 –6 –5 –4
log [drug] (M)
–12
–8
–7
–6
–5
–4
log [drug] (M)
5
0
7
60 nM, Fig. 5; Supplementary Fig. 9). We then assessed whether
c
d
siRNA 2 + 3
NT
(+/–)-TAN-67
5.38
ZINC-3573 interacts with the predicted residues Asp184 and
*
(–)-Cyclazocine
(+)-Morphine
2
.0
*
4.60
*
*
Glu164 using the E164Q and D184N mutations and found that
both eliminated ZINC-3573 agonism at MRGPRX2 (Fig. 5), consis-
tent with the modeling.
(–)-Morphine
3
2
1
Dyn A(1–13)
1
.5
ZINC-3573 as a chemical probe for MRGPRX2
1
.0
To confirm MRGPRX2 selectivity, we tested ZINC-9232 and
ZINC-3573 activity at 315 other human GPCRs using our PRESTO-
Tango GPCRome assay. ZINC-9232 and ZINC-3573 showed
minimal agonist efficacy at receptors other than MRGPRX2 at
–
12
–8 –7 –6 –5 –4
log [drug] (M)
TAN-67
Dyn A 1-13
ZINC-3573
(
–)-Morphine
e
g
f
100
80
60
40
IgE
No IgE
Salvinorin A
DADLE
DAMGO
DMSO
(+/–)-TAN-67
BW373U86
1
0 μM (Supplementary Fig. 10a,b). ZINC-9232 was also screened
3
2
1
against a panel of 97 representative human kinases using the
DiscoveRX KINOMEscan diversity panel; only three kinases
were modestly inhibited, with IC values between 20–30 μM
50
(
Supplementary Fig. 11).
20
These results encouraged us to synthesize both enantiomers of
0
the more potent ZINC-3573, originally supplied as a racemic mix-
ture, in an effort to create a pair of differentially active molecules
that could be used jointly as a chemical probe pair (Supplementary
Note). The R-isomer (3) retained an EC of 740 nM in PRESTO-
–12
–8 –7 –6 –5 –4
log [drug] (M)
TAN-67
DMSO
IONO
(+/–)-TAN-67
(+)-MOR
–)-MOR
h
BW373U86
(+/–)-TAN-67
Sal A
DAMGO
DMSO
50
50
25
0
50
25
0
(
Tango and a similar EC value of 1 μM in the calcium mobilization
50
Dyn A(1–13)
DMSO
assay (Supplementary Fig. 9). The S-isomer (4) had little activ-
ity below concentrations of 100 μM (Supplementary Fig. 9). The
separate activity of the enantiomers makes them highly attractive
probes, as one can distinguish MRGPRX2 activity (owing to the
R-isomer) from general, nonspecific activity because of the scaffold
and its physical properties (due to either isomer’s attributes). Each
enantiomer was further tested using the PRESTO-Tango GPCRome
–12
–8
–7
–6
–5
–4
–12
–8
–7
–6
–5
–4
log [drug] (M)
log [drug] (M)
screening platform (Supplementary Fig. 10c,d), which indicated Figure 6 | MRGPRX2 mediates intracellular calcium release and
that 21 receptors may be activated two-fold or more with (R)- and degranulation in the ꢃAD2 human mast cell line. (a) Aꢊꢃragꢃ
(
S)-ZINC-3573; however, subsequent concentration–response stud- ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ rꢃsꢉꢅꢀsꢃ fꢅr MRGpRX2-sꢃꢇꢃꢄꢁꢆꢊꢃ agꢅꢀꢆsꢁs Zinc-9232 aꢀꢋ
ies showed that no other receptor was substantially activated by Zinc-3573 ꢆꢀ ꢁhꢃ ꢄaꢇꢄꢆꢂm mꢅꢈꢆꢇꢆzaꢁꢆꢅꢀ assay ꢆꢀ lAd2 masꢁ ꢄꢃꢇꢇs
either compound (Supplementary Fig. 11).
(n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ). (b) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ rꢃsꢉꢅꢀsꢃ (n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ)
fꢅr MRGpRX2 ꢉrꢅꢈꢃs Zinc-3573 (R) aꢀꢋ Zinc-3573 (S) ꢆꢀ ꢁhꢃ
β-hꢃxꢅsamꢆꢀꢆꢋasꢃ ꢋꢃgraꢀꢂꢇaꢁꢆꢅꢀ assay ꢆꢀ lAd2 ꢄꢃꢇꢇs. (c). bar graꢉh
MRGPRX2 agonists induce degranulation in mast cells
MRGPRX2 has been implicated in IgE-independent inflammatory ꢋꢃꢉꢆꢄꢁꢆꢀg fꢅꢇꢋ ꢄhaꢀgꢃ ꢉꢃrꢄꢃꢀꢁ ꢋꢃgraꢀꢂꢇaꢁꢆꢅꢀ (ꢈasꢃꢇꢆꢀꢃ ꢆs aꢊꢃragꢃ dMSo
responses in mast cells using peptide ligands and other nonspecific ꢅf aꢇꢇ ꢉꢇaꢁꢃs) ꢆꢀꢋꢂꢄꢃꢋ ꢈy ec80 ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ ꢅf ꢋrꢂg fꢅꢇꢇꢅwꢆꢀg 25 ꢀM
13,15,19
MRGpRX2 sꢆRnA ꢁraꢀsfꢃꢄꢁꢆꢅꢀ. nt, ꢀꢅꢀ-ꢁargꢃꢁꢆꢀg ꢉꢅꢅꢇ; sꢆRnA 2 + 3,
agonists
, prompting us to investigate whether MRGPRX2-
selective agonists induce degranulation and mobilize intracellular MRGpRX2 sꢆRnA ꢉꢅꢅꢇ. Sꢁaꢁꢆsꢁꢆꢄs wꢃrꢃ ꢄaꢇꢄꢂꢇaꢁꢃꢋ ꢂsꢆꢀg ꢁwꢅ-way AnovA
calcium in the LAD2 human mast cell line. ZINC-9232 and ZINC- wꢆꢁh a Sꢆꢋak post hoc ꢁꢃsꢁ (P < 0.05 = *; P = 0.031, 0.031, 0.033, aꢀꢋ 0.017
3
573 induced intracellular calcium release and degranulation in fꢅr tAn, ꢋyꢀꢅrꢉhꢆꢀ A (dyꢀ A), mꢅrꢉhꢆꢀꢃ, aꢀꢋ Zinc-3573, rꢃsꢉꢃꢄꢁꢆꢊꢃꢇy).
LAD2 mast cells at comparable potencies to calcium and β-arrestin n = 3 fꢅr aꢇꢇ ꢃxꢄꢃꢉꢁ Zinc-3573, fꢅr whꢆꢄh n = 2. Aꢇꢇ rꢃꢉꢇꢆꢄaꢁꢃs ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ wꢃꢇꢇs.
assays performed in MRGPRX2-expressing HEK-T cells (Fig. 6a,b). (d) cꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ ꢄꢂrꢊꢃs fꢅr MRGpRX2-aꢄꢁꢆꢊaꢁꢆꢀg ꢅꢉꢆꢅꢆꢋ ꢇꢆgaꢀꢋs
For the stereochemical pair, only (R)-ZINC-3573 promoted degran- aꢀꢋ ꢁhꢃ sꢁrꢂꢄꢁꢂraꢇꢇy rꢃꢇaꢁꢃꢋ, κ-ꢅꢉꢆꢅꢆꢋ rꢃꢄꢃꢉꢁꢅr ꢇꢆgaꢀꢋ (−)-ꢄyꢄꢇazꢅꢄꢆꢀꢃ ꢆꢀ ꢁhꢃ
ulation in LAD2 mast cells (Fig. 6b), consistent with the HEK-T ꢄaꢇꢄꢆꢂm mꢅꢈꢆꢇꢆzaꢁꢆꢅꢀ assay (n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ). (e) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ
in vitro activity. As expected, MRGPRX2 siRNA (short interfering rꢃsꢉꢅꢀsꢃ ꢆꢀ ꢆꢀꢁraꢄꢃꢇꢇꢂꢇar ꢄaꢇꢄꢆꢂm rꢃꢇꢃasꢃ wꢆꢁh MRGpRX2-aꢄꢁꢆꢊaꢁꢆꢀg
RNA) significantly reduced (R/S)-ZINC-3573-induced degranula- (+/−)-tAn-67 aꢀꢋ ꢄaꢀꢅꢀꢆꢄaꢇ ꢅꢉꢆꢅꢆꢋ rꢃꢄꢃꢉꢁꢅr ꢇꢆgaꢀꢋs dAdle, dAMGo,
tion (Fig. 6c; P = 0.01).
saꢇꢊꢆꢀꢅrꢆꢀ A, aꢀꢋ bW373u86 (n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ). (f) bar graꢉh ꢋꢃꢉꢆꢄꢁꢆꢀg
We then considered whether the opioid ligands might activate ꢈasꢃꢇꢆꢀꢃ ꢀꢅrmaꢇꢆzꢃꢋ ꢉꢃrꢄꢃꢀꢁ ꢋꢃgraꢀꢂꢇaꢁꢆꢅꢀ ꢆꢀꢋꢂꢄꢃꢋ fꢅr ꢃaꢄh 10 μM
endogenously expressed MRGPRX2 to induce degranulation in (+/−)-tAn-67, dMSo, aꢀꢋ ꢄaꢇꢄꢆꢂm ꢆꢅꢀꢅꢉhꢅrꢃ ꢆꢅꢀꢅmyꢄꢆꢀ (iono) ꢆꢀ ꢁhꢃ
mast cells. This is important, in part, because of the well-known ꢉrꢃsꢃꢀꢄꢃ ꢅf aꢈsꢃꢀꢄꢃ ꢅf ꢈꢆꢅꢁꢆꢀ-ꢇaꢈꢃꢇꢃꢋ ige aꢀꢁꢆꢈꢅꢋꢆꢃs. (sꢁrꢃꢉꢁaꢊꢆꢋꢆꢀ ꢆꢀ aꢇꢇ
but enigmatic ‘histamine flush’ associated with many opioids, which wꢃꢇꢇs, aꢊꢃragꢃ ꢅf n = 2 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ.) (g) Aꢊꢃragꢃ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ–rꢃsꢉꢅꢀsꢃ
is not due to engagement of classical opioid receptors. We found ꢄꢂrꢊꢃs fꢅr MRGpRX2-aꢄꢁꢆꢊaꢁꢆꢀg ꢅꢉꢆaꢁꢃs (n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ) ꢆꢀ ꢁhꢃ
that (+/−)-TAN-67, (+)-morphine, (−)-morphine, and dynor- β-hꢃxꢅsamꢆꢀꢆꢋasꢃ ꢋꢃgraꢀꢂꢇaꢁꢆꢅꢀ assay. (h) dꢃgraꢀꢂꢇaꢁꢆꢅꢀ ꢄꢅꢀꢄꢃꢀꢁraꢁꢆꢅꢀ
phin A(1–13) produced robust intracellular calcium release in the rꢃsꢉꢅꢀsꢃ wꢆꢁh ꢄaꢀꢅꢀꢆꢄaꢇ ꢅꢉꢆꢅꢆꢋ rꢃꢄꢃꢉꢁꢅr agꢅꢀꢆsꢁs aꢀꢋ MRGpRX2-aꢄꢁꢆꢊaꢁꢆꢀg
mast cells, while the structurally similar κ-opioid receptor agonist (+/−)-tAn-67, (n = 3 ꢆꢀ ꢁrꢆꢉꢇꢆꢄaꢁꢃ). Aꢇꢇ ꢃrrꢅr ꢈars ꢋꢃmꢅꢀsꢁraꢁꢃ s.ꢃ.m.
6
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(
(
−)-cyclazocine, which does not activate MRGPRX2, did not
The micromolar potency of pro-dynorphin peptides at MRGPRX2
Fig. 6d). Correspondingly, structurally unrelated agonists for suggests that in vivo receptor activation might require them to
the κ, δ, and μ opioid receptors (salvinorin A, BW373U86, and be in close proximity to high local concentrations of dynorphin.
DAMGO, respectively) were inactive (Fig. 6e). To determine whether Local synaptic concentrations of neuropeptides can reach millimo-
4
4
MRGPRX2-activating opioids induce degranulation, we first tested lar range , and dynorphin is expressed in MRGPRX2-expressing
4
5–47
1
0 μM (+/−)-TAN-67 in the presence or absence of biotin-labeled regions . At such concentrations, dynorphin could activate syn-
IgE antibodies and found that with or without IgE-biotin antibody aptic GPCRs or those expressed in mast cells, which can localize to
48
activation via streptavidin, the MRGPRX2 ligand (+/−)-TAN-67 nerve terminals . Thus, it is conceivable that MRGPRX2 is exposed
produced 70% degranulation, indicating that MRGPRX2-mediated to activating concentrations of dynorphin in vivo.
degranulation is IgE-independent (Fig. 6f). MRGPRX2 agonists
Structural modeling enabled the discovery of a new scaffold,
(
+/−)-TAN-67, (+)-morphine, (−)-morphine, and dynorphin represented by ZINC-9232 and ZINC-3573, unrelated to classical
A(1–13) all promoted degranulation in LAD2 cells (Fig. 6g), while opioids topologically and structurally. The unusual specificity of
the classical opioid agonists salvinorin A, BW373U86 and DAMGO ZINC-3573 against essentially the entire GPCRome and the relevant
did not (Fig. 6h). 10 μM naloxone pretreatment did not influence kinome, and the availability of an inactive enantiomer, makes this
the EC or efficacy of MRGPRX2 opioid agonists (Supplementary molecule a uniquely useful MRGPRX2 probe. Methodologically, we
50
Fig. 12a,b), whereas MRGPRX2 siRNA significantly attenuated previously used the combination of an initial physical compound
(
+/−)-TAN-67-, dynorphin A(1–13)-, and (−)-morphine-induced screen followed by a much larger docking screen to discover probes
3
degranulation (Fig. 6c; Supplementary Fig. 12c,d). These data sug- for oGPCRs . Identification of ZINC-3573 by a similar approach
gest that MRGPRX2, not the canonical opioid receptors, mediates against a wholly different receptor family suggests this approach has
opioid-induced degranulation in human mast cells.
broad utility in structure-based drug design.
The modeled MRGPRX2 provided intriguing insights to this
receptor’s unique opioid pharmacology. MRGPRX2 shares no
DISCꢀSSIꢄN
Our MRGPRX2 results underscore two major findings. First, the more than 26% sequence identity with the μ-, κ-, δ-, or nociceptin-
oGPCR MRGPRX2 responds to opioid drugs and endogenous opioid receptors, and its modeled orthosteric binding differs from
pro-dynorphin peptides at potentially physiologically relevant that of the canonical opioid receptors. The cationic nitrogen of the
concentrations and mediates opioid-induced degranulation in morphinans is recognized by an aspartate in the opioid receptors,
a human mast cell line. Although this receptor is not in the opi- located in transmembrane (TM) helix 3, position 3.32. The residue
oid receptor family by sequence similarity, its agonism by opioid for cationic nitrogen recognition in MRGPRX2 appears instead to
5
.38
drugs and peptides qualifies it as an atypical opioid receptor that be in TM5, Asp184 (see Supplementary Fig. 6c). Many other
responds to morphinan-based opioids and transmitters. A second recognition residues do not overlap in sequence or structural
5
.38
key result is the structure-based discovery of the selective, submi- placement. Indeed, the entire region surrounding Asp184 and
4
.60
cromolar MRGPRX2 agonist (R)-ZINC-3573. This agonist pro- Glu164 of the MRGPRX2 putative binding site is closed off
3
.33
motes degranulation in mast cells and has no measurable agonism at from ligand contacts in the classical opioid binding site by Tyr
—
over 315 other GPCRs, and the parent scaffold has little activ- a smaller Thr110 in MRGPRX2. These seeming mismatches
ity against 97 representative kinases. The inactive enantiomer reflect that different receptor environments can recognize related
(
S)-ZINC-3573 and the active (R)-ZINC3573 are an effective and ligand chemistry and the capacity for ligands to interact with
26,49
internally controlled probe pair for investigating the biology of receptors from different evolutionary families
this intriguing primate-exclusive receptor. The probes will be G protein coupling.
and different
made available to the community from Sigma-Aldrich (#SML1699
and #SML1700).
Certain caveats merit airing. We have not determined the struc-
ture of MRGPRX2 in complex with any of the ligands discussed
SAR results show MRGPRX2 is distinct from the canonical here, and the modeling that we have used to interpret activity and to
opioid receptors. MRGPRX2 prefers dextro-enantiomers and predict new molecules—however successfully—must remain tenta-
N-methyl-substituted opioid scaffolds, whereas opioid receptors tive. Until the opioid actions at this receptor can be probed in vivo,
21,37
prefer levo-enantiomer opioids
and tolerate a wider array of so, too, must the physiological implications. We note that a prior
N-substituents. In further contradistinction to the canonical opi- study also suggested that MRGPRX2 is a novel “morphine recep-
50
oid receptors, MRGPRX2 is Gα rather than Gα coupled. Thus, tor” that mediates some of morphine’s analgesic activity . Whereas
q
i
MRGPRX2 may be classified as an atypical opioid-recognizing this report certainly shares some similarity with our observations,
receptor, arguably a better example of convergent evolution within we find, in contrast, that MRGPRX2 is more potently activated by
the GPCR family than of the divergent evolution that relates the (+)-, rather than (−)-, morphine, suggesting MRGRPX2 is unlikely
3
7,51
four canonical opioid receptors.
to confer analgesia, as (+)-morphine is devoid of such activity
.
MRGPRX2 activation by opiates may be physiologically and
MRGPRX2 is a novel Gα -coupled opioid-like receptor acti-
q
therapeutically relevant. We found that MRGPRX2, not the canoni- vated by endogenous prodynorphin-derived peptides and opioid
cal opioid receptors, mediates morphine and dynorphin A(1–13)- compounds, including FDA-approved drugs and their metabolites.
induced mast cell degranulation. Morphine and structurally simi- The discovery of selective and relatively potent MRGPRX2 agonist
3
8,39
lar analgesics induce mast cell histamine release
in humans, (R)-ZINC-3573 and its inactive S-isomer provides researchers with
4
0
resulting in pruritus , vasodilation, and hypotension that is poorly a chemical probe pair to specifically modulate this receptor, illumi-
3
8,41
reversed by naloxone ; these effects are not seen for opioid anal- nating its role in pathological reactions such as itch and potentially
3
9
gesics lacking MRGPRX2 activity, such as fentanyl . MRGPRX2 revealing a path for therapeutic design.
may also be involved in the efficacy of sinomenine, which is used
to treat rheumatoid arthritis because of its histamine-releasing rꢀꢁꢀꢂvꢀd 11 augusꢃ 2016; ꢄꢁꢁꢀpꢃꢀd 22 Dꢀꢁꢀmbꢀꢅ 2016;
4
2
pubꢆꢂshꢀd onꢆꢂnꢀ 13 Mꢄꢅꢁh 2017
properties . Dextromethorphan’s potency at MRGPRX2 is also
greater than or equal to reported K values for μ, κ, δ, NMDA,
and σ receptors , suggesting that MRGPRX2 could contribute to MꢁTꢂꢄDS
i
2
1
2
dextromethorphan’s side-effect profile, which includes itch at high Methods, including statements of data availability and any associated
doses . Definitive studies in nonhuman primates are needed to accession codes and references, are available in the online version
4
3
address these hypotheses.
of the paper.
nature CHeMICaL BIOLOGY | AdvAnce online publicAtion | www.ꢀaꢁꢂrꢃ.ꢄꢅm/ꢀaꢁꢂrꢃꢄhꢃmꢆꢄaꢇꢈꢆꢅꢇꢅgy
7

NATꢀRꢁ CꢂꢁMICAꢃ BIꢄꢃꢄGꢅ dOI: 10.1038/nCHeMBIO.2334
article
rꢁfꢁꢅꢁꢃcꢁꢂ
33. Jacobson, M.P., Friesner, ꢀ.A., Xiang, Z. & Honig, B. On the role of the
crystal environment in determining protein side chain conformations.
J. Mol. Biol. 320, 597–608 (2002).
1.
Allen, J.A. & ꢀoth, B.L. Strategies to discover unexpected targets for drugs
active at G protein-coupled receptors. Annu. ꢀev. Pharmacol. Toxicol. 51,
117–144 (2011).
34. Ballesteros, J.A. & Weinstein, H. Integrated methods for the construction of
three-dimensional models and computational probing of structure–function
relations in G-protein-coupled receptors. Methods Neurosci. 25, 366–428 (1995).
35. O’Connor, C. et al. NMꢀ structure and dynamics of the agonist dynorphin
peptide bound to the human ꢁappa opioid receptor. Proc. Natl. Acad. Sci.
USA 112, 11852–11857 (2015).
2
3
4
.
.
.
Overington, J.P., Al-Laziꢁani, B. & Hopꢁins, A.L. How many drug targets are
there? Nat. ꢀev. Drug Discov. 5, 993–996 (2006).
Huang, X.P. et al. Allosteric ligands for the pharmacologically darꢁ receptors
GPꢀ68 and GPꢀ65. Nature 527, 477–483 (2015).
ꢀasꢁ-Andersen, M., Masuram, S. & Schiöth, H.B. e druggable genome:
evaluation of drug targets in clinical trials suggests major shif s in molecular
class and indication. Annu. ꢀev. Pharmacol. Toxicol. 54, 9–26 (2014).
Fredriꢁsson, ꢀ. & Schiöth, H.B. e repertoire of G-protein-coupled receptors
in fully sequenced genomes. Mol. Pharmacol. 67, 1414–1425 (2005).
ꢂroeze, W.ꢂ. et al. PꢀESTO-Tango as an open-source resource for
interrogation of the druggable human GPCꢀome. Nat. Struct. Mol. Biol. 22,
36. Irwin, J.J. & Shoichet, B.ꢂ. Docꢁing screens for novel ligands conferring new
biology. J. Med. Chem. 59, 4103–4120 (2016).
5
6
.
.
37. Jacquet, Y.F., ꢂlee, W.A., ꢀice, ꢂ.C., Iijima, I. & Minamiꢁawa, J. Stereospecific
and nonstereospecific effects of (+)- and (−)-morphine: evidence for a new
class of receptors? Science 198, 842–845 (1977).
38. Baldo, B.A. & Pham, N.H. Histamine-releasing and allergenic properties
of opioid analgesic drugs: resolving the two. Anaesth. Intensive Care 40,
216–235 (2012).
3
62–369 (2015).
7
8
.
.
Ngo, T. et al. Identifying ligands at orphan GPCꢀs: current status using
structure-based approaches. Br. J. Pharmacol. 173, 2934–2951 (2016).
Isberg, V. et al. Computer-aided discovery of aromatic l-α-amino acids as
agonists of the orphan G protein-coupled receptor GPꢀ139. J. Chem. Inf.
Model. 54, 1553–1557 (2014).
39. ꢀosow, C.E., Moss, J., Philbin, D.M. & Savarese, J.J. Histamine release during
morphine and fentanyl anesthesia. Anesthesiology 56, 93–96 (1982).
40. ꢂumar, ꢂ. & Singh, S.I. Neuraxial opioid-induced pruritus: an update.
J. Anaesthesiol. Clin. Pharmacol. 29, 303–307 (2013).
41. Hutchinson, M.ꢀ. et al. Exploring the neuroimmunopharmacology of opioids:
an integrative review of mechanisms of central immune signaling and their
implications for opioid analgesia. Pharmacol. ꢀev. 63, 772–810 (2011).
42. Yamasaꢁi, H. Pharmacology of sinomenine, an anti-rheumatic alꢁaloid from
Sinomenium acutum. Acta Med. Okayama 30, 1–20 (1976).
43. Zajac, M. et al. [ꢀecreational usage of dextromethorphan—analysis based on
internet users experiences]. Przegl. Lek. 70, 525–527 (2013).
44. Scimemi, A. & Beato, M. Determining the neurotransmitter concentration
profile at active synapses. Mol. Neurobiol. 40, 289–306 (2009).
45. Podvin, S., Yaꢁsh, T. & Hooꢁ, V. e emerging role of spinal dynorphin in
chronic pain: a therapeutic perspective. Annu. ꢀev. Pharmacol. Toxicol. 56,
511–533 (2016).
9
1
1
.
Mason, J.S., Bortolato, A., Congreve, M. & Marshall, F.H. New insights from
structural biology into the druggability of G-protein-coupled receptors.
Trends Pharmacol. Sci. 33, 249–260 (2012).
0. Zylꢁa, M.J., Dong, X., Southwell, A.L. & Anderson, D.J. Atypical expansion in
mice of the sensory neuron-specific Mrg G-protein-coupled receptor family.
Proc. Natl. Acad. Sci. USA 100, 10043–10048 (2003).
1. Dong, X., Han, S., Zylꢁa, M.J., Simon, M.I. & Anderson, D.J. A diverse
family of GPCꢀs expressed in specific subsets of nociceptive sensory neurons.
Cell 106, 619–632 (2001).
1
1
2. Lembo, P.M. et al. Proenꢁephalin A gene products activate a new family of
sensory neuron-specific GPCꢀs. Nat. Neurosci. 5, 201–209 (2002).
3. Tatemoto, ꢂ. et al. Immunoglobulin E-independent activation of mast
cell is mediated by Mrg receptors. Biochem. Biophys. ꢀes. Commun. 349,
46. Sweetnam, P.M., Neale, J.H., Barꢁer, J.L. & Goldstein, A. Localization of
immunoreactive dynorphin in neurons cultured from spinal cord and dorsal
root ganglia. Proc. Natl. Acad. Sci. USA 79, 6742–6746 (1982).
47. ꢀojewsꢁa, E., Maꢁuch, W., Przewlocꢁa, B. & Miꢁa, J. Minocycline prevents
dynorphin-induced neurotoxicity during neuropathic pain in rats.
Neuropharmacology 86, 301–310 (2014).
1322–1328 (2006).
1
4. ꢂamohara, M. et al. Identification of MrgX2 as a human G-protein-coupled
receptor for proadrenomedullin N-terminal peptides. Biochem. Biophys. ꢀes.
Commun. 330, 1146–1152 (2005).
1
1
1
5. Subramanian, H. et al. β-Defensins activate human mast cells via Mas-related
gene X2. J. Immunol. 191, 345–352 (2013).
48. Bienenstocꢁ, J. et al. Mast cell/nerve interactions in vitro and in vivo.
Am. ꢀev. ꢀespir. Dis. 143, S55–S58 (1991).
6. ꢀobas, N., Mead, E. & Fidocꢁ, M. MrgX2 is a high potency cortistatin receptor
expressed in dorsal root ganglion. J. Biol. Chem. 278, 44400–44404 (2003).
7. Maliꢁ, L. et al. Discovery of non-peptidergic MrgX1 and MrgX2 receptor
agonists and exploration of an initial SAꢀ using solid-phase synthesis.
Bioorg. Med. Chem. Lett. 19, 1729–1732 (2009).
49. Barelier, S., Sterling, T., O’Meara, M.J. & Shoichet, B.ꢂ. e recognition of
identical ligands by unrelated proteins. ACS Chem. Biol. 10, 2772–2784 (2015).
50. Aꢁuzawa, N., Obinata, H., Izumi, T. & Taꢁeda, S. Morphine is an exogenous
ligand for MrgX2, a G-protein-coupled receptor for cortistatin. J. Cell Animal
Biol. 2, 004–009 (2007).
51. Wu, H.E., Schwasinger, E.T., Terashvili, M. & Tseng, L.F. dextro-Morphine
attenuates the morphine-produced conditioned place preference via the
sigma(1) receptor activation in the rat. Eur. J. Pharmacol. 562, 221–226 (2007).
1
8. Johnson, T. & Siegel, D. Complanadine A, a selective agonist for the
Mas-related G protein-coupled receptor X2. Bioorg. Med. Chem. Lett. 24,
3512–3515 (2014).
1
2
9. McNeil, B.D. et al. Identification of a mast-cell-specific receptor crucial for
pseudo-allergic drug reactions. Nature 519, 237–241 (2015).
0. Southern, C. et al. Screening β-arrestin recruitment for the identification of
natural ligands for orphan G-protein-coupled receptors. J. Biomol. Screen. 18,
ackꢃowlꢁꢀgmꢁꢃꢆꢂ
5
99–609 (2013).
Support was given by National Institutes of Health (NIH) grants U01104974
(B.L.R., B.K.S. and W.K.K.), the NIH Department of Pharmacology Training Grant
(K.L.), a Genentech Foundation Pre-doctoral Fellowship (J.K.), and a PhRMA
Foundation Predoctoral Fellowship (K.L.). We thank the National Institute on Drug
Abuse Drug Supply Program for supplying the morphine and codeine analogs and the
glucuronidated or acetylated metabolites used in this study.
2
2
2
2
2
2
1. Sromeꢁ, A.W. et al. Preliminary pharmacological evaluation of enantiomeric
morphinans. ACS Chem. Neurosci. 5, 93–99 (2014).
2. Wang, M.H. et al. Activation of opioid mu-receptor by sinomenine in cell
and mice. Neurosci. Lett. 443, 209–212 (2008).
3. Nagase, H. et al. e pharmacological profile of delta opioid receptor ligands,
(
+) and (−) TAN-67 on pain modulation. Life Sci. 68, 2227–2231 (2001).
4. White, ꢂ.L. et al. Identification of novel functionally selective κ-opioid
receptor scaffolds. Mol. Pharmacol. 85, 83–90 (2014).
aꢈꢆhoꢅ coꢃꢆꢅibꢈꢆioꢃꢂ
K.L. performed the in vitro pharmacology and 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.K. assisted in the in vitro
small-molecule screening and helped with data and statistical analyses. T.C. performed
in vitro pharmacology experiments. H.N. synthesized (+)-TAN-67 and KNT-127.
F.I.C. synthesized several compounds and advised structure–activity relationship 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.
5. Horn, F. et al. GPCꢀDB: an information system for G-protein-coupled
receptors. Nucleic Acids ꢀes. 26, 275–279 (1998).
6. Lin, H., Sassano, M.F., ꢀoth, B.L. & Shoichet, B.ꢂ. A pharmacological
organization of G protein-coupled receptors. Nat. Methods 10,
1
40–146 (2013).
2
2
2
3
3
7. Irwin, J.J. & Shoichet, B.ꢂ. ZINC--a free database of commercially available
compounds for virtual screening. J. Chem. Inf. Model. 45, 177–182 (2005).
8. Eswar, N. et al. Comparative protein structure modeling using MODELLEꢀ.
Curr. Protoc. Protein Sci. 50, 2.9.1–2.9.31 (2007).
9. Yang, Q. & Sharp, ꢂ.A. Building alternate protein structures using the elastic
networꢁ model. Proteins 74, 682–700 (2009).
Comꢄꢁꢆiꢃg fiꢃꢇꢃciꢇl iꢃꢆꢁꢅꢁꢂꢆꢂ
0. Mysinger, M.M. & Shoichet, B.ꢂ. ꢀapid context-dependent ligand desolvation
in molecular docꢁing. J. Chem. Inf. Model. 50, 1561–1573 (2010).
1. Mysinger, M.M. et al. Structure-based ligand discovery for the protein–
protein interface of chemoꢁine receptor CXCꢀ4. Proc. Natl. Acad. Sci. USA
The authors declare no competing financial interests.
aꢀꢀiꢆioꢃꢇl iꢃfoꢅmꢇꢆioꢃ
Any supplementary information, chemical compound information and source data are
available in the online version of the paper. Reprints and permissions information is
available online at http://www.nature.com/reprints/index.html. Correspondence and
requests for materials should be addressed to B.L.R.
1
09, 5517–5522 (2012).
3
2. Carlsson, J. et al. Ligand discovery from a dopamine D3 receptor homology
model and crystal structure. Nat. Chem. Biol. 7, 769–778 (2011).
8
nature CHeMICaL BIOLOGY | AdvAnce online publicAtion | www.ꢀaꢁꢂrꢃ.ꢄꢅm/ꢀaꢁꢂrꢃꢄhꢃmꢆꢄaꢇꢈꢆꢅꢇꢅgy

ꢄNꢃINꢁ MꢁTꢂꢄDS
-hexosaminidase assay. LAD2 cells were maintained in Stem-Pro34 medium
(GIBCO) supplemented with StemPro34 Supplement (GIBCO), mM
L-Glutamine, 100 U/ml penicillin–streptomycin, and recombinant human stem
cell factor (100 ng/ml) (PeproTech) as described in ref. 52. For the assay, adapted
Cell lines. HTLA cells were a gift from R. Axel (Columbia University) and
were maintained at low passage batches. FLP-IN/T-REX HEK-293-T cells were
purchased from Invitrogen for ease of inducible stable cell line generation and
2
were certified as HEK-T and mycoplasma-free by Invitrogen. FLP-IN/T-REX from ref. 54, cells were incubated in fresh medium for 18 h before the experi-
cells were made into inducible stable cell lines according to the manufacturer’s ment. For testing IgE activation, cells were also incubated with biotin-labeled
instruction. The LAD2 human mast cell line was obtained from cells’ original IgE during this time. Then, cells were washed twice in Tyrode’s buffer (20 mM
52
source lab of A. Kirshenbaum and D. Metcalfe at the Laboratory of Infectious HEPES with 134 mM NaCl, 5 mM KCl, 1.8 mM CaCl , 1 mM MgCl , 5.5 mM
2
2
Diseases at the National Institutes of Health in Bethesda, Maryland and were
glucose, and 0.3% bovine serum albumin, pH 7.4) and seeded at 10,000 cells
maintained as per the instructions. No further characterization of HTLA or per well in V-bottom clear 96-well plates in 90 μl of Tyrode’s buffer. Drugs were
LAD2 cells was performed, as the cells came from the source labs.
diluted at single concentrations or in 8-point concentration–response curves at
a 10× working concentration in Tyrode’s buffer and 10 μl drug, or streptavidin
for biotin-labeled IgE samples, and were added in triplicate to the 90-μl mast cell
suspension. Drugs and cells were incubated for 30 min in a humidified incuba-
Chemical compounds. The nine libraries used in screening were the NCC-1
(
NIH Clinical Collection), NCC-2 (NIH Clinical Collection), NIMH Library,
Tocris, Prestwick, LOPAC, Selleck, Spectrum, and the Roth Lab Collection tor without CO at 37 °C. After drug incubation, plates were centrifuged at 250g
2
(
in-house). Opioid ligands were obtained from either Sigma-Aldrich or
for 5 min, and 30 μl supernatant was added to new 96-well plates containing
10 μl of 10 μM 4-nitrophenyl N-acetyl-β-D-glucosaminide (NAG) diluted in
were synthesized.
0
.1 M citrate buffer (49.5% 0.1M citric acid and 50.5% 0.1M sodium citrate,
Constructs. MRGPRX-Tango plasmids for MRGPRX1, MRGPRX2, or MRGPRX4
pH 4.5). The remaining supernatant was discarded, and cells were lysed by add-
6
were made as was previously described . MRGPRB2 insert was obtained from ing 100 μl of Tyrode’s buffer with 0.1% Triton-X-100 to the pellets and mixing.
Dharmacon cDNA and subcloned into the PRESTO-Tango backbone.
30 μl of lysate was added to 10 μl of 10 μM NAG. All plates were incubated for
0 min in a humidified incubator without CO at 37 °C. Lastly, 100 μl of bicar-
9
2
PRESTO-Tango assay screening. HTLA cells (HEK-T cells stably expressing a
bonate buffer (88 mM Na2CO , 40 mM NaHCO , pH 10) was added to stop the
3
3
β-arrestin2-TEV fusion protein and a tTa-dependent luciferase reporter) were reaction, inducing a color change that was measured on an absorbance plate
maintained in DMEM (Corning) containing 10% FBS, 2 μg/ml puromycin and reader (BMG Labtech POLARstar Omega) at 405 nm. Results were analyzed by
00 μg/ml hygromycin B in a humidified atmosphere at 37 °C with 5% CO2. dividing the released fraction by the total fraction (released + lysed) and multi-
1
In brief, cells were plated to 50% confluency and transfected with a codon-
optimized MRGPRX-Tango construct using the calcium phosphate method . degranulation, average percent degranulation for DMSO-treated wells was sub-
Thenextday, transfectedcellsweretransferredtoglass-bottomed, poly-L-lysine- tracted from drug-treated wells. Total number of dose–response replicates for
plying by 100 to get a percentage of degranulation. To correct for spontaneous
53
coated white 384-well plates at a density of 20,000 cells/well. The following day, each compound is reflected in figures and figure legends.
cells were treated with 10 μM concentrations of small molecules (in quadru-
plicate) diluted in drug buffer (1× HBSS with 20 mM HEPES and 0.3% bovine
serum albumin, pH 7.4) and incubated for 18–24 h. After drug incubation,
medium was removed and 20 μl of Bright-Glo (Promega; diluted 20-fold) was
added to each well and incubated 15 min at room temperature. Luminescence
Short interfering RNA (siRNA) knockdown and qPCR analysis. Using
TransIT 20/20 transfection reagent (Mirus), 25 nM of each siRNA (Dharmacon
#EQ-005666-00-0002, set of four distinct MRGPRX2 siRNAs) or the nontar-
geting siRNA control (Dharmacon #D-001910-10-05) was prepared in 250 μl
was measured on a TriLux luminescence counter and analyzed as relative lumi- of OPTI-MEM with 5 μl TransIT per reaction. After 30 min, the transfec-
nescent units (RLU) using GraphPad Prism. Compounds that increased the tion mixture was added drop-wise to 1.2 million mast cells/well of a 6-well
luminescence signal by two-fold or greater were considered ‘actives’ and were plate and allowed to incubate for 72 h. After 72 h, 200,000 cells were removed
analyzed further using concentration–response curves. More detailed discus- for use in the β-hexosaminidase assay and 1 million cells were assayed using
sion of the PRESTO-Tango assay can be found in ref. 6. Total number of dose–
qRT-PCR analysis with the High-Capacity cDNA Reverse Transcription
response replicates for each compound can be found in Supplementary Kit (Life Technologies) following the manufacturer’s instructions as well as
Table 2. Screening metrics and detailed information related to the screening TaqMan Universal Master Mix II with UNG (Life Technologies) and TaqMan
procedure can be found in Supplementary Table 3.
Gene Expression Assay for MRGPRX2 (Life Technologies #4331182,
Hs00365019_s1) following the manufacturer’s suggested protocol.
Intracellular calcium mobilization assay. We generated a MRGPRX2-
expressing tetracycline-inducible stable cell line containing the MRGPRX2 Significance calculations of siRNA experiments. To assess significance, we
codon-optimized receptor sequence with a N-terminal FLAG tag using the used GraphPad Prism to perform a two-way ANOVA and Sidak post hoc with
FLP-IN/T-REX Core Kit (Invitrogen) following the manufacturer’s instruc- an alpha value of 0.05. Total number of replicates is reflected in figure legends
tions. MRGPRX2 stable cells were maintained in DMEM containing 10% and figures.
FBS, 100 μg/ml hygromycin B, and 15 μg/ml blasticidin. For the calcium
mobilization assay, cells were plated into glass bottomed, poly-L-lysine coated,
black 384 well plates at a density of 20,000 cells/well in medium containing models was generated using PROMALS3D , and homology models were built
% dialyzed FBS, 1 μg/ml tetracycline, 100 IU/ml penicillin and 100 μg/ml with MODELLER-9v8 (ref. 28) using the crystal structure of the κ-opioid
Homology modeling. The alignment for the construction of the MRGPRX2
55
1
streptomycin and incubated 24 h. Following tetracycline induction, medium
was removed and cells were loaded with 20 μl/well of 1× FLIPR Calcium
receptor (PDB code 4DJH) as the template. The homology models were
aligned against the entire MRGPRX and opioid receptor family. The alignment
dye (Molecular Devices) and 2.5 mM probenic acid, for 1 h in a humidified was manually edited to remove the amino and carboxy termini that extended
environment at 37 °C with 5% CO . For mast cell calcium experiments, cells past the template structure, to remove the engineered T4 lysozyme, and to cre-
2
were seeded at a density of 50,000 cells/well in Tyrode’s buffer containing 1× ate different alignments of the flexible and nonconserved second extracellular
calcium dye and incubated for 1 h before treatment and analysis. After dye loop (EL2); the final resulting sequence alignment between the template opioid
loading, baseline was measured for 10 s before drug treatment, and then cells
were treated with 10 μl of 3× concentrated drug in drug buffer (1× HBSS Three models were built from each of 180 elastic network models (ENMs), pro-
with 20 mM HEPES and 2.5 mM probenic acid, pH 7.4) in 16 point concen- duced by the program 3K-ENM , for a total of 540 models built during each
tration response curves from 100 μM to 0.003 nM. Fluorescence was meas- iterative round of model refinement. Although EL2 is substantially shorter in
ured for an additional 120 s and data was analyzed using GraphPad Prism. MRGPRX2 than in other GPCRs, and it lacks the conserved disulfide bond
structure and the MRGPRX receptors is shown in Supplementary Figure 5c.
29
Total number of dose–response replicates for each compound can be found
in Supplementary Table 2.
between TM3 and EL2, an additional 540 models were also built forcing a puta-
tive disulfide between Cys168 in EL2 and Cys180 in TM5.
doi:10.1038/nchembio.2334
nature CHeMICaL BIOLOGY

Model evaluation. Models were ranked on the basis of prioritizing active opioids
stirred for 3 d at room temperature, the reaction mixture was diluted with
(
dextromethorphan) over the rest of the inactive PRESTO-Tango library that was Et O (50 ml) and filtered. The solid was rinsed with Et O (2×). The combined
2
2
used in the β-arrestin screen, as well as over property-matched decoy small mol- Et O solution was cooled to 0 °C and treated with ice and water. The organic
2
ecules. We further insisted that in its docked pose, dextromethorphan engaged in layer was washed with water (2×) and brine (2×). The organic layer was dried
an ion pair between its charged nitrogen and an anionic side chain in MRGPRX2.
The entire PRESTO-Tango library was docked to an analogous 4DJH ligand- yellow solid. H NMR (600 MHz, CDCl ) δ 8.23 (d, J = 2.3 Hz, 1H), 8.12 – 8.04
binding site in the modeled MRGPRX2 receptors for several rounds of iterative
(Na SO ) and concentrated to give the tittle compound (0.82 g, 76%) as pale
2 4
1
3
(m, 2H), 7.58 – 7.48 (m, 3H), 7.45 (s, 1H), 6.84 (d, J = 2.3 Hz, 1H). 13C NMR
binding site refinement. In each round, the top-ranked models were examined (151 MHz, CDCl ) δ 156.2, 149.8, 146.1, 139.2, 136.5, 131.0, 129.2 (2C), 127.5
3
+
for a binding pose that made hydrophobic and electrostatic interactions with
the receptor, including the key positive–negative charge coordination. Residues
within 6 Å of the dextromethorphan pose were then minimized around the
(2C), 105.8, 98.8. HRMS (ESI–TOF) m/z: [M+H] calculated for C H ClN ,
1
2
9
3
230.0485; found: 230.0484.
(R)-N,N-Dimethyl-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidin-
docked ligand with PLOP . The PRESTO-Tango library was then redocked into 3-amine ((R)-ZINC-3573). To a solution of 7-chloro-5-phenylpyrazolo[1,5-a]
this optimized binding site for each model. This refinement continued for several pyrimidine (0.069 g, 0.3 mmol) in dioxane (2 ml) was added DIEA (0.19 ml,
cycles until the top-ranked models all converged to the same dextromethorphan 0.6 mmol), followed by (R)-(+)-3-(dimethylamino)pyrrolidine (0.038 g,
56
pose, with the top-scoring model chosen as the final one. Structural models (PDB
0.33 mmol). The resulting solution was stirred for 16 h at room temperature
files) of characteristic MRGPRX2-modeled complexes (with dextromethorphan before being concentrated and purified by silica gel column to provide the title
1
and ZINC-9232) are shown in Supplementary Data Sets 1 and 2.
compound (0.070 g, 76%). H NMR (600 MHz, CDCl ) δ 8.02 – 7.97 (m, 2H),
7
3
.95 (d, J = 2.3 Hz, 1H), 7.51 – 7.40 (m, 3H), 6.48 (d, J = 2.3 Hz, 1H),
Virtual screens. We used DOCK 3.6 to screen the ZINC database (http://zinc15. 6.12 (s, 1H), 4.30 (t, J = 8.8 Hz, 1H), 4.23 (t, J = 9.8 Hz, 1H), 4.02 (q, J = 9.4 Hz,
docking.org/) (Results). The flexible ligand sampling algorithm in DOCK 1H), 3.83 (t, J = 9.5 Hz, 1H), 2.90 – 2.82 (m, 1H), 2.36 (s, 6H), 2.29 – 2.21 (m, 1H),
3
.6 superimposes atoms of the docked molecule onto binding site matching 2.01 – 1.91 (m, 1H). 13C NMR (151 MHz, CDCl ) δ 157.5, 151.8, 147.9,
3
spheres, which represent favorable positions for individual ligand atoms. Forty- 143.8, 139.2, 129.6, 128.7 (2C), 127.3 (2C), 94.9, 86.2, 65.2, 55.0, 50.1, 44.5
five matching spheres were used, with each respectively starting from the pre- (2C), 30.1. HRMS (ESI-TOF) m/z: [M+H] calculated for C H N , 308.1875;
+
1
8
22
5
vious refinement round’s best pose of dextromethorphan. The degree of ligand
sampling is determined by the bin size, bin-size overlap, and distance tolerance,
set at 0.4 Å, 0.1 Å and 1.5 Å, respectively, for both the matching spheres and the
docked molecules. The complementarity of each ligand pose was scored as the
found: 308.1871.
(S)-N,N-Dimethyl-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidin-3
-amine ((S)-ZINC-3573). The title compound (0.075 g, 81%) was prepared
using the same synthetic procedure for the preparation of (R)-ZINC-3573
1
sum of the receptor–ligand electrostatic and van der Waals’ interaction ener- with (S)-(+)-3-(dimethylamino)pyrrolidine (0.038 g, 0.33 mmol). H NMR
gies and corrected for context-dependent ligand desolvation using QNIFFT (600 MHz, CDCl ) δ 8.05 – 7.97 (m, 2H), 7.95 (d, J = 2.3 Hz, 1H), 7.52 – 7.39
3
5
7
point-charge Poisson-Boltzmann electrostatics models . Partial charges from
(m, 3H), 6.48 (d, J = 2.3 Hz, 1H), 6.12 (s, 1H), 4.30 (t, J = 8.8 Hz, 1H), 4.23
the united-atom AMBER5 force field were used for all receptor atoms; ligand (t, J = 9.8 Hz, 1H), 4.02 (q, J = 9.3 Hz, 1H), 3.83 (t, J = 9.5 Hz, 1H), 2.91 – 2.81
charges and initial solvation energies were calculated using AMSOL (http:// (m, 1H), 2.36 (s, 6H), 2.30 – 2.21 (m, 1H), 2.02 – 1.91 (m, 1H). C NMR (151
8
59,60
13
comp.chem.umn.edu/amsol/). The best scoring conformation of each docked MHz, CDCl ) δ 157.5, 151.8, 147.9, 143.8, 139.2, 129.6, 128.7 (2C), 127.3 (2C),
3
+
molecule was then subjected to 100 steps of rigid-body minimization.
94.9, 86.2, 65.2, 55.0, 50.1, 44.5 (2C), 30.1. HRMS (ESI–TOF) m/z: [M+H]
calculated for C H N , 308.1875; found: 308.1872.
1
8
22
5
Selection of potential ligands for testing. We docked the approximately 3.7
million commercially available molecules of the lead-like subset of the ZINC Code availability. The DOCK3.6 program30,36 is freely accessible to academic
database to the final MRGPRX2 models. The full hit list was automatically fil- labs at http://dock.compbio.ucsf.edu/DOCK3.6/ or http://blaster.docking.org/
tered to remove molecules that possess high internal energy, nonphysical con- for open access use.
formations, which are not well modeled by our scoring function. The reported
rankings reflect this filtering. From the top 0.13% (~5,000 molecules) of the
Data availability. The generated and analyzed raw data sets that support
docked ranking list, 20 compounds were chosen for testing, based on comple- the findings of this study are available from the corresponding author upon
mentarity to the binding site and presence of predicted charge interactions with reasonable request.
4
.60
5.38
Glu164 and Asp184 , mimicking those predicted for dextromethorphan.
5
2. ꢂirshenbaum, A.S. et al. Characterization of novel stem cell factor responsive
human mast cell lines LAD 1 and 2 established from a patient with mast cell
sarcoma/leuꢁemia; activation following aggregation of FcepsilonꢀI or
FcgammaꢀI. Leuk. ꢀes. 27, 677–682 (2003).
Chemistry general procedures. HPLC spectra for all compounds were acquired
using an Agilent 1200 Series system with DAD detector. Chromatography was
performed on a 2.1 × 150 mm Zorbax 300SB-C18 5 μm column with water con-
taining 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic
acid as solvent B at a flow rate of 0.4 ml/min. The gradient program was as
follows: 1% B (0−1 min), 1−99% B (1−4 min), and 99% B (4−8 min). High-
resolution mass spectra (HRMS) data were acquired in positive ion mode using
an Agilent G1969A API-TOF with an electrospray ionization (ESI) source.
Nuclear magnetic resonance (NMR) spectra were acquired with a Bruker DRX-
5
3. Jordan, M., Schallhorn, A. & Wurm, F.M. Transfecting mammalian cells:
optimization of critical parameters affecting calcium-phosphate precipitate
formation. Nucleic Acids ꢀes. 24, 596–601 (1996).
54. Staats, H.F. et al. A mast cell degranulation screening assay for the identification
of novel mast cell activating agents. MedChemComm 4, 88–94 (2013).
5
5. Pei, J., ꢂim, B.H. & Grishin, N.V. PꢀOMALS3D: a tool for multiple protein
sequence and structure alignments. Nucleic Acids ꢀes. 36, 2295–2300 (2008).
6. Jacobson, M.P. Epilepsy in aging populations. Curr. Treat. Options Neurol. 4,
5
1
13
6
00 spectrometer (600 MHz H, 150 MHz C). Chemical shifts are reported in
1
9–30 (2002).
p.p.m. (δ). HPLC was used to establish the purity of target compounds. All
57. Sharp, ꢂ.A. Polyelectrolyte electrostatics: salt dependence, entropic, and
enthalpic contributions to free energy in the nonlinear Poisson–Boltzmann
model. Biopolymers 36, 227–243 (1995).
final compounds had > 95% purity using the HPLC methods described above.
7
-Chloro-5-phenylpyrazolo[1,5-a]pyrimidine. The title compound was
5
8. Meng, E.C., Shoichet, B.ꢂ. & ꢂuntz, I.D. Automated docꢁing with grid-based
energy evaluation. J. Comput. Chem. 13, 505–524 (1992).
6
1
synthesized according to a known literature procedure . To a solution of
-aminopyrazole (2.0 g, 24 mmol) in acetic acid (15 ml) was added ethylben-
3
59. Li, J., Zhu, T., Cramer, C.J. & Truhlar, D.G. New class IV charge model for
zoylacetate (4.7 ml, 27 mmol). The resulting reaction mixture was refluxed
for 3 h before being cooled to room temperature and concentrated. The solid
residue was diluted with EtOAc and filtered to afford 5-phenylpyrazolo[1,5-a]
pyrimidin-7(4H)-one (4.19 g, 83%) as white solid. This intermediate (1.0 g,
extracting accurate partial charges from wave functions. J. Phys. Chem. A
1
02, 1820–1831 (1998).
6
0. Chambers, C.C., Hawꢁins, G.D., Cramer, C.J. & Truhlar, D.G. Model for
aqueous solvation based on class IV atomic charges and first solvation shell
effects. J. Phys. Chem. 100, 16385–16398 (1996).
4
.7 mmol) was dissolved in POCl (5.0 ml, 55 mmol). To the resulting solution
61. Paruch, ꢂ. et al. Discovery of Dinaciclib (SCH 727965): a potent and selective
inhibitor of cyclin-dependent ꢁinases. ACS Med. Chem. Lett. 1, 204–208 (2010).
3
was added pyridine (0.25 ml, 3.1 mmol) at room temperature. After being
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doi:10.1038/nchembio.2334