Discovery of β2 adrenergic receptor ligands using biosensor fragment screening of tagged wild-type receptor

Article abstract of DOI:10.1021/ml400312j

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization. However, the screening of fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel, selective, high affinity antagonists of human β2 adrenoceptor.

Full text of DOI:10.1021/ml400312j

Technology Note
pubs.acs.org/acsmedchemlett
Discovery of β2 Adrenergic Receptor Ligands Using Biosensor
Fragment Screening of Tagged Wild-Type Receptor
Tonia Aristotelous,†,○ Seungkirl Ahn,‡,○ Arun K. Shukla,‡,○ Sylwia Gawron,† Maria F. Sassano,§
Alem W. Kahsai,‡ Laura M. Wingler,‡ Xiao Zhu,‡ Prachi Tripathi-Shukla,‡ Xi-Ping Huang,§ Jennifer Riley,†
Jeremy Besnard,† Kevin D. Read,† Bryan L. Roth,§,∥ Ian H. Gilbert,† Andrew L. Hopkins,*,†
́
́
Robert J. Lefkowitz,*,‡,⊥,# and Iva Navratilova*,†
†Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1
5EH, United Kingdom
‡Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, United States
§NIMH Psychoactive Drug Screening Program, Department of Pharmacology, The University of North Carolina Chapel Hill School
of Medicine, Chapel Hill, North Carolina 27759, United States
∥Department of Pharmacology and Division of Chemical Biology and Medicinal Chemistry, The University of North Carolina Chapel
Hill School of Medicine, Chapel Hill, North Carolina 27759, United States
⊥Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, United States
#Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, United States
S
* Supporting Information
ABSTRACT: G-protein coupled receptors (GPCRs) are the primary
target class of currently marketed drugs, accounting for about a
quarter of all drug targets of approved medicines. However, almost all
the screening efforts for novel ligand discovery rely exclusively on
cellular systems overexpressing the receptors. An alternative ligand
discovery strategy is a fragment-based drug discovery, where low
molecular weight compounds, known as fragments, are screened as
initial starting points for optimization. However, the screening of
fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane
proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening
that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel,
selective, high affinity antagonists of human β2 adrenoceptor.
KEYWORDS: Fragment screening, G-protein coupled receptors, surface plasmon resonance, β2 adrenoceptor
atoms). The low molecular weights of fragments usually result in
significantly lower affinity hits, compared to HTS hits; however,
the interactions can be very ligand efficient2 as the fragments can
adopt optimum orientations in the active site. Conventional
ligand displacement assay methods measuring the displacement
of radiolabeled and fluorescent-labeled ligands have been used to
G-protein coupled receptors (GPCRs) are the principal class of
drug targets. The conventional approach to the discovery of
GPCR ligands has focused on the high-throughput screening
(HTS) of cellular systems overexpressing the receptors against
very large collections of compounds (ten to hundreds of
thousands of compounds), with typical molecular weights
ranging from 350 to 500 Da (27 to 38 non-hydrogen atoms).
In contrast, fragment screening is now established as an
important new approach to drug discovery.1 The principle of
fragment-based drug discovery is that a relatively small number of
low molecular weight fragments can represent large areas of
chemical space. However, fragment screening has not been
routinely applied to membrane proteins. This is particularly
limiting, as around a quarter of current drug targets of approved
medicines are GPCRs. Fragment-based drug discovery starts
with the screening of a small library, consisting often of only
several hundred to a couple of thousand low molecular weight
compounds, called fragments. Fragments are usually in the
molecular weight range of 100 to 300 Da (8 to 23 non-hydrogen
3
fragment screening of histamine-H4 and adenosine-A3 recep-
tors,4 respectively, in cell-based assays. However, for fragment
screening there are significant advantages to exploiting highly
sensitive label-free, biophysical screening techniques in order to
detect potential low affinity fragment hits. Currently, surface
plasmon resonance (SPR) has become a dominant technology
for fragment screening.5 The advantage of SPR based screening is
that it has unparalleled potential not only to design screening
procedures to identify both orthosteric and allosteric ligands6 but
Received: August 14, 2013
Accepted: September 3, 2013
© XXXX American Chemical Society
A
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Figure 1. SPR sensorgrams of (a) an antagonist alprenolol and an agonist fenoterol, and (b) fragments A to E interacting with β2 adrenoceptor. Red
lines represent kinetic fit. The right-side inserted graphs for fragments D and E represent equilibrium fits.
also more importantly to use more sophisticated targets such as
protein complexes, which are otherwise not feasible in cellular
systems.7
The β2 adrenoceptor has been the prototypical GPCR for the
development of new methods. The β2 adrenoceptor was the first
ligand-binding GPCR to be purified from cell membranes; the
first to be cloned and sequenced;16 provided the first high-
resolution crystal structures;17−19 provided the first example of
structure-based discovery of novel GPCR ligands by virtual
screening;20 and was the first receptor to be crystallized in
complex with its G protein, Gs.21 Hence, the β2 adrenoceptor
was selected as the ideal candidate GPCR for novel biophysical
methods development.
A human β2 adrenoceptor construct containing a FLAG tag at
the N-terminus and histidine 10 (His-10) tag at the C-terminus
was generated for baculovirus expression in Sf 9 cells (Supporting
Information Figure S1). The receptor was solubilized and
purified as described before.22 The β2 adrenoceptor was
captured via His-10 tag on NTA sensor chip.
In recent years, biophysical techniques have advanced to face
the challenge of studying membrane bound GPCRs. Thermo-
stabilized mutated GPCRs, developed for crystallization, have
been shown to be suitable for biophysical fragment screening, by
SPR and nuclear magnetic resonance (NMR).8−10 However, the
process of engineering conformational stability results in GPCR
constructs that have been shown to lack the full range of wild-
type pharmacology.11−13 Ideally, biophysical fragment screening
of the wild-type sequence GPCRs, which retain the full range of
pharmacology,7,14,15 may have advantages for ligand discovery
over mutated, thermostabilized GPCRs. We have developed a
biosensor fragment screening protocol that enables cell-free,
label-free fragment screening that directly measures fragment
interactions with the nonstabilized, purified wild-type GPCR. We
exemplify the method by the discovery of novel antagonists of
the human β2 adrenoceptor.
To establish whether the captured β2 adrenoceptor is
pharmacologically active on the surface, binding of an agonist
(fenoterol) and an antagonist (alprenolol) was measured (Figure
1a). The measured affinity of alprenolol (KD = 790 pM)
B
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ACS Medicinal Chemistry Letters
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Table 1. Kinetic Parameters (ka, kd), Affinity (KD), and Ligand Efficiency (LE) (kcal/mol/non-hydrogen atom) Values for
Alprenolol, Fenoterol, and Fragments A to E Binding to β2 Adrenoceptor, Collected in the DDM (Dodecylmaltoside) Detergent
Measured Using Surface Plasmon Resonance (SPR) at 10 °C, and Radioligand Competition (ki) for β1 and β2 Adrenoceptors for
a
Fragments A to E at Room Temperature
SPR
competition binding (ki)
compd
ka (M−1 s−1
)
kd (s−1
)
KD
LE
β2
β1
alprenolol
8.11( 0.02) × 105
5.45( 0.09) × 105
5.06( 0.03) × 105
1.16( 0.02) × 106
6.62( 0.07) × 105
NA
6.4( 0.05) × 10−4
7.6( 0. 1) × 10−2
8.94( 0.04) × 10−3
1.08( 0.02) × 10−1
5.47( 0.05) × 10−2
NA
790 ( 60) pM
139.0 ( 0.9) nM
17.6 ( 0.1) nM
93.4 ( 0.7) nM
82.5 ( 0.7) nM
22.2 ( 0.7) μM
3.5 ( 0.1) μM
0.65
0.40
0.48
0.54
0.54
0.40
0.50
fenoterol
A
B
C
D
E
177.8 ( 16.8) nM
342.8 ( 22.6) nM
191.4 ( 14.2) nM
14.1( 1.2) μM
1467 ( 75.9) nM
963.4 ( 64.7) nM
152.0 ( 9.7) nM
9.9 ( 0.7) μM
NA
NA
15.0 ( 1.4) μM
12.9 ( 0.8) μM
a
The errors reported for the SPR data represent the SD from duplicates and for radioligand binding are the mean SE obtained from three
independent experiments done in duplicates.
corresponds very well to the binding affinity measured in native
membrane using radioligand binding (ki = 1 nM).23 Fenoterol
exhibited affinity KD = 139 nM, which is also in very good
agreement with measured affinity by radioligand binding (ki =
126 nM).24 These data suggest that immobilization of the
purified β2 adrenoceptor on the SPR surface does not alter its
pharmacological properties and therefore represents a viable
approach for in vitro drug screening.
the β2 adrenoceptor in the presence of the MNG detergent
(Supporting Information Table S2 and Figure S4). The
structures of the confirmed hits are shown in Figure 2.
As a proof of principle, a library consisting of 656 fragments5
was screened against the immobilized β2 adrenoceptor at one
concentration. The average molecular weight (MW) of the
fragment library is MW = 187 (equating to 13 non-hydrogen
(heavy) atoms) with fragment sizes varying from 94 Da (7 heavy
atoms) to 341 Da (24 heavy atoms). To reduce the nonspecific
binding, especially at high concentrations that could be a source
of false positives when screening fragment libraries, it is
important to find a suitable reference surface. The reference
surface should ideally be captured in the same manner as the
target protein but in an inactive state of ligand binding. Receptors
with pharmacologically blocked binding sites have previously
been demonstrated as highly suitable reference surfaces for
GPCR analysis by SPR.6 The β2 receptor was preincubated with
a slow-off-rate agonist compound BI-16710725 to create a
control/reference target receptor (i.e., ligand binding site-
blocked state), which was immobilized by the method described
above. A third channel was used as a blank reference surface. To
evaluate screening data, single-point concentrations were read
for each of the fragments before the end of the injection.
Examples of collected data are shown in Supporting Information
Figure S2a,b. Each sensorgram was then inspected against all
three surfaces (target, reference target, and blank). Examples for
two of the hit compounds are shown in Supporting Information
Figure S2c,d. Eighty-one compounds were then further selected
and screened at six concentrations, ranging from 300 to 1.2 μM.
A total of five fragment hits were confirmed: fragments A to E
with affinities ranging from KD = 17 nM to KD = 22 μM (Table
1). Fragments A to E were screened at concentrations adjusted to
the affinity of each fragment in duplicate (Figure 1b).
Figure 2. Chemical structures of the validated β2 adrenoceptor
fragment hits A to E and the synthesized fragment analogues F to L.
In order to further confirm these potential hits, we measured
the affinities of the fragments A to E in a radioligand competition
binding assay. Moreover, the radioligand competition binding
assay also tests whether these fragments occupy the orthosteric
ligand binding pocket of the receptor. All five fragments showed
specific competitive inhibition of [125I]-cyanopindolol (CYP)
binding with ki values, which are close to the affinities observed
by SPR (Table 1 and Figure 3a). This data suggest that these
fragments occupy the classical orthosteric binding pocket of the
β2 adrenoceptor and that they are bonafide receptor ligands
capable of binding to the receptor in native membranes. As the
ligand binding pocket of the β1 adrenoceptor and the β2
adrenoceptor are highly conserved and there are already a series
of nonselective ligands described in the literature, we tested
whether these fragments also bind to the β1 adrenoceptor
(Figure 3b) in a competition binding assay with radioligands.
Interestingly, fragment A exhibited about 10-fold selectivity for
Fragment hits A to E were further tested for binding to the β2
adrenoceptor in the presence of MNG detergent. Minimal
differences in the affinities were observed compared to data
obtained using DDM detergent suggesting a very limited
influence of the different detergents on binding activity of the
compounds. (Supporting Information Table S1 and Figure S3).
In addition, fragments A and B were resynthesized (Supporting
Information) and their high affinities reconfirmed by SPR against
C
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ACS Medicinal Chemistry Letters
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Figure 3. Radioligand competition binding and functional assays for the β2 adrenoceptor with fragments A to E. (a,b) Dose-dependent competition
binding curves of the fragments for the β2 adrenoceptor (a) and β1 adrenoceptor (b). (c,d) Stimulatory dose response curves of the fragments obtained
from cAMP production (c) and β-arrestin recruitment (d) assays. (e,f) Inhibitory effects of the fragments at 10 μM on isoproterenol-stimulated dose
responses in cAMP production (e) and β-arrestin recruitment (f) assays. All of the data points represent mean SE obtained from three independent
experiments done in duplicate. Dose response curves for each compound were obtained using the nonlinear iterative curve-fitting computer program
Prism. ISO, isoproterenol; CYP, cyanopindolol; ICI, ICI-118,551 (a β2 adrenoceptor-specific antagonist); CGP, CGP-20712A (a β1 adrenoceptor-
specific antagonist).
the β2 adrenoceptor compared to the β1 adrenoceptor.
However, the other four fragments exhibited nonselective
binding to the β2 adrenoceptor and the β1 adrenoceptor
(Table 1). In addition, the selectivity of fragments A to E were
further tested by screening the compounds against a panel of 27
GPCRs consisting of the α1 and α2 adrenoceptors, the serotonin
receptors, the dopamine receptors, and the histamine receptors
(Supporting Information Table S3). Fragment A is relatively
selective for the β2-adrenoceptor with off-target affinities against
only three other receptors ki < 1 μM (5-HT2B ki = 407 nM; 5-
HT2C ki = 965 nM; histamine H1 ki = 399 nM) and measured
binding activity against a further three receptors with affinities
within 10-fold of the radioligand displacement ki (α2A
adrenoceptor ki = 1202 nM; 5HT6 ki = 1262 nM; β1
adrenoceptor ki = 1467 nM).
Next, we tested the functional activity of these fragments in
cell-based signaling assays to investigate whether these fragments
are blockers or agonists for the β2 adrenoceptor. Interestingly,
none of these fragments activated either the G protein coupling,
monitored through elevation of the cAMP level or β-arrestin
recruitment to the β2 adrenoceptor (Figure 3c,d). Rather, all of
these fragments inhibited isoproterenol-induced responses in
both the cAMP production and β-arrestin recruitment assays
(Figure 3e,f). For the β-arrestin recruitment assay, the C-
terminus of the β2 adrenoceptor is replaced with the C-terminus
of the V2 vasopressin receptor in order to increase the signal-to-
D
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ACS Medicinal Chemistry Letters
Technology Note
noise ratio while retaining the ligand binding properties of the
native β2 adrenoceptor. The relative inhibition for these
fragments corresponded well with their affinities for the β2
adrenoceptor. Fragments D and E led to modest inhibition of
isoproterenol-induced cAMP production and β-arrestin recruit-
ment, and only at high concentrations, reflective of their low
affinity for the receptor (Supporting Information Figure S5a,b).
In order to further confirm the specificity of these ligands for the
β adrenoceptors, we tested these fragments on another Gs
coupled receptor, the arginine-vasopressin type 2 receptor
(AVPR2). However, we did not observe any detectable agonistic
or blocking activity of these fragments on either cAMP
production or β-arrestin recruitment in the AVPR2 system,
suggesting that these are β adrenoceptor selective ligands
(Supporting Information Figure S6a−c). Interestingly, none of
the fragments from this small library exhibited agonistic activity
although our SPR assay described here does have the capability of
identifying classical small molecule agonists as demonstrated by
the measurement of fenoterol binding.
Removal of the benzene ring entirely to change the 4-piperazine-
quinoline (F) to a 4-piperazine-pyridine (L) reduces the affinity
by 400-fold, but L retains a high ligand efficiency (KD = 141 600
nM; LE = 0.44).
In summary, we have established the utility of biosensor-based
fragment screening of wild-type GPCRs by the discovery of
novel, high affinity, antagonists of the β2 adrenoceptor. We have
demonstrated that fragment screening by SPR can be undertaken
on tagged, native GPCRs without the need for extensive protein
engineering. The advantage of the method is that by screening
tagged native receptors, without the need for introducing
stabilizing mutations, the pharmacology of the wild-type receptor
can be maintained. The process of engineering conformational
thermostability of a GPCR has been shown to markedly affect the
pharmacology of mutant receptors compared to wild-type
receptors.11,12 Engineering conformational stability requires the
receptor to be trapped in a conformation induced by a test ligand
to produce either an agonist or inverse agonist/antagonist
conformation.12,13 For example, antagonist-conformation stabi-
lized adenosine A2A receptors are incapable of binding agonists
with an appreciable affinity, vice versa antagonist binding to
agonist-conformation stabilized adenosine A2A receptor display
weaker affinity.11,12 In contrast, biophysical fragment screening
of wild-type GPCRs retain the full breadth of pharmacology in
measuring agonists and antagonists7,14,15 and thus provide the
opportunity for discovering compounds with diverse mecha-
nisms and potentially novel binding sites.6
To elucidate the features responsible for the high affinity of
fragment A, a limited number of analogues were prepared and
tested using the SPR β2 adrenoceptor binding assay in the MNG
detergent (Table 2; Supporting Information Figure S7 and Table
Table 2. Structure−Activity Relationship Table for the
Fragments A, B, C, and F to L Interacting with the β2
Adrenoceptor, Collected in the MNG Detergent, Measured
Using Surface Plasmon Resonance (SPR)
The SPR fragment screening method not only provides a
useful new approach to the discovery of novel GPCR ligands, but
it also presents unique opportunities to screen for ligands against
biased signaling conformations of the receptors as well as
receptor signaling complexes.
fragment
A
R2
R6
R7
KD
LE
A
B
C
F
G
H
I
NH
NH
NH
NH
NH
NH
NCH3
CF2
O
CF3
CH3
H
CH3
H
H
39.0 ( 0.1) nM
72.5 ( 0.4) nM
76.5 ( 0.7) nM
348.3 ( 0.6) nM
84.2 ( 0.2) nM
8 ( 0.1) μM
0.45
0.54
0.54
0.52
0.46
0.33
0.34
0.22
NA
H
H
Cl
H
H
H
ASSOCIATED CONTENT
■
CF3
H
H
H
S
H
CF3
H
* Supporting Information
CF3
CF3
CF3
CH3
CH3
CH3
1.8 ( 0.1) μM
67.1 ( 0.5) μM
no binding
Details of the experimental procedures, author contributions,
and the acknowledgements. This material is available free of
charge via the Internet at http://pubs.acs.org.
J
H
K
L
H
NH
141.6 ( 0.7) μM
0.42
AUTHOR INFORMATION
■
S4), to give initial data about binding requirements. 4-Piperazine-
quinoline was prepared as an undecorated core for comparison
(fragment F). The undecorated F is also a high affinity fragment
with a high ligand efficiency (KD = 348 nM ; LE = 0.52 kcal/mol/
non-hydrogen atom). The following initial structure−activity
relationships are observed: (i) addition of a substituent at the R2
position (B, Me; G, CF3) gave increases in potency, while
maintaining high ligand efficiency; (ii) a substitution in the 6-
position (compare A, R6 = CH3, with G, R6 = H) loses 2-fold in
potency but retains ligand efficiency, suggesting no significant
interaction at this vector; (iii) a chloro substitution in the 7-
position (compare C, R7 = Cl, with F, R7 = H) gave a 4-fold
increase in potency and retained ligand efficiency, while the
larger CF3 substituent (H) gave a significant reduction in both
potency and ligand efficiency, possibly indicating limited scope
for substitution at this position; (iv) the 4-position NH group of
the piperazine ring (substituent A) is a major contributor to
affinity. Replacement of the 4-position NH of fragment A with
NMe (fragment I; KD = 1800 nM) or a CF2 (fragment J; KD = 67
000 nM) leads to a loss in activity of 50-fold and 2000-fold,
respectively. The replace with the secondary amine group (A)
with an aliphatic ether oxygen (K) leads to ablation of activity.
Corresponding Authors
*(I.N.) E-mail: i.hopkinsnavratilova@dundee.ac.uk.
*(R.J.L.) E-mail: lefko001@receptor-biol.duke.edu.
*(A.L.H.) E-mail: a.hopkins@dundee.ac.uk.
Author Contributions
○T.A., S.A., and A.K.S. contributed equally to this work.
Funding
This work is funded by the MSD Scottish Life Sciences Fund (to
I.N. and I.H.G), in part by grants HL16037 and HL70631 from
the National Institutes of Health (to R.J.L.) and NIH contracts
and grants supporting drug discovery and receptor pharmacol-
ogy (to B.L.R.). R.J.L. is an Investigator of the Howard Hughes
Medical Institute. B.L.R. also received support from the Michael
Hooker Chair of Pharmacology. The work is funded in part by
the Innovative Medicines Initiative’s K4DD consortium under
grant agreement no 115366 (I.N. and A.L.H.) and benefits from
the Wellcome Trust Strategic Awards to the University of
Dundee (WT083481 and 100476/Z/12/Z).
Notes
The authors declare no competing financial interest.
E
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