Structural and Functional Analysis of G Protein-Coupled Receptor Kinase Inhibition by Paroxetine and a Rationally Designed Analog

Article abstract of DOI:10.1124/mol.113.089631

ABSTRACT Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein-coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanisms underlying paroxetine selectivity among GRKs. We directly measured the KD for paroxetine and assessed its mechanism of inhibition for each of the GRK subfamilies and then determined the atomic structure of its complex with GRK1, the most weakly inhibited GRK tested. Our results suggest that the selectivity of paroxetine for GRK2 largely reflects its lower affinity for adenine nucleotides. Thus, stabilization of off-pathway conformational states unique to GRK2 will likely be key for the development of even more selective inhibitors. Next, we designed a benzolactam derivative of paroxetine that has optimized interactions with the hinge of the GRK2 kinase domain. The crystal structure of this compound in complex with GRK2 confirmed the predicted interactions. Although the benzolactam derivative did not significantly alter potency of inhibition among GRKs, it exhibited 20-fold lower inhibition of serotonin reuptake. However, there was an associated increase in the potency for inhibition of other AGC kinases, suggesting that the unconventional hydrogen bond formed by the benzodioxole ring of paroxetine is better accommodated by GRKs. Copyright

Full text of DOI:10.1124/mol.113.089631

1521-0111/85/2/237–248$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.113.089631
Mol Pharmacol 85:237–248, February 2014
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Structural and Functional Analysis of G Protein–Coupled
Receptor Kinase Inhibition by Paroxetine and a Rationally
Designed Analog
Kristoff T. Homan, Emily Wu, Michael W. Wilson, Puja Singh, Scott D. Larsen,
and John J. G. Tesmer
Life Sciences Institute and the Departments of Pharmacology and Biological Sciences (K.T.H., E.W., P.S., J.J.G.T.), and
Vahlteich Medicinal Chemistry Core and the Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan
(M.W.W., S.D.L.)
Received September 17, 2013; accepted November 11, 2013
ABSTRACT
Recently we identified the serotonin reuptake inhibitor parox- reflects its lower affinity for adenine nucleotides. Thus, sta-
etine as an inhibitor of G protein–coupled receptor kinase 2 bilization of off-pathway conformational states unique to GRK2
(GRK2) that improves cardiac performance in live animals. will likely be key for the development of even more selective
Paroxetine exhibits up to 50-fold selectivity for GRK2 versus inhibitors. Next, we designed a benzolactam derivative of
other GRKs. A better understanding of the molecular basis of paroxetine that has optimized interactions with the hinge of the
this selectivity is important for the development of even more GRK2 kinase domain. The crystal structure of this compound
selective and potent small molecule therapeutics and chemical in complex with GRK2 confirmed the predicted interactions.
genetic probes. We first sought to understand the molecular Although the benzolactam derivative did not significantly alter
mechanisms underlying paroxetine selectivity among GRKs. We potency of inhibition among GRKs, it exhibited 20-fold lower
directly measured the KD for paroxetine and assessed its inhibition of serotonin reuptake. However, there was an associ-
mechanism of inhibition for each of the GRK subfamilies and ated increase in the potency for inhibition of other AGC kinases,
then determined the atomic structure of its complex with suggesting that the unconventional hydrogen bond formed by
GRK1, the most weakly inhibited GRK tested. Our results the benzodioxole ring of paroxetine is better accommodated by
suggest that the selectivity of paroxetine for GRK2 largely GRKs.
inhibition of GRK2, achieved via adeno-associated virus ad-
ministration of the C-terminal portion of GRK2 (bARKct),
Introduction
G protein–coupled receptor (GPCR) kinases (GRKs) initiate
effectively restores a normal phenotype and bAR signaling in
the termination of GPCR signaling via the phosphorylation
cellular and animal models of heart failure (Rockman et al.,
of cytoplasmic loops and carboxy-terminal tails of activated
1998; Akhter et al., 1999; Raake et al., 2013). Small molecule
GPCRs, which targets these receptors for deactivation through
inhibitors of GRK2 would also serve as important clinical
arrestin binding and clathrin-mediated endocytosis (Gurevich
therapeutics as well as useful biochemical probes to under-
et al., 2012). Proper regulation of GPCRs by GRKs is critical
stand GRK2 function in living cells (Shirakawa, 2009). Re-
for normal cellular signaling. For example, during heart fail-
cently, we identified the selective serotonin reuptake inhibitor
ure GRK2 is overexpressed, which leads to the uncoupling of
b adrenergic receptors (bARs) from normal sympathetic con-
trol (Ungerer et al., 1993, 1994). In fact, dominant negative
(SSRI) paroxetine, a Food and Drug Administration-approved
drug, as a relatively selective inhibitor of GRK2 (Thal et al.,
2012). Paroxetine was reported to exhibit an IC50 of 20 mM
and up to 50-fold selectivity over other GRKs such as GRK1
This work was supported by the National Institutes of Health National
and GRK5, which represent the GRK1 and GRK4 subfamilies,
respectively (Mushegian et al., 2012). Structural analysis of
the GRK2·paroxetine complex indicated that the drug occu-
pies the adenine, ribose, and polyphosphate binding subsites
in the active site (Fig. 1A). The residues that paroxetine
interact with in the active site are highly conserved among
Heart, Lung, and Blood Institute [Grants HL071818 and HL086865] to J.J.G.T.;
and the American Heart Association [Grant N014938] to K.T.H. Use of the
Advanced Photon Source was supported by the US Department of Energy,
Office of Science, Office of Basic Energy Sciences, under Contract No. DE-
AC02-06CH11357; and use of LS-CAT Sector 21 was supported by the
Michigan Economic Development Corporation and Michigan Technology Tri-
Corridor Grant [085P1000817].
dx.doi.org/10.1124/mol.113.089631.
ABBREVIATIONS: AMPPNP, adenosine 59-(b,g-imido)triphosphate; AST, active site tether; bΑR, b adrenergic receptor; bROS, bovine rod outer
segments; CCG-206584, 5-{[(3S,4R)-4-(4-fluorophenyl)piperidin-3-yl]methoxy}isoindolin-1-one hydrochloride; DDM, n-dodecyl-b-D-maltoside;
DMSO, dimethyl sulfoxide; GPCR, G protein–coupled receptor; GRK, G protein–coupled receptor kinase; HPLC, high-performance liquid
chromatography; PDB, Protein Data Bank; PKA, protein kinase A; PKC, protein kinase C; RH, regulator of G protein signaling homology; RMSD,
root-mean-square-deviation; SSRI, selective serotonin reuptake inhibitor.
237

238
Homan et al.
Fig. 1. Structure of paroxetine bound to GRK2 and of inhibitors used in this study. (A) Paroxetine in the active site of GRK2 in the GRK2·paroxetine–
Gbg complex (PDB ID 3V5W). The kinase domain large lobe is colored yellow and the small lobe green. Paroxetine is drawn as a stick model with carbons
colored cyan, oxygens red, and nitrogens blue. Three hydrogen bonds, including an unconventional C–O hydrogen bond, are shown as black dashed lines
with associated distances. Chemical structures of paroxetine (B) and its benzolactam derivative CCG-206584 (C) are shown.
GRKs, and thus the mechanism underlying its selectivity than other AGC kinases. Together, these results provide
for GRK2 could not be divined from this structure alone. important new insights into how paroxetine and its deriva-
tives function as selective inhibitors of GRKs.
However, because paroxetine stabilizes the kinase domain of
GRK2 in a conformation that has not been observed in other
reported GRK2·inhibitor complexes (Thal et al., 2011; Tesmer
et al., 2012), this structure could serve as a unique platform
for the development of GRK2 selective inhibitors distinct from
other classes of currently known GRK2 inhibitors as well as
from other protein kinase A, G, and C (AGC kinase) family
members.
Materials and Methods
Protein Purification. Rhodopsin in bovine rod outer segments
(bROS) was prepared as previously reported (Papermaster, 1982).
Bovine GRK11–535, bovine and human GRK2S670A, bovine GRK5, and
human palmitoylation deficient GRK6 (pal2) were purified via
a common procedure consisting of Ni-NTA affinity, Source15S, and
tandem S200 size exclusion chromatography as previously described
(Lodowski et al., 2006; Thal et al., 2012). Bovine Gbg-C68S mutant
(Gbg), which is not geranylgeranylated and, thus, is not membrane
associated, was expressed in High Five cells and purified via Ni·NTA
affinity, MonoQ, and tandem S200 size exclusion chromatography as
previously reported (Thal et al., 2012).
Biolayer Interferometry. GRKs were biotinylated with 400 mM
EZ-Link HPDP-Biotin (ThermoScientific, Waltham, MA) for 2 hours
at room temperature or overnight at 4°C prior to purification via
tandem S-200 size exclusion chromatography to isolate only properly
folded biotinylated protein. A FortéBio (Menlo Park, CA) Octet RED
system was used to measure the binding of small molecules to each
GRK. Streptavidin-coated tips were soaked for at least 15 minutes to
rehydrate the streptavidin prior to protein loading. Biotinylated
GRKs (0.05 mg/ml) in assay buffer (20 mM HEPES pH 7.0, 5 mM
MgCl2, and 0.025% DDM [n-dodecyl-b-D-maltoside]) were then bound
to the streptavidin tips for 30 minutes and washed in buffer for 10
minutes to remove any unbound kinase. DDM was included to help
disrupt any potential small molecule aggregates, and the concentra-
tion used does not significantly inhibit GRK activity (data not shown).
These conditions were designed to be similar to those used for kinetic
assays. The protein-loaded tips were then sequentially placed in a
buffer supplemented with 1% dimethyl sulfoxide (DMSO) for 1 minute
With the long-term goal of developing more selective inhib-
itors of GRK2, we sought to determine the molecular basis for
the selectivity of paroxetine for GRK2 by directly determining
the affinity of paroxetine for various GRKs, its inhibition con-
stants and mechanisms of inhibition for GRK1 and GRK2, and
its atomic structure in complex with GRK1, the GRK most
weakly inhibited by paroxetine. These results suggest that
paroxetine traps the kinase domain of GRKs in a conformation
similar to that used to bind ADP and that the selectivity of
paroxetine among GRKs is driven primarily by differences in
their affinities for adenine nucleotides, in particular ADP. To
probe the role of an unusual hydrogen bond formed by the
benzodioxole ring of paroxetine in the GRK active site, we
modeled and then synthesized a benzolactam derivative of
paroxetine (CCG-206584; 5-{[(3S,4R)-4-(4-fluorophenyl)piperidin-
3-yl]methoxy}isoindolin-1-one hydrochloride) that would have
optimized hydrogen bonds with the hinge region of GRK2
(Fig. 1, B and C). This alteration did not lead to more potent
inhibition of the GRKs, but did exhibit a 20-fold reduction in
SSRI activity, confirming that kinase inhibition can readily be
uncoupled from “off target” effects. We then solved the crystal
structure of the GRK2·CCG-206584 complex, which confirmed
our design principles. However, other AGC kinases exhibited
one order of magnitude increased potency of inhibition
relative to the parent compound. Thus, GRKs seem better
to reach
a stable baseline, then in buffer supplemented with
paroxetine and 1% DMSO for 3 minutes, and then placed back into
the baseline buffer to allow dissociation to occur for 5 minutes.
Changes in refractive index were analyzed with FortéBio’s Data
suited to accommodate the benzodioxole ring of paroxetine Analysis 7.0, and averaged reference sensor measurements were

Inhibition of GRKs by Paroxetine Analogs
239
used for baseline subtraction prior to data processing. The association
were subjected to an elliptical mask with major and minor axes
and dissociation curves were individually fit using GraphPad Prism corresponding to the highest resolution data limits (Lodowski et al.,
(GraphPad Software, Inc., La Jolla, CA) using a two-phase model. The 2003). Indexing, integration, and scaling were performed with HKL2000
final kon and koff rate constants were constrained to be shared among (Otwinowski and Minor, 1997). A molecular replacement solution was
all data sets for each individual GRK and used to calculate the
ensemble dissociation constants (KD 5 koff/kon).
achieved with the Phaser module of CCP4 using PDB ID 3C50 as a search
model (McCoy et al., 2007; Winn et al., 2011). Refinement was performed
Kinetic Assays. GRK kinetic assays were conducted in a buffer with the Refmac5 module of CCP4 and model building was conducted
containing 20 mM HEPES pH 7.0, 2 mM MgCl2, and 0.025% DDM with
with Coot (Murshudov et al., 1997; Emsley and Cowtan, 2004).
50 nM GRK and 500 nM ROS in 5-minute reactions. The low salt con- Differential crystal contacts involving each of the regulator of G protein
centration and DDM were used to maximize GRK activity and disrupt signaling homology (RH) domains leads to conformational differences in
small molecule aggregates from forming, respectively. For mechanism the loops linking helices a6, a7, and a8. In chain A, the active site tether
of inhibition by paroxetine with respect to ATP, the concentration of region is completely ordered due to unique lattice contacts, whereas the
ATP was varied 1–100 mM, and the concentration of paroxetine was analogous region of chain B exhibits a similar degree of order as observed
varied 4–1000 mM. Reactions were quenched with SDS loading buffer, in other GRK1 ADP complexes (Singh et al., 2008). The final model was
separated via SDS-PAGE, dried, and exposed with a phosphorimaging validated with MolProbity (Chen et al., 2010) prior to deposition of
screen prior to quantification via Typhoon imager, as previously re- coordinates and structure factors (PDB ID 4L9I).
ported (Thal et al., 2012). Data were analyzed and inhibition curves
GRK2 CCG-206584–Gbg Structure Determination. Human
were fit via GraphPad Prism. The most appropriate model of inhibition GRK2 and Gbg were mixed in a 1:1 ratio and concentrated to a final
was determined using the Akaike Information Criteria by comparing total protein concentration of 4.5 mg/ml in the presence of 1 mM CCG-
competitive and noncompetitive models of inhibition. Protein kinase C 206584 (from a 50 mM stock in DMSO) and 2 mM MgCl2. Crystals
(PKC) assays were conducted with the PKCa kinase enzyme system
(Promega, Madison, WI) in which 0.1 mg PKCa was added to 1 mg
CREBtide substrate (KRREILSRRPSYR), PKC lipid activator, and
were obtained via the vapor diffusion method using hanging drops
consisting of 0.8 ml protein solution added to an equal volume of well
solution, which consisted of 1.2 M NaCl, 100 mM 4-morpholineetha-
50 mM ATP for 30 minutes. After the initial reaction, ADP-Glo Reagent nesulfonic acid 7.0, and 9% PEG3350. Crystals appeared in approx-
was added to the reaction and allowed to incubate for an additional imately 4 days and continued to grow in size for several weeks. During
40 minutes. Finally the kinase detection reagent was added and allowed harvesting the crystals were cryoprotected through the addition of
to incubate for 30 minutes and the luminescence was measured with
a BMG Labtech PHERAstar (Ortenberg, Germany) imaging system.
Protein kinase A (PKA) assays were performed with the ADP-Glo
system using 0.1 mg PKA and 1 mg kemptide substrate (LRRASLG), and
50 mM ATP for 30 minutes prior to addition of kinase detection reagent
and imaging on a Pherastar system.
25% ethylene glycol to the harvested crystals prior to flash freezing in
liquid N2. Diffraction data were collected at Advanced Photon Source
on LS-CAT beamline ID-D at a wavelength of 1.1272 Å. The diffraction
data collected were highly anisotropic, with reflections extending to
2.4 and 3.5 Å spacings in the highest and lowest resolution directions,
respectively. The data were subjected to an elliptical mask with major
Fluorescence Polarization Displacement Assay. Each kinase and minor axes corresponding to the highest resolution data limits
was first incubated with BODIPY TR-ADP (Invitrogen, Carlsbad, CA)
before scaling (Lodowski et al., 2003). Indexing, integration, and
in assay buffer (20 mM HEPES pH 8, 150 mM NaCl, 5 mM MgCl2 and scaling were performed with HKL2000 (Otwinowski and Minor,
2 mM dithiothreitol) for 10 minutes at room temperature in the dark.
Note that higher ionic strength is used than in the Octet Red and
kinetic assays, but because inhibitor binding is not expected to be
1997). A molecular replacement solution was achieved with the
Phaser module of CCP4 using PDB ID 3V5W as a search model
(McCoy et al., 2007; Winn et al., 2011; Thal et al., 2012). Refinement
strongly salt dependent, such should not prevent comparing relative was performed with the Refmac5 module of CCP4 and model building
effects among GRKs in each assay. The GRK-fluor mixture was then was conducted with Coot (Murshudov et al., 1997; Emsley and
incubated with various concentrations of small molecule compounds Cowtan, 2004; Winn et al., 2011). In the GRK2·CCG-206584 complex,
in a 96-well plate (Corning, Corning, NY; black plate). The plate was
incubated at room temperature for an additional 10–15 minutes in
the dark before measuring millipolarization on a PHERAstar plate
there are several minor structural differences from the analogous
GRK2·paroxetine complex, such as in the a5–a6 loop of the RH
domain and ordering of the b1–b2 hairpin loop in the pleckstrin
reader (excitation at 575 nm and emission at 620 nm). The concen- homology domain, likely due to differences in crystal packing
tration of kinase used during competition assays [3.5 mM bovine GRK1, environments. The final model was verified with MolProbity (Chen
4 mM human GRK2, and 4 mM human GRK6 (pal2)] corresponds to the et al., 2010) prior to deposition (PDB ID 4MK0).
KD estimated from equilibrium direct binding fluorescence polarization
Chemistry. Chemical names follow Chemical Abstracts Service
assays in which each GRK was titrated against a fixed concentration nomenclature. Starting reagents and solvents were purchased from
of BODIPY TR-ADP (25, 50, and 100 nM, respectively) wherein Fisher Scientific (Hampton, NH) and (3S,4R)-tert-butyl 4-(4-fluorophe-
nonspecific binding was determined by addition of 20 mM EDTA. nyl)-3-(hydroxymethyl)piperidine-1-carboxylate (1) was obtained from
GraphPad Prism was used to fit curves according to a one site com- Astatech (Bristol, PA). 5-Hydroxyisoindolin-1-one was purchased from
petition with ligand depletion model wherein the KD variable was Sphinx Chemical. All chemicals were used without purification.
shared between each individual experiment for each GRK.
Reactions were monitored by thin layer chromatography using pre-
GRK1·Paroxetine Crystal Structure Determination. Parox- coated silica gel 60 F254 plates. Silica gel chromatography was per-
etine (from a 10 mM stock dissolved in water) and MgCl2 were added to a formed with silica gel (220–240 mesh) obtained from Silicycle (Quebec
7–8 mg/ml bGRK1535 protein solution to attain a final concentration of 5
City, QC, Canada). NMR spectra were recorded on a Varian (Wash-
and 2 mM, respectively. Crystals were obtained via vapor diffusion using
ington, DC) 400 MHz spectrometer. Chemical shifts are reported in d
hanging drops consisting of 1.0 ml of protein and 1.0 ml of well solution, (parts per million) by reference to the hydrogenated residues of deu-
which consisted of 1 M NaCl, 100 mM 4-morpholineethanesulfonic acid terated solvent as internal standard CDCL3: d 5 7.28 (1H-NMR). Mass
pH 5.75, and 12–15% PEG3350. Crystals appeared in approximately
3 days and continued to grow in size for at least 1 week. During
spectra were recorded on a Micromass Liquid Combustion Technology
time-of-flight (Waters Corporation, Milford, MA) instrument utilizing
harvesting, the crystals were cryoprotected by addition of 25% ethylene the electrospray ionization mode. The purity of the compounds was
glycol to the drops prior to flash freezing in liquid nitrogen. Diffraction
data were collected at the Advanced Photon Source on the LS-CAT
beamline ID-G at a wavelength of 1.0793 Å. The diffraction data collected
was anisotropic with reflections extending to 2.3 and 3.4 Å spacings in the
highest and lowest resolution directions, respectively. As such, the data
assessed via analytical reverse phase high-performance liquid chroma-
tography (HPLC) with a gradient of 10–90% acetonitrile:water over 6
minutes (C18 column, 3.5 mm, 4.6 Â 100 mm, 254 nm detection).
(3S,4R)-Tert-Butyl
4-(4-Fluorophenyl)-3-[(Tosyloxy)meth-
yl]piperidine-1-Carboxylate (2). p-Toluenesulfonyl chloride (0.03 g,

240
Homan et al.
0.16 mmol) was added to a 0°C solution of compound 1 (0.05 g, 0.16
mmol) in dry 1:1 tetrahydrofuran/pyridine (2 ml). The resulting
mixture was stirred for 40 minutes at 0°C then for 2 hours at room
temperature. The starting material was consumed by thin layer chro-
matography and HPLC. The mixture was concentrated on a rotary
evaporator and dried under high vacuum for 1 hour. The resulting
brown oil was used without further purification.
GRK2 with greater affinity than to other GRKs. To inves-
tigate whether differences in the binding or dissociation
kinetics of paroxetine contribute to differences in its IC50
values, we used biolayer interferometry, wherein the GRK is
immobilized on a waveguide and changes in refractive index are
measured as a function of time after addition or dilution of a small
(3S,4R)-Tert-Butyl 4-(4-Fluorophenyl)-3-{[(1-Oxoisoindolin-
5-yl)oxy]methyl}piperidine-1-Carboxylate (3). Cesium carbonate
(0.69 g, 2.1 mmol) was added to a room temperature solution of
5-hydroxyisoindolin-1-one (0.32 g, 2.11 mmol) in dry dimethylformamide
(2 ml). The resulting mixture was stirred 30 minute at room temperature
before adding a solution containing compound 2 (0.33 g, 0.71 mmol) in
dimethylformamide (2 ml). The resulting mixture was heated at 65°C for
1 hour and then cooled to 50°C and stirred overnight. The reaction mixture
was then cooled to room temperature and extracted 2Â with equal volumes
of ether. The combined ether extracts were washed with 1 N NaOH (2Â),
saturated NaCl (1Â), and dried (MgSO4). After evaporation in vacuo, the
crude oil was purified by flash chromatography (MeOH/CH2Cl2 gradient).
Obtained compound 3 (0.1 g, 0.23 mmol, 31.9% yield) was a yellow oil.
HPLC purity: 92% (tR 5 7.4 minutes). 1H-NMR (400 MHz, chloroform-d) d
7.68 (d, J 5 8.5 Hz, 1H), 7.23 (m, 1H), 7.12 (ddd, J 5 8.0, 5.3, 2.3 Hz, 2H),
7.04–6.88 (m, 2H), 6.83 (dd, J 5 8.4, 2.2 Hz, 1H), 6.73 (d, J 5 2.1 Hz, 1H),
4.48 (m, 1H), 4.32 (s, 2H), 4.21 (m, 1H), 3.72 (dd, J 5 9.4, 2.9 Hz, 1H), 3.57
(dd, J 5 9.4, 6.6 Hz, 1H), 2.90–2.47 (m, 3H), 2.22–1.86 (m, 1H), 1.86–1.53
(m, 2H), 1.47 (s, 9H). Electrospray ionization in the positive mode mass
spectrometry m/z 385.1 (M1H1-t-butyl).
CCG-206584. HCl (4 M) in dioxane (0.5 ml, 2 mmol) was slowly
added to a solution of compound 3 (0.08 g, 0.182 mmol) in methylene
chloride. The mixture was stirred 2 hour at room temperature before
concentrating. The residue was triturated in EtOAc and ether and
then filtered to obtain the HCl salt of 5-{[(3S,4R)-4-(4-fluorophe-
nyl)piperidin-3-yl]methoxy}isoindolin-1-one (0.04 g, 0.12 mmol, 68%
yield) as a white hygroscopic solid. HPLC purity: 100% (tR 5 4.4
minutes). 1H-NMR (400 MHz, DMSO-d6) d 8.94 (s, 2H), 8.28 (s, 1H),
7.48 (d, J 5 8.2 Hz, 1H), 7.36–7.03 (m, 3H), 7.03–6.73 (m, 2H), 4.22 (s,
2H), 3.78–3.57 (m, 2H), 3.57–3.40 (m, 1H), 3.36 (d, J 5 12.4 Hz, 1H),
3.11–2.73 (m, 3H), 2.08–1.62 (m, 3H). Electrospray ionization in the
positive mode mass spectrometry m/z 341.1 (M1H1).
Thermal Denaturation Studies. Thermal denaturation assays
were conducted using a ThermoFluor (Johnson & Johnson, New
Brunswick, NJ) plate reader as previously described in a buffer containing
20 mM HEPES pH 7.0, 5 mM MgCl2, 2 mM dithiothreitol, and 1 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid with
0.2 mg/ml final concentration of GRK and 100 mM anilinonaphthalene-
8-sulfonic acid (Thal et al., 2012). The 3-[(3-cholamidopropyl)di-
methylammonio]-1-propanesulfonic acid detergent was used as pre-
viously reported to reduce the presence of aggregates in the experiment.
Serotonin Reuptake Inhibition. Human embryonic kidney 293
cells stably transfected with the serotonin reuptake transporter were
cultured in Dulbecco’s modified Eagle’s medium media supplemented
with 10% fetal bovine serum and 1% PenStrep. For each reaction, 105
cells were added to each well of a 100 mm 24-well plate that had been
coated with 0.05 mg/ml poly-D-lysine. Serotonin transporters were
blocked through a 1-hour preincubation with compound, which was
then washed 3Â with a buffer containing 25 mM HEPES pH 7.4,
120 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, and 1.3 mM
KH2PO4. Next, 100 nM total 5-hydroxytryptamine (20 nM [3H]5HT)
was added to the wells to initiate a 10-minute reaction that was
terminated by 3 washes of cold phosphate-buffered saline prior to
lysis of the cells with 1% SDS. Lysed cells were added to 5 ml of
scintillation cocktail, and tritium counts were recorded. Data were
plotted and inhibition curves fit with GraphPad Prism.
Fig. 2. Quantitative measurement of ligand binding to GRKs. Rates of
paroxetine association (A) and dissociation (B) using GRKs representing
each GRK subfamily (GRK1, GRK2, and GRK5), as measured by biolayer
interferometry on an Octet RED. Both sets were best fit by a biphasic
model that was interpreted to be comprised of both specific and nonspecific
interactions between paroxetine and the protein. Only the specific
portions of kon (1600 6 120, 1200 6 110, and 1100 6 62 M21·sec21 for
GRK1, GRK2 and GRK5, respectively) and koff (0.021 6 0.0003, 0.008 6
0.0002, and 0.015 6 0.0002 second21 for GRK1, GRK2, and GRK5,
respectively) were used to calculate KD values (13, 7.3, and 14 mM,
respectively). Error bars represent the S.E.M. for 3 independent experi-
ments performed in duplicate. C, Fluorescence polarization competition
binding experiments revealed a 30-fold higher affinity binding of ADP to
GRK1 (1.4 mM) versus GRK2 (46 mM). Error bars represent S.E.M. for 2
Results
The most straightforward explanation for selective in-
hibition of GRK2 by paroxetine would be if the drug binds to independent experiments performed in triplicate.

Inhibition of GRKs by Paroxetine Analogs
241
TABLE 1
Interaction of small molecules with GRKs
Values represent mean 6 S.E.M.
GRK1
GRK2
GRK5
GRK6
mM
KD (paroxetine)
13
7.3
14
N.D.
N.D.
Ki (paroxetine) (versus ATP)
KM (ATP)
78 6 10
3.2 6 0.5
1.4 6 0.2
49 6 15
0.55 6 0.2
0.34
3.5 6 0.9
8.8 6 0.5
46 6 16
110 6 20
N.D.
N.D.
N.D.
N.D.
N.D.
0.16
N.D.
KD (ADP) (FP)
11 6 2
190 6 7
0.30 6 0.15
0.49
KD (AMPPNP) (FP)
KD (balanol) (FP)
IC50 (balanol) a
0.074 6 0.07
0.042
FP, measured by fluorescence polarization (this study); N.D., not determined.
aFrom Tesmer et al., 2010.
molecule. The technique can determine rates of association (kon), weakly and strongly inhibited GRKs we have tested, re-
spectively (Table 1). A caveat in interpreting these assays is
that they cannot be run under strict Michaelis-Menten con-
ditions, and, because the reaction is bireactant, the resulting
inhibition constants are approximations that also depend on
the concentration of and KM for rhodopsin, as well as changes in
affinity for one substrate that occur upon binding the other.
The estimated Ki value for GRK2 (3.5 mM) is 20-fold lower than
for GRK1 (78 mM), similar to the 50-fold difference measured
for their IC50 values (Table 1). Surprisingly, the inhibition data
for GRK1 and GRK2 was best fit by different models. Namely,
the mechanism of paroxetine inhibition was noncompetitive for
GRK1 (R2 5 0.75, .99.9% probability according to the Akaike
Information Criteria) and competitive for GRK2 (R2 5 0.71,
.99.9% probability) with respect to ATP.
dissociation (koff), and binding constants (e.g., KD 5 koff/kon).
The kon and koff rates of paroxetine for GRK1, GRK2, and
GRK5, representing each of the three vertebrate GRK
subfamilies (Mushegian et al., 2012), were similar (Fig. 2, A
and B; Table 1), although approximately 2-fold higher affinity
was measured for GRK2. Thus, the selectivity we observed in
our kinetic assays is not entirely accounted for by preferential
association of the drug with GRK2.
Next, we turned to enzymatic assays in which we measured
GRK-mediated phosphorylation of light-activated bROS where-
in the concentration of ATP was varied versus several parox-
etine concentrations to determine the inhibition constants (Ki)
for GRK1 (Fig. 3A) and GRK2 (Fig. 3B), which are the most
Considered along with the bio-layer interferometry data, these
results suggest that differences in affinities of these GRKs for
competing substrates or products may be chiefly responsible for
selectivity observed in enzymatic assays. We therefore deter-
mined the KD of the ATP analog adenosine 59-(b,g-imido)
triphosphate (AMPPNP) and ADP, representing the pre- and
posthydrolysis states of the phosphotransfer reaction, using
a fluorescence polarization assay in which we monitored the
displacement of BODIPY-TR ADP from the enzyme (Fig. 2C;
Table 1). GRK2 exhibited an approximately twofold higher KD
for AMPPNP than GRK1 (106 versus 49 mM), but a 30-fold
higher KD for ADP than GRK1 (46 versus 1.4 mM). Thus, during
the catalytic cycle GRK2 retains ADP much less efficiently than
GRK1, and thus is more susceptible to inhibition by paroxetine
during catalysis. Interestingly, balanol exhibits ∼10-fold selec-
tivity for GRK2 based on IC50 values (Tesmer et al., 2010) and
has 5- to 6-fold higher affinity for GRK2 as measured in the
BODIPY-TR ADP displacement assay (Table 1). Thus, the
selectivity exhibited for GRK2 by compounds chemically un-
related to paroxetine does not seem as strongly dependent on the
relative affinity of GRKs for ADP.
Next, we determined the crystal structure of GRK1 in
complex with paroxetine using diffraction data extending to
2.3 Å spacings in the highest resolution direction (Table 2).
The complex forms an asymmetric dimer in the asymmetric
unit of the crystals using the same RH domain-mediated
twofold interface as observed in all prior structures of wild-
type GRK1. The two GRK1 molecules exhibit a Ca root-mean-
square-deviation (RMSD; 492 atomic pairs) of 0.69 Å for the
entire molecule, and 0.47 Å (323 atomic pairs) when just the
kinase domain structures are compared. Strong electron
density for paroxetine is observed in the active sites of each
Fig. 3. Inhibition kinetic assays with GRK1 and GRK2. Paroxetine was
determined to inhibit GRK1 (A) noncompetitively (.99% likelihood), with
a Ki of 78 mM versus ATP, whereas GRK2 (B) is inhibited competitively
(.99% likelihood), with a Ki of 3.5 mM versus ATP. Error bars represent
S.E.M. for 3 independent experiments performed in duplicate.

242
Homan et al.
TABLE 2
Crystallographic statistics
Low completeness values reflect the fact that an elliptical mask was applied prior to scaling was used to accommodate
highly anisotropic diffraction data (Lodowski et al., 2003). Without the mask, data had 82.4% overall completeness and
82% in the highest resolution shell for 4MK0 and 100% overall completeness and 100% completeness in the highest
resolution shell for 4L9I.
Protein Complex
GRK2·CCG-206584–Gbg
GRK1·Paroxetine
X-ray source
LS-CAT ID-D
1.1272
19.99-2.4 (2.44-2.4)
C2221
a=61.2, b=240.9, c=212.0
30794
LS-CAT ID-G
1.0793
29.9-2.32 (2.36-2.32)
P 212121
a=66. 8, b=122.1, c=152.9
54845
Wavelength (Å)
Dmin(Å)
Space group
Cell constants
Unique reflections
Rsym (%)
10.6 (39.0)
49.9 (1.0)
61.7/4.0 (7.0/3.0)
5.0 (2.8)
10.9 (94.9)
36.9 (0.8)
49.7/1.8 (5.4/2.0)
9.1 (9.2)
Completeness (%)
I/sI
Redundancy
Refinement resolution (Å)
Total reflections used
RMSD bond lengths (Å)
RMSD bond angles (°)
Est. coordinate error (Å)
Ramachandran plot
Most favored, outliers (%)
Rwork
20.0-2.4 (2.47-2.4)
29,079
29.9-2.32 (2.38-2.32)
51,966
0.004
0.009
0.875
0.409
1.4
0.30
94.1, 0.1
96.7, 0.1
19.5 (38.3)
18.8 (24.5)
Rfree
24.6 (81.1)
22.9 (28.8)
Protein atoms
8235
7894
Water molecules
Inhibitor atoms
147
25
309
48
Average B-factor (Å2):
Protein
55.3
23.4
55.9
19.4
Inhibitor
50
20.8
MolProbity score
MolProbity Ramachandran outlier
MolProbity Cb deviations
MolProbity bad backbone bonds
MolProbity bad backbone angles
PDB ID
1.28 (100th percentile)
1.37 (100th percentile)
1
0
0
0
1
0
0
0
4MK0
4L9I
kinase domain (Fig. 4, A and B) in a conformation essentially GRK1 active site tether forms no direct contacts with the
identical to that of paroxetine bound to GRK2. In both chains, drug. The piperidine ring of paroxetine seems better ordered
the kinase domain adopts a partially closed conformation that in the GRK1 structure, and its nitrogen forms hydrogen bonds
most closely resembles those of GRK1 in complex with ADP with the side chain of Asp271 and the backbone oxygen of
such as in PDB IDs 3C50 (Singh et al., 2008), 3C4Z (Singh Glu318. In GRK2, the piperidine nitrogen forms a hydrogen
et al., 2008), and 3QC9 (Huang et al., 2011) [Z-scores of 25.4, bond to a water molecule and to GRK2-Ala321 (analogous to
25.2, and 20.0, respectively, using the PDBeFold server GRK1-Glu318). This difference likely results from the slightly
(Krissinel and Henrick, 2004)]. The kinase domains of 3C50 different conformation of these kinase domains. The phos-
and 3C4Z exhibit a Ca RMSD of 0.64 Å RMSD (322 atomic phate binding loops in the structures of the GRK1·paroxetine
pairs) and 0.65 Å (326 atomic pairs), respectively, versus and GRK2·paroxetine complexes also adopt distinct confor-
chain A of the paroxetine complex. The kinase domain in the mations, with atoms within the phosphate binding loop of the
GRK1·paroxetine complex is, however, in a slightly different GRK2·paroxetine up to 0.9 Å closer to the bound drug than in
conformation, and a 3° rotation of the large lobe relative to the the GRK1·paroxetine structure (Figs. 4C and 7B). However,
small lobe is required to achieve the best alignment with the neither conformation is more collapsed than the analogous
ADP complexes. Interestingly, the GRK2 kinase domain in loop in the GRK1·ADP complex, likely a consequence of its
complex with paroxetine (Thal et al., 2012) is also more interactions with the diphosphate moiety of the nucleotide
similar to that of GRK1·ADP (2.3 Å RMSD; 435 atomic pairs) (Fig. 4C). Despite these differences, the buried accessible
than to those of other reported GRK2 structures. Thus, par- surface area of paroxetine in the GRK1 and GRK2 structures
oxetine seems to stabilize GRKs in a conformation similar are nearly identical: 280 and 270 Å2, respectively, consistent
to their ADP-bound state. Unfortunately, the structure of a with our observations that paroxetine exhibits similar KD
GRK2·ADP complex is not currently available to confirm this values toward each enzyme.
prediction.
In the crystal structure of the GRK2·paroxetine–Gbg com-
Several structural differences are evident upon comparison plex (Thal et al., 2012), one of the most striking interactions
of the structures of GRK1 and GRK2 in complex with par- between GRK2 and paroxetine are two specific hydrogen
oxetine (Fig. 4C). A 5° rotation of the large lobe of GRK2 bonds formed between the backbone atoms of residues in the
relative to the small lobe is required to attain the same degree hinge of the kinase domain and an oxygen and a carbon in
of closure of the kinase domain in the GRK1·paroxetine the small molecule (Fig. 1A). Carbon-oxygen hydrogen bonds
complex. In GRK2, there are hydrophobic interactions formed are unusual and have lower free energy than conventional
between paroxetine and the active site tether loop, but the hydrogen bonds (Horowitz and Trievel, 2012). Therefore,

Inhibition of GRKs by Paroxetine Analogs
243
substitution of the benzodioxole ring with a similar sized
aromatic system, such as a benzolactam, might yield a more
potent inhibitor due to the formation of two strong hydrogen
bonds with the hinge. The benzolactam derivative of parox-
etine (CCG-206584) was synthesized in a straightforward
three-step synthesis from commercially available alcohol (1)
embodying both of the requisite stereocenters (Scheme 1).
Tosylation of the alcohol and base-catalyzed displacement of
the resulting tosylate (2) with 5-hydroxyisoindolin-1-one pro-
vided N-Boc benzolactam (3). Cleavage of the Boc group under
acidic conditions then afforded the HCl salt of the desired
analog (Fig. 1C).
To evaluate whether CCG-206584 was capable of interact-
ing with GRKs in a manner similar to that of paroxetine, we
first measured its ability to thermostabilize GRKs from each
of the three vertebrate subfamilies. CCG-206584 caused small
but statistically significant increases in the melting points of
GRK1 and GRK5 relative to paroxetine (Fig. 5A): (2.3°C for
GRK1, 0.8°C for GRK2, and 0.8°C for GRK5), consistent with
the formation of improved hydrogen bonds. We next evaluated
the potency of inhibition by CCG-206584 for GRK1, GRK2,
and GRK5 using two substrates: bROS and the soluble sub-
strate tubulin (Fig. 5, B and C; Table 3). There were no sig-
nificant changes in the affinity of CCG-206584 relative to
paroxetine for any of the GRKs. We also tested the potency of
inhibition against PKA and PKC, representing the broader
AGC kinase family. Paroxetine was determined to have IC50
values of 45 and 220 mM and CCG-206584 to have IC50 values
of 2.6 and 26 mM for PKA and PKC, respectively (Fig. 6A).
Thus, the introduction of conventional hydrogen bonds in
CCG-206584 increased potency of inhibition of other AGC
kinase family members and decreased selectivity for GRKs.
Development of the paroxetine scaffold into a kinase inhib-
itor must also coincide with reducing the “off-target” effect of
serotonin reuptake inhibition. Previous studies have probed
the structure-activity relationship of derivatives of SSRIs
such as paroxetine (Marcusson et al., 1992; Mathis et al.,
1992, 1994), but a benzolactam derivative of paroxetine has
not been reported. To assess how the alterations made to the
paroxetine scaffold in CCG-206584 change the SSRI function
of paroxetine, human embryonic kidney 293 cells stably
transfected with the serotonin transporter were used to com-
pare the inhibition of serotonin reuptake by paroxetine and
CCG-206584 (Fig. 6B). CCG-206584 was ∼20-fold less able to
from 2.8 to 3.2 Å are formed with main chain atoms in the hinge (Thr265
and Met267) and the large lobe (backbone of Glu318 and side chain of
Asp271). (C) The conformation of the kinase domain in the structure of the
GRK1·paroxetine complex (light blue) strongly resembles its conformation
in the GRK2·paroxetine (PDB ID 3V5W, green) and GRK1·ADP (PDB ID
3C4Z, beige) complexes. The active site tether (AST) loop of Chain A in the
GRK1·paroxetine complex is more distant from the drug than in GRK2
complex and forms no direct contacts, and its phosphate-binding loop
(P-loop) is displaced furthest from the ligand binding site relative to the
other two structures. For example, there is a 2.3 Å displacement of the Ca
atom of Gly196 in the P-loop between the ADP and paroxetine complexes
of GRK1, likely due to engagement of the P-loop by the polyphosphate
tail of ADP in the GRK1·ADP structure. Atoms in the P-loop of the
GRK1·paroxetine structure are displaced by up to 0.9 Å away from the
drug relative to the GRK2·paroxetine structure. ADP from the 3C4Z
structure (stick model with carbons colored beige) is shown for reference.
Both ADP and paroxetine form analogous hydrogen bonds with backbone
atoms of Thr265 and Met267 in the hinge of the GRK1 kinase domain.
Fig. 4. Structural analysis of the GRK1·paroxetine complex. (A) Overview
of the complex. Paroxetine binds to GRK1 in the active site at the interface
of the small and large lobes of kinase domain (light blue). The RH domain
(darker blue) is shown for reference. An omit map calculated at 3 s (green
mesh) is shown for paroxetine (stick model with carbons colored yellow,
nitrogens blue and fluorines teal) within the active site. (B) Detailed view
of paroxetine within the GRK1 active site. Hydrogen bonds formed by
paroxetine (black dashed lines terminating in gray spheres) that range

244
Homan et al.
Scheme 1. Synthesis of CCG-206584. a,
TsCl, tetrahydrofuran, pyr; b, 5-hydroxyi-
soindolin-1-one, Cs2CO3; c, HCl, dioxane.
inhibit serotonin uptake (IC50 5 38 6 8 nM) compared with
paroxetine (IC50 5 2 6 0.3 nM), demonstrating that SSRI
activity can readily be uncoupled from GRK inhibition.
Recently we identified the SSRI paroxetine as an inhibitor
of GRK2 that exhibits 50-fold selectivity over other GRKs
(Thal et al., 2012). Paroxetine was shown to effectively
penetrate into cells, where it inhibits GRK2-dependent phos-
phorylation of an activated GPCR with an IC50 of ∼35 mM,
simulates increased contraction in isolated mouse cardiomyo-
cytes, and generates more powerful contraction of the heart
without associated changes in heart rate in live animals
(Thal et al., 2012). A better understanding of the origins of
paroxetine’s selectivity for GRK2 would assist in the design of
improved, more selective chemical probes based on the
paroxetine scaffold, itself being a successful Food and Drug
Administration-approved drug since 1992. A common expla-
nation for selectivity is differences in binding or dissociation
kinetics due to distinct interactions with the target protein.
However, bio-layer interferometry experiments showed that
paroxetine binds to GRK1, GRK2, and GRK5 similarly with
∼10 mM affinity, although highest affinity was for GRK2 (Fig.
2, A and B; Table 1). Although unexpected, this result is
consistent with paroxetine binding in the active site of the
kinase domain where sequence and structural identity among
GRKs is very high. We therefore hypothesized that the ∼50-
fold selectivity for GRK2 based on IC50 values is due to
differences in the ability of the various GRKs to bind their
nucleotide substrates and/or products, which would compete
for the same site as paroxetine. Kinetic analysis of GRK1,
the most weakly inhibited GRK, and GRK2, the most
strongly inhibited, revealed that the two enzymes exhibit
a 20-fold (78 versus 3.5 mM, respectively) difference in their
Ki values (Table 1), approximately fivefold lower than their
respective IC50 values. As expected for an inhibitor that
binds in the active site, paroxetine exhibits a competitive
mode of inhibition for GRK2 versus ATP (Fig. 3B). However,
paroxetine inhibition of GRK1 is best fit with a noncompet-
itive model with respect to ATP (Fig. 3A). A noncompetitive
mode of inhibition for a drug that binds in the active site is
not without precedent in the AGC kinase family of enzymes.
For example, staurosporine acts as a noncompetitive in-
hibitor of PKC (Ward and O’Brian, 1992), although crystal
structures of the drug in complex with PKC (Xu et al., 2004)
or PKA (Prade et al., 1997) demonstrate that the staur-
osporine binding site overlaps with that of ATP. The dif-
ference in mode of GRK inhibition may reflect different rate
limiting steps in these GRKs or that assumptions made in
deriving Michaelis-Menten parameters are not fully valid, as
necessitated by the assay conditions used for GRKs.
To confirm our rational design approach, we also de-
termined the crystal structure of the GRK2·CCG-206584–
Gbg complex (Fig. 7; Table 2). Despite a different space group
and unique crystal packing relative to the structure of the
GRK2·paroxetine–Gbg complex, the overall conformation of
each structure is quite similar with an all atom RMSD for
kinase domains of 0.79 Å (606 residues aligned using the
PDBeFold Server). Comparison of paroxetine (bound to either
GRK1 or GRK2) and CCG-206584 in the active site illustrate
a highly conserved configuration (Fig. 7B). There is a small
internal rotation between the two small molecules that
results in the atoms of the benzolactam ring of CCG-206584
shifting by up to 0.5 Å, which likely results from the formation
of conventional hydrogen bonds between CCG-206584 and
the backbone of GRK2-Asp272 and Met274 (Fig. 7, A and B).
These bonds have distances of 2.8 and 2.7 Å, respectively,
instead of 2.9 and 3.4 Å for their equivalents in the paroxetine
complex. The backbone carbonyl of GRK2-Met274 also seems to
rotate to avoid a steric clash with the benzolactam carbonyl.
The hydrogen bond formed between the piperidine ring of each
inhibitor and the backbone of GRK2-Ala321 is also maintained.
Discussion
The development of small molecule inhibitors for protein
kinases has been of longstanding interest because of the
regulatory roles played by these enzymes in signaling net-
works. However, to date there have been relatively few
clinically useful kinase inhibitors beyond blockbuster receptor
tyrosine kinase inhibitors such as imatinib (Nagar et al.,
2002) or iressa (Yun et al., 2007). Several GRKs have also
emerged as targets for the development of small molecule
inhibitors, which would ultimately serve to increase the sen-
sitivity of GPCRs to extracellular signals. Potent AGC kinase
inhibitors known to be effective against GRKs include the
natural products staurosporine (Ward and O’Brian, 1992),
sangivamycin (Loomis and Bell, 1988), and balanol (Tesmer
et al., 2010). However, the utility of these compounds is
limited by their lack of selectivity and/or toxicity. Even more
recently identified compounds synthesized by Takeda Phar-
maceuticals (Osaka, Japan), which exhibit selective inhibition
of GRK2 over other GRKs (Thal et al., 2011) and AGC family
kinases, have not progressed toward clinically relevant
treatments (Ikeda et al., 2007).

Inhibition of GRKs by Paroxetine Analogs
245
Fig. 6. Comparison of the potency of paroxetine and CCG-206584 for non-
GRK targets. (A) CCG-206584 (CCG) is ∼20Â and ∼10Â more potent
inhibitor of PKA and PKC, respectively, relative to paroxetine (pax). Data
shown are representative of 5 experiments for PKA and 3 experiments for
PKC, performed in duplicate. The error bars represent standard deviation.
(B) CCG-206584 inhibits serotonin (SERT) uptake by human embryonic
kidney (HEK) 293 cells stably expressing the serotonin transporter ∼20-
fold less potently than paroxetine. The data are representative of 5
experiments performed in triplicate, with error bars showing standard
deviation of the experiment.
ability of the nonhydrolyzable ATP analog AMPPNP and the
product ADP to displace a fluorescently labeled ADP ligand
and showed that GRK2 has twofold higher KD for AMPPNP
and 30-fold higher KD for ADP over GRK1 (Fig. 2C; Table 1).
Therefore at steady state, paroxetine has to compete with
more tightly bound ATP and/or ADP in the case of GRK1. This
observation is consistent with the fact that, despite the use of
saturating conditions, we have not yet crystallographically
resolved adenine nucleotides in the active site of GRK2,
whereas GRK1 and GRK6 readily cocrystallize in complex
with adenine nucleotides. The binding of rhodopsin to GRK1
is reported to increase the affinity of ATP binding (Pulver-
muller et al., 1993). Therefore, during the catalytic cycle,
preferential interaction of rhodopsin with GRK1 [KM 5 4 mM;
Fig. 5. CCG-206584 selectivity and potency for GRKs. (A) CCG-206584
(CCG) thermostabilizes GRKs to a greater extent than paroxetine (pax).
The melting temperature (Tm) was determined from the inflection point of
sigmoidal curves fit to thermal denaturation data. Data shown are
collected from 4 experiments performed in triplicate, with error bars
showing S.E.M. Significant increases in the thermal stabilization of CCG-
206584 relative to paroxetine were observed for GRK1 (P , 0.0001) and Vmax 5 700 nmol/min/mg (Palczewski et al., 1988)], which
GRK5 (P = 0.016). (B) Inhibition of representative members of the 3 GRK
represents a physiologic pair, rather than with GRK2 [KM
5
subfamilies (GRK1, GRK2 and GRK5) in response to CCG-206584 using
the soluble substrate tubulin. Data shown are representative of at least 4
experiments performed in duplicate, and the error bars represent standard
deviation in the experiment shown. (C) Analogous assays using light-
activated rhodopsin in rod outer segments as the substrate. Data shown
are representative of at least 3 experiments performed in duplicate, and
the error bars represent S.D. of the experiment shown.
6 mM; Vmax 5 72 nmol/min/mg (Benovic et al., 1987)] may also
render it more difficult for ligands such as paroxetine to
inhibit activity. However, this does not seem to be the case for
the inhibitor balanol, which exhibits consistent IC50 versus
KD values among the GRKs tested (Table 1).
The crystal structure of the GRK1·paroxetine complex is
generally consistent with the above conclusions. The orienta-
To further support the hypothesis that differential affinities tion of the large and small lobes of the kinase domain of the
of GRK1 and GRK2 for ATP and/or ADP may be responsible GRK1·paroxetine complex is most similar to those exhibited
for the observed selectivity of paroxetine, we monitored the by ADP bound complexes of GRK1. The conformation of the

246
Homan et al.
TABLE 3
Inhibition of GRK phosphorylation of tubulin and rhodopsin by
paroxetine and CCG-206584
Errors represent the S.E.M. from n experiments performed in duplicate.
Paroxetine
CCG-206584
plogIC50 6 S.E.M.
n
plogIC50 6 S.E.M.
n
Tubulin
GRK1
GRK2
GRK5
GRK1
GRK2
GRK5
3.8 6 0.04
5.6 6 0.04
3.9 6 0.05
3.5 6 0.07a
4.7 6 0.04a
3.6 6 0.03a
7
9
4
3.8 6 0.06
5.6 6 0.03
4.0 6 0.06
3.7 6 0.4
5.2 6 0.04
4.1 6 0.07
5
7
6
3
5
4
Rhodopsin
aFrom Thal et al., 2012.
paroxetine seems to drive the kinase domain of GRKs into
a conformational state similar to that used to bind the product
ADP, the ligand for which GRK1 and GRK2 exhibit the
greatest difference in affinity (Table 1). Despite several dif-
ferences in specific contacts with the drug, the total buried
surface area for paroxetine is quite similar in GRK1 and
GRK2, consistent with their similar kon and koff values mea-
sured by Octet Red (Fig. 2, A and B).
Our analysis also provides new insight into the molecular
basis for highly selective inhibition (.1000-fold) of GRK2 by
the compounds produced by Takeda Pharmaceuticals Inc.
(Thal et al., 2011). These molecules stabilize GRK2 in
a conformation that is not expected to be along the normal
GRK2 reaction coordinate. Such may account, at least in part,
for the enhanced selectivity of these compounds. Notably, an
RNA aptamer highly selective for GRK2 also trapped the
kinase domain of GRK2 in a conformational state that is likely
unique to the GRK2 subfamily of GRKs (Tesmer et al., 2012).
Thus, achieving more selective inhibition of GRK2 may re-
quire the development of paroxetine analogs that induce more
“off-pathway” conformational states.
We hypothesized that the benzodioxole ring of paroxetine
and its relatively weak interactions with the hinge of the
kinase domain, an element that helps dictate the relative
orientation of the large and small lobes, may be an important
driver of selectivity. By substitution of a benzolactam, we
predicted that we would introduce two strong hydrogen bonds
between the small molecule and the hinge region of GRK2
without disrupting the overall configuration of the kinase
domain. However, CCG-206584 yielded only subtle increases
in thermostability for GRK1, GRK2, and GRK5, and no sig-
nificant increase in potency (Table 3). Thus, incorporation of
a conventional hydrogen bond in place of the carbon-oxygen
hydrogen bond did not yield a better GRK inhibitor. Instead,
the potency of inhibition of PKA and PKC was increased about
10-fold (Fig. 6A). However, we confirmed a 20-fold decrease in
Fig. 7. Crystallographic analysis of the GRK2·CCG-206584 complex. (A)
Electron density Fo-Fc omit map contoured at 3s (green mesh) of CCG-
206584 bound to the active site of GRK2 (gray cartoon). The compound
makes specific hydrogen bonds to backbone atoms of Asp272, Met274, and
Ala321, shown as black dashed lines. (B) Superposition of the active sites
of the GRK2·paroxetine–Gbg, GRK2·CCG-206584–Gbg, and GRK1·par-
oxetine complexes based on alignment of their small lobes. The small and
large lobes of the kinase domain in the GRK2·paroxetine complex are
colored green and yellow, respectively, and the carbons of paroxetine are
colored cyan. The GRK1 complex is colored blue, with blue carbons in
paroxetine. All molecules bind in a similar conformation in the active site.
The rationally designed CCG-206584 undergoes a small rotation toward potency of serotonin reuptake inhibition for CCG-206584
the hinge region of GRK2 that allows it to form 2.7 and 2.8 Å hydrogen
versus paroxetine (Fig. 6B). Thus, it is possible to select
bonds with Asp272 and Met274, respectively. (C) Superposition of the
against SSRI activity, an “off target” effect, while retaining
GRK2·balanol complex (PDB ID 3KRW) with that of GRK2·CCG-206584–
potency of GRK2 inhibition.
Gbg by pair fitting key Ca residues in the active site (GRK2 residues 201,
272, 273, 274, and 321). GRK2 in complex with balanol is drawn in the
color beige. Despite distinct relative conformations adopted by the large
and small lobes of each kinase, both CCG-206584 (dark gray carbons) and
balanol (pink carbons) form analogous hydrogen bonds (balanol green and
CCG-206584 dark green) to backbone atoms in the hinge.
Using X-ray crystallography, we showed that the overall
conformation of the GRK2 kinase domain is similar upon
binding either inhibitor (Fig. 7, A and B) and that both
compounds bind in similar configurations in the active site.
Furthermore, the chemical environment occupied by parox-
kinase domain in the GRK2·paroxetine–Gbg complex is also etine in complex with GRK1 or GRK2 is highly conserved with
more similar to those of the GRK1·ADP complexes than it is to that occupied by CCG-206584 (Fig. 7B). The strong electron
any other reported GRK structure. Thus, the binding of density present in the omit maps of CCG-206584 (Fig. 7A)

Inhibition of GRKs by Paroxetine Analogs
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site of GRK2 relative to paroxetine, although comparison of
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ole ring may be preferable to a benzolactam, at least if GRK
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Authorship Contributions
Participated in research design: Homan, Larsen, Tesmer.
Conducted experiments: Homan, Wu, Singh.
Contributed new reagents or analytic tools: Wilson.
Performed data analysis: Homan, Singh.
Wrote or contributed to the writing of the manuscript: Homan,
Larsen, Tesmer.
in
G protein-coupled receptor kinase activation. J Biol Chem 283:
14053–14062.
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of human G protein-coupled receptor kinase 2 in complex with the kinase inhibitor
balanol. J Med Chem 53:1867–1870.
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inhibition of g protein-coupled receptor kinase 2 by a selective RNA aptamer.
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J, Gao E, and Cheung JY et al. (2012) Paroxetine is a direct inhibitor of g protein-
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a

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Address correspondence to: Dr. John J. G. Tesmer, Life Sciences Institute,
The University of Michigan, 210 Washtenaw Ave. Rm 3425, Ann Arbor, MI
48109. E-mail: tesmerjj@umich.edu