Professional Documents
Culture Documents
http://jpet.aspetjournals.org/content/suppl/2010/08/26/jpet.110.173005.DC1.html
0022-3565/10/3353-572–579$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 335, No. 3
Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 173005/3638461
JPET 335:572–579, 2010 Printed in U.S.A.
Jonathan D. Violin, Scott M. DeWire, Dennis Yamashita, David H. Rominger, Lisa Nguyen,
Kevin Schiller, Erin J. Whalen, Maxine Gowen, and Michael W. Lark
Trevena Inc., King of Prussia, Pennsylvania
Received July 15, 2010; accepted August 25, 2010
ABBREVIATIONS: GPCR, G protein-coupled receptor; AngII, angiotensin II; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2
receptor; SII, 1Sar, 4Ile, 8Ile-angiotensin II; ARB, angiotensin receptor blocker; eNOS, endothelial nitric-oxide synthase; MAPK, mitogen-activated
protein kinase; FAK, focal adhesion kinase; MAP, mean arterial pressure; PRSW, preload recruitable stroke work; PV, pressure volume; ESPVR,
end systolic pressure volume relationship; ANOVA, analysis of variance; HEK, human embryonic kidney; MEM, modified Eagle’s medium; FBS,
fetal bovine serum; IP1, inositol monophosphate; RVU, relative volume unit; ERK, extracellular signal-regulated kinase; U2-OS, U2-osteosarcoma;
siRNA, small interfering RNA; TRV120023, Sar-Arg-Val-Tyr-Lys-His-Pro-Ala-OH; TRV120026, Sar-Arg-Val-Tyr-Tyr-His-Pro-NH2; TRV120027,
Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH.
572
AT1R -Arrestin Bias Improves Cardiac Function 573
2007; Tran et al., 2009; Zidar et al., 2009). These “biased -Arrestin Recruitment. The PathHunter protein complemen-
ligands” are agonists when assaying some receptor functions, tation assay (DiscoveRx Corporation, Fremont, CA) was performed
but they are antagonists or even inverse agonists when as- according to the manufacturer’s protocol and read for chemilumines-
saying other receptor functions (Violin and Lefkowitz, 2007; cent signaling on a PheraStar reader (BMG Labtech, Durham, NC).
Kenakin, 2009). One of the most studied GPCRs in this In brief, complementary halves of -galactosidase were genetically
fused to the carboxyl termini of the AT1R and -arrestin2. When
regard is the angiotensin II type 1 receptor (AT1R). This
cotransfected, the two fusion proteins serve as a proximity sensor;
receptor engages all three classes of -arrestin function: de- when -arrestin2 translocates to active receptor, the -galactosidase
sensitization (Violin et al., 2006), internalization (Kule et al., fragments interact to form a functional enzyme, which is detected by
2004), and signaling (Barnes et al., 2005; DeWire et al., 2008; a chemoluminescent substrate.
Ahn et al., 2009). It was also one of the first receptors for Inositol Monophosphate Accumulation. IP1 was measured
which a -arrestin biased ligand was described: 1Sar, 4Ile, with the IP-One Tb HTRF kit (Cisbio, Bedford, MA). Plates were
8
Ile-angiotensin II (SII) induces -arrestin recruitment, re- read on a PheraStar reader using a time-resolved fluorescence ratio
ceptor internalization, and -arrestin-mediated signals with- (665 nm/620 nm).
out activating G protein coupling (Wei et al., 2003; Ahn et al., Immunoblotting. U2-OS or HEK-293 cells stably expressing the
2004; Kim et al., 2009). Unfortunately, this peptide displays rat AT1aR were grown in MEM supplemented with 10% FBS. After
overnight serum starvation, cells were stimulated with 1 M angioten-
low receptor binding affinity (Holloway et al., 2002), which
sin II, valsartan, TRV0100027, TRV0100023, or vehicle for 5 min.
has made in vivo characterization of its effects challenging. Lysates were immunoblotted with phospho-Y416-Src, phospho-T202/
However, ex vivo studies revealed that SII promotes contrac- Y204-ERK, or phospho-S1177-eNOS antibodies per the manufacturer’s
ECG-derived indices of conduction (PQ, QRS) and repolarization confirmed by whole-cell radioligand binding studies, which
duration (QT/QTcF) were measured. showed that TRV120027, but not the ARB losartan, re-
Statistics. Statistics were assessed by using Prism version 5 duced angiotensin II binding sites in HEK cells overex-
(GraphPad Software Inc., San Diego, CA). Schild analysis fitting the pressing human AT1R (Supplemental Fig. 1B).
Schild model of competitive antagonism to a series of dose-response
We chose TRV120027, the more potent ligand, for more
curves with varying amounts of antagonist was also performed with
thorough characterization. Escalating concentrations of
Prism software.
TRV120027 shifted the EC50 of angiotensin II-evoked G
protein coupling without suppressing maximal efficacy,
Results consistent with a purely competitive mechanism of action
The AT1R Is Capable of Potent, Robust Efficacy by (Fig. 1D). Fitting the data to a Schild model of competitive
-Arrestin Biased Ligands. To identify new, higher- antagonism (Lew and Angus, 1995) supported this conclu-
potency -arrestin biased ligands, we initiated an iterative sion (Schild slope ⫽ 1.04 ⫾ 0.06), with an estimated Kd of
evaluation of custom-synthesized peptides based on SII, 19 nM. This is consistent with radioligand binding studies with
125
using assays of G protein coupling (inositol monophos- I-angiotensin II, which showed a Ki for TRV120027 of 16 nM
phate accumulation) and -arrestin recruitment to the (Supplemental Table 1). These studies also showed that
AT1R (enzyme complementation) to characterize the po- TRV120027 has apparent first-order binding, with a kon of 3.9 ⫻
tency, efficacy, and -arrestin bias of new compounds at 106 M⫺1 䡠 min⫺1, koff of 4.7 ⫻ 10⫺2 min⫺1, corresponding to a
overexpressed human AT1R. This search led to the iden- residence half-time of 16 min, similar to that of losartan. The
Fig. 1. TRV120027 is a highly potent -arrestin biased ligand at the AT1R. A–C, -arrestin2 recruitment (black circles) was measured by
chemiluminescent -galactosidase activity, and G protein coupling (red squares) was measured by IP1 accumulation, in HEK cells expressing human
AT1R for angiotensin II (A), the ARB valsartan (B), and TRV120027 (C). D, TRV120027 preincubated at escalating concentrations shifts the
angiotensin II dose-response curve for IP1 accumulation, consistent with competitive antagonism. Data are means ⫾ standard error for three
independent experiments.
AT1R -Arrestin Bias Improves Cardiac Function 575
Fig. 2. -Arrestin biased ligands stimulate selective signaling in comparison with an unbiased agonist and an unbiased antagonist in U2-OS cells
overexpressing rat AT1aR. A–D, activation by phosphorylation was measured by immunoblot with phospho-specific antibodies for ERK1/2 (A),
p38 (B), Src (C), and eNOS (D) activation. E, -arrestin2 siRNA prevents biased ligand-stimulated eNOS phosphorylation compared with a
control siRNA. Data are means ⫾ standard errors of three to seven independent experiments. ⴱ, p ⬍ 0.05 versus vehicle and valsartan and ⴱⴱ,
Hemodynamics
Heart rate (bmp) 371 ⫾ 6 356 ⫾ 5 351 ⫾ 8 359 ⫾ 5 369 ⫾ 4
Mean arterial pressure (mm Hg) 103 ⫾ 3 92 ⫾ 3* 84 ⫾ 4* 72 ⫾ 5* 88 ⫾ 3
Stroke volume (l) 26.3 ⫾ 3.1 25.2 ⫾ 4 27.1⫾4 26.2 ⫾ 5.1 29.6 ⫾ 4
End diastolic volume (RVU) 15.6 ⫾ 1.6 15.5 ⫾ 1.6 15.3 ⫾ 1.5 15.4 ⫾ 1.7 15.5 ⫾ 1.7
End systolic pressure (mm Hg) 118 ⫾ 4 112 ⫾ 7 99 ⫾ 8 87 ⫾ 9* 104 ⫾ 8
End diastolic pressure (mm Hg) 6⫾2 6⫾2 6⫾3 5⫾2 7⫾2
Systolic function
dP/dtmax (mm Hg/s) 6060 ⫾ 198 5494 ⫾ 325 4697 ⫾ 365* 3755 ⫾ 248* 5304 ⫾ 316
Ees, ESPVR slope (mm Hg/RVU) 45 ⫾ 1 51 ⫾ 1 54 ⫾ 2* 60 ⫾ 4* 48 ⫾ 1
PRSW (SWU/RVU) 67 ⫾ 6 60 ⫾ 2 59 ⫾ 1 58 ⫾ 4 58 ⫾ 4
Stroke work (mm Hg ⫻ RVU) 110 ⫾ 3 104 ⫾ 5 93 ⫾ 13 66 ⫾ 13* 87 ⫾ 13
Normalized PRSW (1/RVU) 0.62 ⫾ 0.1 0.59 ⫾ 0.03 0.67 ⫾ 0.09 0.93 ⫾ 0.1 0.69 ⫾ 0.08
Diastolic function
dP/dtmin (mm Hg/s) ⫺6333 ⫾ 73 ⫺5602 ⫾ 167 ⫺4921 ⫾ 288* ⫺4036 ⫾ 295* ⫺5699 ⫾ 429
Tau (s) 12 ⫾ 1 12 ⫾ 1 12 ⫾ 0 12 ⫾ 1 11 ⫾ 0
Cardiac conduction
PQ (ms) 41 ⫾ 4 41 ⫾ 4 41 ⫾ 4 42 ⫾ 5 42 ⫾ 4
Fig. 6. TRV120027 reduces blood pressure while increasing cardiac performance in normal rats. A, PV loop analysis in normal rats infused with either
TRV120027 or telmisartan revealed similar effects on MAP. B, neither compound substantially changes heart rate. C, TRV120027 increased, whereas
telmisartan decreased, load-independent cardiac performance, as measured by the slope of ESPVR. D, telmisartan, but not TRV120027, significantly
reduced stroke volume. Data shown are mean ⫾ standard errors from three independent experiments with three to four animals per group. ⴱ, p ⬍ 0.05
versus baseline, as measured by ANOVA with the Bonferroni multiple comparison test.
578 Violin et al.
A
Vehicle Angiotensin II Valsartan Losartan
Supplementary Figure 1. TRV120027 stimulates receptor internalization. (A) rat AT1aR fused
to cyan fluorescent protein and expressed in HEK-293 cells exposed to vehicle, angiotensin II, ARBs
(valsartan, losartan, or telmisartan) or β-arrestin biased ligands (TRV120023 or TRV120027). (B)
Downregulation of cell-surface human AT1R binding sites in response TRV120027 or losartan at 37°C. Data
are the mean total [125I]-angiotensin II binding ± SEM from 2-3 independent experiments.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics
A B
C D
E F
A B
A B
C D
Supplementary Methods
Selectivity Profiling
The following receptors and ion channels were tested for TRV120027 binding by radioligand
displacement - ExpresS Profile (Cerep, Potiers, France): (human) Adenosine A1, A2A and A3, Adrenergic
β1 and β2, Angiotensin-II AT1, AT2, Bradykinin B2, Cannabinoid CB1, Cholecystokinin CCK1 (CCKA),
Dopamine D1, and D2Short, Endothelin ETA, GABA, Galanin GAL2, Chemokines CXCR2, CCR1,
Histamine H1 and H2, Melanocortin MC4, Melatonin MT1 (ML1A), Muscarinic M1, M2 and M3,
Neurokinin NK2 and NK3, Neuropeptide Y1 and Y2, Neurotensin NTS1 (NT1), Opioid δ2DOP), Opioid
µ(MOP), Opioid NOP (ORL1), Prostanoid TP (TXA2/PGH2), Serotonin 5-HT1A, 5-HT2A, 5-HT2B , 5-HT3
, 5-HT5a , 5-HT6 and 5-HT7, Vasoactive intestinal peptide VPAC1 (VIP1) and V1a, Norepinephrine
transporter, Dopamine transporter, Serotonin 5-HT transporter, APJ (apelin), Angiotensin-converting
enzyme ACE; (rat) Adrenergic α1 and α2, Benzodiazepine BZD, Serotonin 5-HT1B, Opioid κ (KOP),
Ca2+ channel (L verapamil site), K+ channels KV and SKCa Na+ channel (site 2), Cl- channel (GABA-
gated); (murine) Somatostatin sst (non-selective).
Rat pharmacokinetics
During a 2-hour IV infusion, 10 ug/kg/min TRV120027 rapidly reached a steady-state concentration of
246 ng/mL (266 nM), and upon ending infusion revealed a half-life of 1.5 minutes with low volume of
distribution of 208 mL/kg in rats. The high clearance indicates that infusion is the optimal route of
administration to elicit pharmacological effects, and given the high affinity of TRV120027 for the AT1R,
predicts a pharmacologically effective dose of 0.1-10 ug/kg/min. The rapid clearance and volume of
distribution also suggest a rapidly reversible effect limited to the vascular space.
Rats were intraperitoneally (IP) anesthetized to effect with sodium pentobarbital (78.8 ± 1.5 mg/kg; 63.4
– 90.0 mg/kg). Following induction, the animals were prepared for surgery, endotracheally intubated (via
a tracheotomy), and mechanically ventilated (~90 breaths/min, ~2.5ml tidal volume with a 95% O2/ 5%
CO2 mixture) via an adjustable small animal ventilator (model 680; Harvard Apparatus). Anesthesia was
sustained with routine sodium pentobarbital injections (total, 28.0 ± 1.8 mg/kg IP) until completion of the
study. Temperature was continuously monitored throughout the duration of the surgical/experimental
procedures via a rectal probe, and was maintained within the physiological range via a heated
temperature-controlled (closed-loop) small animal surgical table/control unit (model 03-01; Vestavia
Scientific). Subsequently, transthoracic needle electrodes forming a single-lead ECG (lead II) were
placed. The right carotid artery was isolated, dissected free from the surrounding tissue, and cannulated
with a 2F high-fidelity micromanometer catheter (SPR-838; Millar Instruments). In order to determine
left-ventricular pressure this catheter was advanced retrogradely across the aortic valve, and into the LV.
Similarly, in order to record arterial pressures, a 2F high-fidelity micromanometer catheter (SPR-320;
Millar Instruments) was inserted into the right femoral artery, and advanced into the abdominal aorta.
Finally, indwelling catheters were placed in the left femoral and jugular veins for the administration of the
vehicle/control/test articles and of angiotensin-II (respectively). The animals were kept/placed in dorsal
recumbence until the end of the experiment.
After instrumentation and hemodynamic stabilization for approximately 10 min, baseline data were
collected (i.e., pre-dosing data, PRE). Subsequently, the animals were treated with the test/vehicle/control
articles and following hemodynamic stabilization, as well as data collection the peak cardiovascular
responses to eight single-dose AT-II injections (IV bolus) were evaluated. Throughout the experiments,
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics
analog signals were digitally sampled (1000Hz) and recorded continuously with a data acquisition system
(IOX; EMKA Technologies). Heart rate (HR), and systemic/left-ventricular hemodynamic/mechanical
indices were measured both before/after dosing, as well as during the AT-II dose-response generation.
Mean (MAP) arterial pressure and left-ventricular hemodynamic/mechanical parameters (ESP, EDP,
dP/dt max/min, time-constant of relaxation – tau, contractility-index – Vmax) were measured from the
aortic/left-ventricular pressure signals (respectively). Meanwhile, the following ECG-derived indices/
parameters were measured both before and after vehicle/control/test-article administration only: HR, RR,
PQ, QRS, QT, and Fridericia’s rate-corrected QT (QTcF) 1. Data were acquired digitally (IOX; EMKA
Technologies), and analyzed offline (IOX/ECG Auto; EMKA Technologies).
Healthy, acclimatized, male Sprague-Dawley rats (Harlan Laboratories, IN; 283 – 392g) were
intraperitoneally (IP) anesthetized to effect with sodium pentobarbital (~60mg/kg). Following induction,
the animals were prepared for surgery, endotracheally intubated (via a tracheotomy), and mechanically
ventilated (~90 breaths/min, ~2.5ml tidal volume with a 95% O2/ 5% CO2 mixture) via an adjustable
small animal ventilator (model 680; Harvard Apparatus). In-dwelling catheters were placed in a femoral
and a jugular vein for the administration of either anesthetic agents or vehicle/control/test articles
(respectively). Anesthesia was sustained with a continuous sodium pentobarbital infusion (~ 10 mg/kg/h
IV) until completion of the study. Once a proper anesthetic regime was established and verified (e.g.,
nonresponsive to toe pinch), the animals were paralyzed with pancuronium (1 mg/kg/h IV bolus).
Temperature was continuously monitored throughout the duration of the surgical/experimental procedures
via a rectal probe, and was maintained within the physiological range via a heated temperature-controlled
(closed-loop) small animal surgical table/control unit (model 03-01; Vestavia Scientific).
Subsequently, transthoracic needle electrodes forming a single-lead ECG (lead II) were placed. The right
carotid artery was isolated, dissected free from the surrounding tissue, and cannulated with a 2F high-
fidelity conductance/micromanometer catheter (SPR- 838; Millar Instruments). In order to simultaneously
determine left-ventricular pressure and volume (via conductivity; MPCU-200, Millar Instruments), this
catheter was advanced retrogradely across the aortic valve, and into the LV. The thoracic cavity was
opened via a right-thoracotomy, and a non-constricting hydraulic vessel-occluder (3mm; In Vivo Metrics)
filled with distilled water was securely placed around the inferior vena cava. In order to record arterial
pressures, a 2F high-fidelity micromanometer catheter (SPR-320; Millar Instruments) was inserted into a
femoral artery. Finally, the animals were placed in dorsal recumbency until the end of the experiment.
After instrumentation and hemodynamic stabilization, baseline data was collected. Subsequently,
escalating-dose infusions of the test/control article(s) were started, and measurements were taken once a
steady state was reached at each concentration (~10min). Finally, an additional set of measurements was
obtained during the washout period (~5-7min after the end of infusion). Meanwhile, in the vehicle group,
measurements were at baseline, just before the end of a timematched PBS (TRV-vehicle) infusion (i.e.,
~40-45min after infusion onset), and immediately after an acute inotropic challenge with dobutamine
(VEH+DOB). Left-Ventricular Mechanics: Once a steady hemodynamic state was reached at a given
time-point, left-ventricular pre-load was acutely reduced by means of brief (~8-10 beats) vena cava
occlusions (via transient inflation of the vessel occluder) in order to generate a family of pressure-volume
curves/loops; approximately three occlusions were performed at each time-point, allowing for
hemodynamic recovery between tests. The resulting left-ventricular pressure and volume data were
analyzed both on- and off-line (IOX/ECG Auto; EMKA Technologies) in order to generate relationships
representing the contractile and energetic state of the myocardium. At the end of each experiment,
conductance signals (in relative volume units, RVU) were calibrated in-vitro with warm blood, as recently
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics
reviewed/summarized by Pacher et al. 2, in order to derive absolute left-ventricular volumes (in μL). In
short, blood was freshly collected, heparinized, and used to fill cylindrical wells of known
volume/dimensions (calibration cuvette model 910-1048; Millar Instruments); three wells with outer
diameters (OD) of 2, 3, and 4mm were used. Subsequently, average conductance values were obtained
from each blood-filled cylinder. In order to derive a calibration function (slope/intersect), the resulting
conductivity values were linearly regressed against the “known” volume of the wells; a unique function
(correction) was obtained for each animal. Throughout the experiments, analog signals were digitally
sampled (1000Hz) and recorded continuously with a data acquisition system (IOX; EMKA
Technologies). Digitally acquired signals were analyzed on/offline (IOX/ECG Auto; EMKA
Technologies). The following ECG-derived indices/parameters were measured offline with the aid of
pattern-recognition software (ECG Auto; EMKA Technologies): HR, RR, PQ, QRS, QT, and Fridericia’s
rate-corrected QT (QTcF) 1. Mean arterial (MAP) and endsystolic / end-diastolic left-ventricular (ESP,
EDP) pressures were measured. Meanwhile, left-ventricular mechanical/geometrical indices were
obtained from the pressure (dP/dt max/min, time-constant of relaxation – tau, contractility-index – Vmax)
and volume (ESV, EDV, SV, dV/dt) signals. In addition, the following measurements were derived from
left-ventricular pressure-volume data (PV loops) generated during brief periods of preload reduction:
Pressure volume area (PVA), and Stroke Work (SW), End-systolic (ESPVR) and end-diastolic (EDPVR)
pressure volume relationships. From the end-systolic PV-relationship, estimates of the left-ventricular
unstressed volume (volume-axis intercept, Vo) and elastance (slope, Ees), End systolic pressure and end-
diastolic volume relationship (Arterial Elastance,Ea), Slope of the stroke work (SW) and end-diastolic
volume (linear) relationship, also called preload recruitable stroke work (PRSW).
HEK cells stable expressing rat AT1AR-mCFP and rat b-arrestin2-mYFP were produced under selection
with G418 (500ug/ml) and Zeocin (Invitrogen, 150ug/ml). Cells were plated on glass the night before the
experiment, and transferred to a phenol-red free culture media prior to the experiment. Individual dishes
were stimulated with 1uM compound for 30 minutes at 37C, and post-stimulation images were taken
using an epifluorescence microscope. Slidebook (Intelligent Imaging Innovations) software suite was
used for image analysis.
HEK-293 cells with stable expression of the human AT1R were harvested by centrifugation at 400xg for
30min at 4°C, washed once with a balanced salt solution, re-pelleted, and the pellet flash frozen in liquid
nitrogen. The cell pellets were stored at -80°C until processed for membranes. Pellets were resuspended
in buffer (50 mM HEPES, 2 mM EDTA pH 7.4 containing fresh protease inhibitors - Complete Brand
protease tablets from Roche Diagnostics (Indianapolis, IN) and subjected to nitrogen cavitation with a
Parr Cell Disruption Bomb (Parr Instrument Co., Moline, IL) at 1000 psi for 20 min on ice. Ruptured
cells were sedimented at 500g for 10 min at 4°C and the supernatant containing cellular membranes was
washed twice at 48,000g for 15 min. cell pellets were re-suspended at 4oC in 10 volumes of ice-cold
buffer A and cavitation, placed on ice. To remove large particles, a low speed centrifugation (500xg for
30 min at 4oC) was performed, followed by high-speed centrifugation (48,000xg for 45 min at 4oC),
resuspension in buffer plus protease inhibitor cocktail, and a final high speed centrifugation at (48,000g
for 45 min at 4oC). A dounce homogenizer was used to resuspend the final pellet using ice-cold buffer.
The membrane suspension was passed through a 23G needle, aliquoted, and stored at -80oC. Total
protein concentration of the membrane preparation was determined with a Coomassie Plus Reagent Kit
from Pierce Biotechnology (Rockford, IL) using bovine serum albumin as the standard.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics
Membranes were diluted in assay buffer (50 mM Hepes, 150 mM NaCl, 5mM MgCl2 pH 7.2 at 23oC) to a
concentration of 10-20 µg protein/well. Assays were initiated by the addition of 97 µl of membrane
suspension to 200 µl of [125I]-Angiotensin II ([125I]-ANGII, specific activity 2200 Ci/mmol; PerkinElmer
Life and Analytical Sciences, Boston, MA), at 0.5-1 times Kd and various concentrations of inhibitors in
buffer plus a cocktail of protease inhibitors and 0.02% BSA to reduce non-specific binding. Compounds
were diluted in DMSO and tested at a final concentration of 1% DMSO (determined to be non-
detrimental to the assay). Binding assays were performed in duplicate in polypropylene 96 well plates
(Costar Corp., Cambridge, MA). Nonspecific binding was defined in the presence of 1µM saralasin.
Competition assays were performed at 23°C for 3-4 hours to allow adequate time for compounds and
radioligand to reach equilibrium for binding. The separation of bound from free radioligand was
accomplished by rapid vacuum filtration of the incubation mixture over GF/B uni-filter
(polyethylenamine-treated) plates (Perkin Elmer, Waltham, MA) using a Brandel cell harvester (Brandel,
Gaithersburg, MD). Filters were washed 2 times with 0.3 ml of ice-cold phosphate buffered saline pH 7.0
containing 0.01% Triton X100. Radioactivity on the filters was quantified using a MicroBeta TriLux
Liquid Scintillation Counter (PerkinElmer Life and Analytical Sciences, Waltham, MA). In radioligand
time course experiments, designed to determine unlabeled compound kinetic (association and
dissociation) rate constants, radioligand, membranes and unlabeled test compound were added to the
wells at various time points (0-4 hr) and the assay wells harvested simultaneously.
AT1R downregulation was measured as loss of cell surface angiotensin II binding sites following
methods previously described 3,4. Briefly, HEK-293 cells, with stable expression of human AT1R(2x 105
cells per well) in 24-well plates (Poly-D-Lysine treated) were stimulated with either Opti-MEM (vehicle),
angiotensin II, TRV0120027 or losartan for 30 minutes at 37°C. Surface-bound ligands were removed by
a gentle acid wash (50 mM glycine-150 mM NaCl, pH 3) for 10 min at 4°C, which did not affect
subsequent receptor binding. After rinsing several times, a whole cell radioligand binding assay was
performed (3-5 hours at 4°C) to quantify receptors remaining cell surface receptors. [125I]-ANGII at a
concentration of 0.1 to 0.2 nM was added in buffer containing 150 mM NaCl, 5 mM MnCl2, 0.1%
DMSO and 0.02% bovine serum albumin. Total binding was measured with the addition of assay buffer
and nonspecific binding was defined in the presence of 1 µM saralasin. After 3-5 h incubation at 4°C,
cells were solubilized with 0.5 M NaOH-0.05% SDS, and total radioligand bound was quantified by
addition of scintillation fluid using a Microbeta TriLux scintillation counter. Downregulation was
examined bycomparing binding in the presence of TRV027 or losartan to control.
Apparent binding affinities, Ki = IC50/ (1+ [Ligand]/Kd) were performed using the nonlinear iterative
curve-fitting computer program GraphPad PRISM (San Diego, CA). The association and dissociation rate
constants of unlabeled ligands were determined using a previously described method 5,6 in which
association of a radiolabeled ligand is measured in the presence of a fixed concentration(s) of unlabeled
test ligand. The model assumes that the ligands bind in a competitive manner according to simple
bimolecular reactions.
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa
Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics
References
1. Fridericia LS (1920) Die systolendauer im elektrokardiogramm bei normalen menschen und bei
herzkranken. Acta Med Scand 53:469-486
2. Pacher P, Nagayama T, Mukhopadhyay P, Bátkai S, Kass DA (2008) Measurement of cardiac
function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc
3(9):1422-34
3. Modrall JG, Nanamori M, Sadoshima J, Barnhart DC, Stanley JC, Neubig RR. (2001) ANG II
type 1 receptor downregulation does not require receptor endocytosis or G protein coupling. Am J
Physiol Cell Physiol 281:C801-C809
4. Feng YH, Ding Y, Ren S, Zhou L, Xu C, and Karnik SS (2005) Unconventional homologous
internalization of the angiotensin II Type-1 receptor induced by G-Protein–independent signals.
Hypertension 46:419-425
5. Motulsky H and Mahan LC (1984) The kinetics of competitive radioligand binding predicted by
the law of mass action. Mol Pharmacol 25:1–9
6. Hoare SRJ and UsdinTB (2000) Tuberoinfundibular peptide (7-39) [TIP(7-39)], a novel,
selective, high-affinity antagonist for the parathyroid hormone-1 receptor with no detectable
agonist activity. J Pharmacol Exp Ther 295:761–770
Selectively engaging β-arrestins at the AT1R reduces blood pressure and increases cardiac performance
Jonathan D. Violin Ph.D., Scott M. DeWire Ph.D., Dennis Yamashita Ph.D., David H. Rominger, Lisa Nguyen, Kevin Schiller, Erin J. Whalen Ph.D., Maxine Gowen Ph.D., and Michael W. Lark Ph.D
The Journal of Pharmacology and Experimental Therapeutics
Supplementary Table 1. Kinetic parameters of TRV120027 and Losartan binding to the human AT1Rin
HEK293 cell membranes. Time course of [125I]-ANGII association binding with the hAT1 receptor in
HEK membranes to determine unlabeled compound kinetics was measured as described under
Supplementary Methods. Association time course data in the presence of unlabeled ligand were fitted
according to data analysis to obtain apparent values for kon and koff, respectively the association and
dissociation rate constants of the unlabeled ligand. The following parameters determined independently
for [125I]-ANG II were held constant in the analysis: [L], k1 8.3x 10 7mol/L-1 ⋅ min-1, k2 0.013 min-1 along
with [I] tested. Values presented are mean ± SEM from two independent experiments.
Systolic Function
dP/dt max (mmHg/s) 5,487 ± 940 3,736 ± 141* 3,515 ± 186* 3,127 ± 236* 3,224 ± 223*
Ees, ESPVR slope (mmHg/RVU) 30 ± 2 25 ± 2 22 ± 4 20 ± 4* 20 ± 4*
PRSW (SWU/RVU) 65 ± 3 53 ± 8 38 ± 10 43 ± 3 52 ± 8
Stroke Work (mmHg * RVU) 176 ± 23 112 ± 2* 94 ± 4* 107 ± 7* 123 ± 4*
normalized PRSW (1/RVU) 0.38 ± 0.04 0.48 ± 0.08 0.39 ± 0.08 0.41 ± 0.06 0.42 ± 0.07
Diastolic Function
dP/dt min (mmHg/s) 6,333 ± 816 -4,357 ± 141* -4,020 ± 215* -3,468 ± 345* -3,687 ± 390*
Tau (s) 13 ± 1 14 ± 1 14 ± 2 15 ± 2 15 ± 1
Cardiac Conduction
PQ (ms) 44 ± 2 43 ± 2 43 ± 2 43 ± 2 44 ± 1
QRS (ms) 16 ± 1 16 ± 1 16 ± 1 15 ± 0 16 ± 1
QT (ms) 77 ± 4 76 ± 4 78 ± 5 76 ± 4 78 ± 3
QTcF (ms) 139 ± 5 136 ± 4 138 ± 4 135 ± 2 136 ± 3
Systolic Function
dP/dt max (mmHg/s) 6,099 ± 372 10,608 ± 1345*
Ees, ESPVR slope (mmHg/RVU) 30 ± 8 42 ± 6
PRSW (SWU/RVU) 103 ± 10 120 ± 15
Stroke Work (mmHg * RVU) 279 ± 43 294 ± 43
normalized PRSW (1/RVU) 0.39 ± 0.1 0.43 ± 0.05
Diastolic Function
dP/dt min (mmHg/s) -7,262 ± 508 -8,917 ± 755
Tau (s) 13 ± 0 10 ± 0
Cardiac Conduction
PQ (ms) 42 ± 2 40 ± 1
QRS (ms) 16 ± 1 15 ± 1
QT (ms) 64 ± 3 65 ± 2
QTcF (ms) 117 ± 6 126 ± 5