Development of inhibitors of heterotrimeric Gαi subunits

Article abstract of DOI:10.1016/j.bmc.2014.04.035

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gα_i by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α₂-adrenoceptor (α₂AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gα_i. Molecular docking was used to identify potential molecules that bind to and stabilize the Gα_i-GDP complex by directly interacting with both Gα_i and GDP. Gα_i-GTP and Gα_q- GDP were used as a computational counter screen and Gα_q-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gα_i. Several compounds showed Gα_i specific inhibition and were able to block α₂AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.

Full text of DOI:10.1016/j.bmc.2014.04.035

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of inhibitors of heterotrimeric Gai subunits
Kathryn M. Appleton a, , Kevin J. Bigham a, , Christopher C. Lindsey a, Starr Hazard b, Jonel Lirjoni a,
Stuart Parnham c, Mirko Hennig c, Yuri K. Peterson a,
⇑
a Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425, United States
b Department of Pharmacology, Medical University of South Carolina, Charleston, SC 29425, United States
c Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors
(GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/
14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate
they function as selective GDIs for Gai by competing for the GPCR and Gbc and preventing GDP release. We
modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell
Received 27 January 2014
Revised 10 April 2014
Accepted 20 April 2014
Available online xxxx
permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses
Keywords:
G-protein
NMR
G-protein coupling to the receptor and completely blocked
cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gai
Molecular docking was used to identify potential molecules that bind to and stabilize the G i–GDP com-
plex by directly interacting with both G i and GDP. G i–GTP and G q–GDP were used as a computational
counter screen and G q–GDP was used as a biological counter screen. Thirty-seven molecules were tested
using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct
interaction with G i. Several compounds showed Gai specific inhibition and were able to block 2AR
mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of G subunits
a2-adrenoceptor (a2AR) mediated decreases in
.
a
Synthetic chemistry
Drug discovery
Guanine nucleotide dissociation inhibitor
GPCR
Pertussis toxin
Docking
cAMP
a
a
a
a
a
a
a
has the advantage of circumventing the upstream component of GPCR-related signaling in cases of over-
stimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
which facilitates the exchange of GDP for GTP on the G
Once activated, the heterotrimer G and b subunits dissociate
and signal to their downstream effectors. The G-protein signal is
then terminated when GTP, bound to G , is hydrolyzed to GDP
and GDP-bound G re-associates with Gb
There are four major subclasses of heterotrimeric alpha sub-
units, G i, G s, G q, and G 12/13. Of these, a large number of vital
hormones such as epinephrine, dopamine, acetylcholine, somato-
statin, and angiotensin all signal through the Gai pathway.1
is best described as being the inhibitory isoform of G that sup-
a subunit.
a
c
Heterotrimeric G-proteins are a ubiquitously expressed class of
proteins that transduce signals from activated G-protein coupled
receptors (GPCRs). In its inactive form, the G-protein heterotrimer,
a
c.
a
composed of G
with G in the GDP bound state. When a GPCR is activated by
agonist binding, the receptor undergoes a conformational change
a and bc subunits, is found at the plasma membrane
a
a
a
a
a
Gai
a
Abbreviations: AC, adenylyl cyclase;
a2AR, a2-adrenoceptor; cAMP, cyclic
presses adenylate cyclase activity leading to decreased cAMP accu-
mulation.2–4 Other effector proteins coupled to Gai include c-Src,
ERK1/2, phospholipase-C (PLC), and monomeric GTPases.5–12 In
recent years, GPCR signaling cascades have come to light as pri-
mary mediators of inflammatory and oncogenic signaling playing
critical roles in invasion, metastasis, and uncontrolled growth.13,14
Misregulated signaling at the level of the G-protein is also a major
component of multiple cancers leading to the potential that Gai
inhibitors could serve a therapeutic niche.11,15,16
adenine monophosphate; Fsk, forskolin; GDI, guanine nucleotide dissociation
inhibitor; GDP, guanine diphosphate; GPCRs, G-protein coupled receptor; GPR, G-
protein regulatory; GTP, guanine triphosphate; GTPc
S, guanosine 50-O-[gamma-
thio]triphosphate; NMR, nuclear magnetic resonance; PTX, pertussis toxin; PLC,
phospholipase-C; SAR, structure–activity relationship; STD, saturation transfer
difference; Tc, Tanimoto coefficient.
⇑
Corresponding author at present address: Dept. of Drug Discovery and
Biomedical Sciences, College of Pharmacy, Medical University of South Carolina,
280 Calhoun St., QF415, Charleston, SC 29425, United States. Tel.: +1 843 792 3691.
E-mail address: petersy@musc.edu (Y.K. Peterson).
These authors had equal contribution.
http://dx.doi.org/10.1016/j.bmc.2014.04.035
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Appleton, K. M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.035

2
K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
The principal laboratory tool for inhibiting the G
ai subunit is per-
2.2. Cell Culture
tussis toxin (PTX), an enzyme complex produced by the bacterial
pathogen Bordetella pertussis. The toxin is taken into the cell via
endocytosis where the active enzyme subunit, the S1 A-protomer,
ADP-ribosylates the GDP-bound Gai subunit of heterotrimeric
G-proteins.17,18 ADP-ribosylation of the alpha subunit locks the
entire heterotrimer in its inactive state and prevents activation by
agonist-stimulated receptors.19 PTX blocks the mitogenic effects of
GPCR activation by hormones such as epinephrine, sphingosine-1-
phosphate (S1P), lysophosphatidic acid (LPA), cytokines, and seroto-
nin.15,20–23 As an enzymatic modifier, PTX efficacy is slow requiring
overnight incubations leading to compensatory mechanisms. Alter-
native inhibitors would be useful when acute pharmacological study
is required or the monomeric subunit only needs to be inhibited.
An alternative strategy used endogenously by cells to inhibit
GloSensor HEK293 cells (Promega, Inc.) were maintained at
37 °C in a 5% CO2 atmosphere and maintained in Dulbecco’s
Modified Eagle Medium (ThermoScientific #SH30243.01) supple-
mented with 10% fetal bovine serum and 50
was added for selection.
lg/mL hygromycin B
2.3. GTP
cS Exchange Assay
[
35S]GTP
c
S
(1250 Ci/mmol) (21.8 Ci/mmol) were purchased
S binding assays were gener-
from Dupont/NEN (Boston, MA). GTP
c
ally conducted as described.39
Gai (100 nM) was preincubated for
20 min at 24 °C in the presence and absence of GPR peptides.
Binding assays (duplicate determinations) were initiated by addi-
the G
containing the G-protein regulatory (GPR) or GoLoco motifs bind to
ai family subunits and function as GDP dissociation inhibitors
a
subunit is to prevent nucleotide exchange. Cellular proteins
tion of 0.5
volume = 50
l
M GTP
c
S (4.0 ꢀ 104 dpm/pmol) and incubations (total
l
L) continued 30 min at 24 °C. Reactions were termi-
G
nated by rapid filtration through nitrocellulose filters (S&S BA85)
with 4 ꢀ 4 ml washes of stop buffer (50 mM Tris–HCl, 5 mM MgCl2,
1 mM EDTA, pH 7.4, 4 °C). Radioactivity bound to the filters was
determined by liquid scintillation counting. Nonspecific binding
(GDI) by preventing nucleotide exchange and subunit activa-
tion.24–26 GPR/Goloco motif-containing proteins in humans include
RGS12/14, GPSM1 (AGS3), GPSM2 (LGN), GPSM3 (AGS4 or G18.1b),
GPSM4 (PCP2), Rap1GAP, and WAVE1.26–30 These proteins bind to
was defined by 100 lM GTPcS.
and stabilize the GDP-bound inactive state of Gai, prevent nucleo-
tide exchange, and block heterotrimer oligomerization.24,25,31–35 To
date, there are no conclusive reports of an endogenous GDI for any
of the other heterotrimeric alpha subunit families beyond Gai
2.4. Computer infrastructure
Molecular manipulations and imaging was performed on
desktop computers using freeware and MOE.2011 (CCG). Computa-
tionally intensive docking was accomplished with the MUSC Com-
putational Biology Resource Center’s Cluster. The CBRC cluster
consists of sixteen Dell PE1950 dual quad core nodes running RHEL
4.3 and managed by Platform Computing Open Cluster Stack soft-
ware with LAVA scheduler. Initial in silico screening was per-
formed against the neutral clean lead 280,357 compound subset
of the 2009 ZINC database (http://zinc.docking.org/).40,41
which underscores the critical nature of regulating the Gai. GPR/
Goloco motif-containing proteins have been implicated clinically
in organism development, addiction, and cancer where they are
central in the regulation of cell division and differentiation.36–38
The GPR consensus peptide represents the conserved amino
acids found among the eight human proteins that contain a GPR
motif.25 The 28 amino acid GPR motif contains 12 conserved
residues arranged within a minimum 20mer consisting of charged
and hydrophobic microdomains.25 The core conserved residues
that are required for activity corresponds to: EE-FF-LL—Q–
RMDDQR.35 An X-ray crystal structure of the GPR/Goloco domain
of RGS14 supports hydrophobic interactions with the amino termi-
2.5. Protein targeting
X-ray coordinates came from the PDB 2OM2 file (G
and 2RGN (G
q-GDP).43 Both proteins had GDP-Mg2+ as well as a
a
i1-GDP)42
nal residues of the peptide with a hydrophobic patch on G
ionic interactions between the carboxy terminal residues of the
peptide and G i. Also of critical importance the conserved arginine
ai, and
a
peptide from either RGS14 or p63RhoGEF complex respectively.
PDB file 1AGR was selected as a transition state analog mimicking
a
in the core of the peptide has a direct interaction with the bound
nucleotide. These three points of contact create an elegant and spe-
cific mechanism to create a stable four part complex of G-protein,
nucleotide, Mg2+, and GDI. Like endogenous GPR domains, the GPR
the activated state of Gai1-GTP and was used as a negative control
to remove compounds that bound to either state in the docking
simulations. In order to better mimic the natural state of the
G-protein the sulfur from the Xray GSP was replaced with an
oxygen. Receptor viewing and editing was done with CHIMERA
1.4. Receptor setup was based on Dock suggested setting. The
selected_spheres file was manually edited to further focus the
docking procedure. The bound RGS14 peptide and p64RhoGEF as
well as any waters were removed prior to docking. Before analysis
or simulations, proteins were protonated at pH 7.4 and structures
energy minimized with heavy atoms constrained. The positions of
GDP and Mg2+ were used as positional seeds to set up the initial
compound search.
consensus peptide competes for binding of AGS3 to G
GTP binding to G i, blocks receptor coupling of G i, and stabilizes
the GDP-bound conformation of Gai
ai, inhibits
a
a
25,35
.
Here we report the TAT-GPR peptide development strategy and
in vitro effects on cAMP. In addition to being an intriguing probe to
study G-protein pharmacology, the TAT-GPR peptide also serves as
a baseline and platform to develop small molecule GDIs. The bio-
chemistry of the GPR domain allows us to exploit native mecha-
nisms in the design of synthetic mimetics. We therefore go on to
report the strategy, discovery, and validations of small molecules
GDIs designed to mimic the GPR peptide interaction with Gai
and GDI biological effects.
2.6. Screening Docking Simulations
Ligand screening was performed with UCSF DOCK version 6.3.44
DOCK was compiled from source with MPICH2.1 that in turn was
compiled with INTEL C and FORTRAN compilers (v 10.1). DOCK
used default GRID settings, vdw_AMBER_parm99 energy field
and 1000 maximum poses per sphere. For initial screening, all
ligands were evaluated in rigid mode. The ligand-receptor energy
was scored with the unmodified GRID method of DOCK. Both
electrostatic and vDW parameters were calculated. Molecules were
ranked according to this rigid docking GRID score with 500
2. Experimental section
2.1. GPR Peptides
Peptides were synthesized and purified by Bio-Synthesis, Inc.
(Lewisville, TX) and United Peptide Corp (Herndon, VA), and pep-
tide mass verified by matrix-assisted laser desorption ionization
mass spectrometry.
Please cite this article in press as: Appleton, K. M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.035

K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
maximum poses per sphere. Best scoring compounds were re-
docked in flexible mode, using the same parameters as the rigid
docking simulations leading to 210 top predicited compounds.
Additional analysis of the high scoring hits and pharmacophore
development were accomplished with MOE 2012(CCG). The top
(254 nm), followed by staining the plate and drying on a hot plate.
The stain was made with 25 grams, of phosphomolybdic acid, 10
grams of cerium sulfate, 60 mL H2SO4, and 940 mL H2O. The potas-
sium permanganate stain was made with 200 mL H2O, 1.33 grams
KMnO4, 13.33 grams of K2CO3, and 4 mL of 5% NaOH. Chromatog-
raphy was performed by Teledyne ISCO. In reactions where water
was not present by solvent, reagent or by-product, the vessels were
flame-dried under a slow argon flow. A slight positive pressure of
dry argon was maintained via rubber septa seal during the course
of the reaction. The argon stream originated from a regulated high
pressure argon tank and was used without further drying. All
reactions were stirred with a Teflon-coated magnetic stir bar and
stir plate. Removal of solvents was typically done using a Buchi
rotary evaporator, Model number 011 connected to house vacuum
line. The condenser was cooled by a Haake B3 circulator chilled to
0 °C cold thumb. All reaction solvents were purchased as anhy-
drous with sure seal tops. 1H NMR spectra were recorded at
400 MHz on a Bruker spectrometer. All chemical shifts were
reported from tetramethylsilane with the solvent resonance of
CDCl3 (7.27 ppm), DMSO-d6 (2.5 ppm). Mass spectra were recorded
on a Finnegan LCQ Advantage Max. Reverse phase analytical runs
were done on an Agilent 1100 HPLC 95:5 H2O w/ 0.1% TFA/MeCN
w/ 0.1% TFA ꢂ100% MeCN w/ 0.1% TFA on a phenomenex Luna 3
micron C18 100 angstrom column 20 ꢀ 4 mm, with a flow rate of
1.0 mL/minute.
210 compounds were then docked to G
a ai1-GTP-
i1-GDP-Mg2+, G
Mg2+, and G q-GDP-Mg2+. Docking was performed in screening
a
mode where the receptor (in this case the trimeric system of G-
protein, nucleotide, and magnesium) was held rigid and the ligands
were allowed to flex. MOE docking used Amber12:EHT forcefield
and Born solvation model. Simulations were focused on the nucle-
otide by selecting the magnesium ion as the ligand placement
strategy. Initial placement calculated 60 poses per molecule using
triangle matching placement with London dG scoring, the top 30
poses were then refined using forcefield placement and Affinity
dG scoring. The 0990 focused docking simulations used the same
structural pbd file and docking as in in the DOCK6 simulations.
In this case, initial placement calculated 100 poses using triangle
matching with London dG scoring.The top 50 poses were then
refined using forcefield refinement and Affinity dG scoring.
2.7. Protein expression and purification
Human G
a
i1 was expressed using a plasmid containing the Pro-
i1 inserted into pPAL7 and
finity eXactÒ (BioRad)45 construct of G
a
stably transfected into Escherichia coli BL21 cells. BL21 cells were
grown at 28 °C in LB media made using 15.5 g/L of Difco™ 2ꢀ YT
(ThermoScientific #244020) plus 7.5 g/L NaCl and 50 mg/L ampicil-
2.10. 0990CL synthesis
lin. G
a
i1 expression was induced with 30
l
M isopropyl-thiogalac-
To a flame dried flask cooled under argon is added the amine
(1.0 equiv), the aryl halide (1.1 equiv), and triethylamine
(1.5 equiv) The mixture is then taken up in 1.4 mL of ethanol
(stored over 4 angstrom mol. sieves). The vessel is sealed, and then
heated to 120 °C for a period of 4 days which was determined
empirically to give the optimum yield. The contents are concen-
trated and purified via basic alumina using dichloromethane/
Methanol (97:3) 1H NMR (CDCl3) d 7.92 (d, 8.28 Hz, 1H), 7.79 (m,
2H), 7.68 (m, 2H), 7.51 (m, 3H), 7.25 (m, 1H), 4.50 (s, 1H), 1.87
(s, 3H), 1.39 (s, 6H). Mass calcd for C21H21N5, 343.18. Observed,
344.3 (M+1). The final yield for 0990CL synthesis was ꢁ40%. Con-
gener synthesis is provided in the Supplementary methods.
tose when OD600 was ꢁ0.6. Temperature was reduced to 25 °C at
induction, and induction was carried out 12–16 h. Cells were har-
vested at 3200g for 18 min. Gaq was expressed in Hi5 insect cells
using G
aq baculovirus (generous gift of Steven Graber WVU) at 5ꢀ
MOI for 48 h.46
Cell pellets were lysed using 100 mM NaPO4, pH 7.2 supple-
mented with 150 lM GDP, 5 mM b-mercaptoethanol, Roche Inhib-
itor CocktailÒ, and 1 mM phenylmethylsulfonyl fluoride. Cell
lysates were sonicated and then centrifuged at 100,000g for
30 min. Supernatants were removed and filtered using 0.45 lm
syringe filter. Gai1 was then purified according to the Profinity
eXactÒ protocol. Gaq was partially purified by first isolating the
low speed pellet (10,000g) after and lysis followed by isolation
using high (50 k) and low (30 k) molecular weight cut-off filters
(Amicon Ultracel UFC805024 and UFC803024).
2.11. STD NMR
Samples for STD NMR experiments were prepared with 34
lM
protein and 500 M ligand. STD NMR data were collected at
l
2.8. Real time nucleotide exchange assays
298 K on a Bruker Avance III 600 MHz NMR spectrometer equipped
with a 5 mm cryogenically-cooled QCI-inverse probe and using a
standard STD pulse sequence47,48 with 30 ms 8.3 kHz spin lock to
minimize background protein resonances. Solvent suppression
was achieved using the excitation sculpting scheme.49 Saturation
of the protein signals was performed using a train of 10, 20 or 59
selective 56 dB Gaussian pulses of 50 ms duration, totaling a
0.51, 1.02 or 3.01 s saturation time. The on-resonance frequency
was set up at ꢂ0.5 ppm, and the off-resonance one was applied
to ꢂ17.16 ppm. STD spectra were acquired with a total of 2048
Real time nucleotide exchange assays were performed in 96-well
clear flat bottom plates (Corning 3596). 600 nM purified Gai1 or Gaq
was incubated with inhibitor compounds in 10 mM HEPES, 100 mM
NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 7.4 (BODIPY Buffer) for 30 min,
and 250 nM of the non-hydrolyzable fluorescent BODIPY FL GTP
(Invitrogen) was added to initiate nucleotide exchange. Final well
volumes were 50 L. Test compounds were dissolved in DMSO
and final vehicle concentration was 61% v/v. 1 M unlabeled GTP
cS
l
l
cS
was added after 20 min to assess protein viability. Relative
fluorescence units (RFU) were measured using a SpectraMax M5
(Molecular Devices) plate reader. Fluorescence was measured at
approximately four minute intervals at 475 nm excitation, 525 nm
emission, and 515 nm cut-off wavelengths for one hour.
transients in addition to 8 dummy scans. Longitudinal T1
relaxation times were determined from an inversion recovery
experiment processed and analyzed using Topspin 3.1. STD
enhancements at tsat = 3 s compensated for perturbations from
differential ligand proton T1-relaxation rates were determined
following the procedure outlined by Kemper et al.50
2.9. Synthetic chemistry
2.12. Transfections
Reactions were monitored by analytical thin-layer chromatog-
raphy on EM-Science hard layer silic gel-60F-254 plates cut into
1 ꢀ 2.5 cm pieces. Visualization was effected by ultraviolet light
For the cAMP GloSensor HEK293 cells, 1
receptor plasmid DNA was added to a final volume of 500
l
g of a2 adrenergic
ll of
Please cite this article in press as: Appleton, K. M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.035

4
K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
OptiMEM in tube ‘A’, and 2.5
final volume of 500 ll of OptiMEM in tube ‘B’. Tubes A and B were
l
l of lipofectamine were added to a
in presence of PTX, as demonstrated by the absence of enhanced
forskolin (Fsk) induced cAMP signal in presence of PTX. Cells were
pre-treated with vehicle, PTX (100 ng/mL, 24 h), or TAT-GPR
vortexed briefly and incubated at room temperature for 5 min. Fol-
lowing incubation, tube B was added to tube A, inverted twice to
mix, and incubated at room temperature for 40 min. The 1 mL mix-
ture of DNA, lipofectamine, and OptiMEM was added to a 15 mL
conical tube containing 1 mL pre-warmed OptiMEM and 3 mL of
serum-free MEM. Following medium aspiration and 1ꢀ PBS wash,
the 5 mL mixture was added to each 10 cm plate and incubated
overnight.
(0.1
GPR peptides, cells were treated with 250 nM Fsk and 10
the 2AR selective agonist UK14304. Stimulation with UK14304
reduced the Fsk-stimulated cAMP, and this effect was suppressed
by PTX. TAT-GPR was very effective at preventing the G i mediated
lM, 1.0
lM and 10.0
l
M, 20 min). After pre-treatment with
l
M of
a
a
inhibition of cAMP stimulated by UK14304, resulting in cAMP levels
that were similar to Fsk alone. Also of interest, TAT-GPR but not
PTX, was able to raise basal cAMP levels compared to vehicle treat-
ment (p = 0.009 between vehicle and GPR basal).
2.13. cAMP measurements
These data demonstrate TAT-GPR can selectively block Gai reg-
Cyclic AMP was measured in GloSensor cAMP HEK293 cell line
ulation of GPCR mediated adenylyl cyclase activity. In the only
in vivo experiment using GDIs to date, the TAT-GPR peptide repli-
cates the phenotype of chronic cocaine-treatment by inducing
altered craving, locomotor behavior, and increased glutamate
transmission when TAT-GPR is infused into the prefrontal cortex
of rats.38,60,61 Although TAT-GPR is very useful as a pharmacologic
probe, as a peptide it has several liabilities in development as a
potential therapeutic. The GPR consensus peptide has a molecular
weight of 3.2 kDa and a pI of 4.1. TAT-GPR is a 40mer with a mass
of 4847 Da, a pI of 10.9, and an instability index of 108. We there-
fore set out to discover small molecule GDIs.
(Promega, Inc.) in 96-well clear bottom plates using a FLIPRTETRA 51
.
For PTX treated wells, PTX was added 24 h prior to the assay to a
final concentration of 100 ng/mL. The day of the assay (72 h after
transfection), cells were pre-equilibrated in GloSensor cAMP
reagent and pre-incubated with test compounds for 20 min prior
to initiating the assay. Forskolin and/or UK14304 were added
simultaneously and relative luminescence units (RLU) were
recorded every 2 s for 600 reads.
3. Results
3.1. The permeable GPR peptide
The GPR peptide is a unique and practical way to study the bio-
3.2. Identification of small molecule heterotrimeric GDIs
Studies with the GPR peptide and GPR/Goloco domain proteins
have taught us that the GDI interaction minimally only needs three
points of contact between the GDI and G-protein. Therefore, a
small molecule GDI drug only needs to bind the nucleotide and
the protein, and each additional interaction is a gain for affinity
and selectivity. The initial phase of small molecule identification
was performed via computational docking using the X-ray crystal
chemical properties of the GPR motif, Gai, and GPCR function
in vitro and in vivo. However, a major obstacle to observing the
GPR peptide effects within a cell is the inherent lack of peptide per-
meability through cell membranes. In order to facilitate the entry
of the GPR peptide into cells, membrane permeable amino acid
sequence tags were explored (Fig. 1A). Numerous membrane per-
meable sequence tags allow passage of usually impermeable pro-
teins and peptides through the cell membrane.52 Two chemically
distinct sequences were chosen for comparison. A basic peptide
sequence derived from the TAT (aa 48–59, NP_057853)
(GRKKRRQRRRPP) protein from HIV.53,54 TAT fusions (TAT-SIRK)
structure of Gai1–GDP, the ZINC small molecule database of
280,000 molecules, and DOCK6.3.40,42,62 The intent was to identify
molecules that interacted with both the protein and embedded
nucleotide and therefore inhibit nucleotide exchange. This docking
study used the inactive conformation crystal structure of
to a Gb
c
binding peptide were able to dissociate G
a
ib
c
heterotri-
Ga
i1–GDP (PDB: 2OM2)42 and docked small molecules to a region
mers while stimulating ERK, JNK, PLC and Ca2+ release.32,55 We also
explored a hydrophobic signal-sequence based peptide derived
from the FGF related oncogene K-FGF (7–21, NP_001998) (AAVALL-
PAVLLALLA) found in humans.56 The permeable leader sequences
were fused to the amino terminus of the consensus GPR peptide
to create a synthetic, cell-permeable GPR peptide.
proximal to the nucleotide binding pocket (Fig. 3A and B). The crys-
tal structure of G i–GTP (active conformation; PDB: 1AGR) was
a
used as a counter screen to ensure subunit state selectivity of the
identified potential inhibitor molecules.63 The crystal structure of
Ga
q–GDP (PDB: 2RGN)43 was used as another counter screen to fil-
ter virtual hits and ensure subunit isoform selectivity. Only 10
The inhibition of GTPcS binding to purified G-protein by the
compounds out of 280,000 interacted with G q–GDP with a pre-
a
modified GPR peptides was then compared to the unmodified
GPR peptide (Fig 1B). A major concern was the possibility that mod-
ification of the GPR peptide would perturb the secondary structure
of the peptide, rendering it less active. Both of the tagged peptides
dicted binding energy <ꢂ1000 kcal. After ranking and filtering, a
total of 210 molecules emerged from the flexible docking after
passing thresholds of ꢂ1000 kcal and represented a virtual hit rate
of ꢁ0.08% (Fig. 3C). We further validated the DOCK6 based
simulations using MOE as an alternate simulation methodology.
We redocked the top 210 compounds to the top 210 compounds
inhibited GTPcS binding to purified Gai with slightly lower potency
than observed with the unmodified GPR consensus peptide. FGF-
GPR peptide modification exhibited a half-log right shift in IC50
for Gai while the TAT-GPR peptide exhibited an apparent affinity
similar to the unmodified GPR peptide. TAT modification remained
more potent than the FGF modification and, therefore, the TAT-GPR
peptide was chosen a better candidate for further studies.
to G
3D). The large scatter and high (poor) scores for G
q–GDP indicated that DOCK6 and MOE were in agreement to
enrich for compounds that reproducibly interacted with the
i1–GDP nucleotide site.
The specific G i1–GDP hits were rank ordered according to
a
i1–GDP–Mg2+, G
a
i1–GTP–Mg2+, and G
a
q–GDP–Mg2+ (Fig
i1–GTP and
a
Ga
Ga
We then assessed the intracellular effects of Gai GDIs on 2nd
a
messenger cAMP levels using the
the
2AR in multiple cellular backgrounds as well as in vivo.57–59
Note that due to a presumed deficiency of specificity of the GPR
motif across G i isoforms, it is not specifically known if G i1 versus
i2 (or both) is coupling to the 2AR within our system. 2AR
expression was optimized to prevent G-protein switching to Gas
a
2AR (Fig. 2). G
a
i1 associates with
binding energy and grouped into 65 molecularly similar chemical
clusters using a Tanimoto similarity coefficient (Tc) of 60%.64
Fifteen representative molecules representing one-quarter of the
possible clusters were selected to sample diverse chemical space.
These compounds were then validated for their ability to regulate
a
a
a
Ga
a
a
Gai and Gaq.
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K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Figure 1. The permeable GPR peptides and their effect of on GTP
c
S binding to purified G
ai. (A) Amino acid sequences of GPR peptides. GPR motif consensus residues are
colored in red and membrane permeable tags are colored in blue. (B and C) GTP
c
35S (500 nM) binding to purified G
ai1 (100 nM) was measured in the absence and presence
of peptides. Data are expressed as the percent of specific binding (ꢁ5 pmol) observed in the absence of peptide and represent the mean SD derived from two experiments
performed in duplicate. The TAT-GPR data in B has been previously published.38
Figure 2. Effect of G
mL 2AR receptor. Cells were pre-treated prior to the experiment with 100 ng/mL PTX (24 h) or 0.1
treated with 250 nM Forskolin (Fsk) or Fsk and 10 M of the
2AR agonist UK14304. ⁄⁄t Test between vehicle and TAT-GPR was significant with p = 0.009. Data was derived
ai inhibitors PTX and TAT-GPR on
a2AR/G
ai mediated cAMP suppression. cAMP levels were measured in HEK293 GloSensor cells transfected with 0.2
lg/
a
l
M and 10 M TAT-GPR peptide (GPR, 20 min). Cells were subsequently
l
l
a
from triplicate determinates from three independent experiments.
3.3. Validation of small molecule Gai GDIs
wavelengths by using an excitation of 475 nm and monitored
emission at 525 nm with a 515 nm cut-off. Although this modifica-
tion only left a 10 nm observation window (515 to 525 nm) and
decreased the overall signal intensity, we found that it improved
the ability to search for low affinity interactions while decreasing
potential fluorescence interference.
Real time nucleotide exchange assays were performed on small
molecules obtained from the docking screen using the non-hydro-
lyzable fluorescent nucleotide analog BODIPY FL GTP
improve sensitivity of the assay, we modified the experimental
c
S.65,66 To
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6
K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 3. Computational protein docking for the discovery of G-protein inhibitors. (A) Grid and placement spheres of Gai used for site targeted docking simulations. (B)
Ribbon diagram G
i1–GDP–Mg2+. Ribbons are colored according to chain termini. (C) Top 210 compounds (from 280,000) rank ordered from ZINC neutral DOCK6 docking to
i1–GDP–Mg2+. (D) MOE based re-docking of top 210 compounds to G i1–GDP–Mg2+, G i1–GTP–Mg2+, and G q–GDP–Mg2+. Data shows scores for 30 poses per molecule.
a
G
a
a
a
a
We tested the 15 compounds in the primary G
a
i1 and Gaq
90:10) exhibiting a deprotonated nitrogen N10 and a sp3-hybrid-
exchange assay (Table 1). The exchange assay demonstrated a con-
centration-responsive reduction in fluorescence for several com-
pounds compared to vehicle control reactions (Fig. 4A and B &
ized carbon C30. Compound 8005, which showed no inhibition of
Ga
i nucleotide exchange, was used as a negative control. Figure 5B
and D show the aliphatic and aromatic regions of reference spectra
of a mixture of 0990CL and G i1 in the presence of excess GDP. The
Fig. S1). Four compounds showed selectivity for G
Selectivity was best demonstrated by compounds 0990 and 4630
which demonstrated selectivity for G i and resulted in a maximum
a
i1 over Gaq
.
a
STD NMR spectra recorded with varying saturation times (tsat = 1
and 3 s) revealed several points of contact on 0990CL when bound
a
endpoint fluorescence reduction of 38 8% and 34 3%, respec-
tively (Fig. 4C & Fig. S1). Compounds 8005, 8770, and 4799 were
selective for Gaq with 8005 showing a maximum reduction of
endpoint fluorescence of 27 7% respectively (Fig. 4D & Fig. S1).
Compounds 2967, 6715, and 1026 were not selective and showed
to G
ual 0990CL protons reflect the proximity to the binding site of
i1. However, the T1-relaxation of individual protons and
ai1 (Fig. 5B and D). The relative STD enhancements for individ-
Ga
exchange kinetics can further complicate the analysis of the bind-
ing epitope.50,68 Therefore, we explicitly accounted for differential
longitudinal relaxation times of 0990CL protons ranging from
2.284 for the aromatic H400 to 0.422s measured for the aliphatic
20-Me (Fig 5A).50 Normalized STD enhancements (setting one H30
proton to 100%) at a saturation time of tsat = 3 s are summarized
in Figure 5C. The largest STD effects can be observed for the two
aliphatic H30 protons followed by the two 20-Me and the 40-Me
groups. In the aromatic region, more moderate interactions of the
inhibition of nucleotide exchange with both G
The remainder of the compounds did not affect nucleotide
exchange activity. Compounds that showed selectivity for G i1
ai1 and Gaq (Table 1).
a
and also a reduction in end-point fluorescence greater than 10%
were retained for further testing. This threshold criterion was
chosen in order to explore greater chemical space and chemotypes
in the early phase of screening. We also re-synthesized 0990 to
confirm structure and purity for all further experiments (Supple-
mentary methods, in-house synthesized compound is specifically
named 0990CL).
compound with G
tems involving protons 20 through 60 and H5, 6, 7 and 8. Inspection
of mixtures of G i1 in its GDP-bound state together with 0990CL
ai were also observed for both aromatic ring sys-
a
To confirm binding of 0990CL to Gai in solution we used satu-
ration transfer difference (STD) NMR. STD NMR is a robust tech-
nique allowing direct measurement of ligand binding to target
proteins through ligand signals that appear in STD experiments
due to saturation transfer from the target protein while in the
ligand’s bound state.47,67 STD NMR confirmed that compound
and 8005 revealed that all of the signals of the 8005 negative con-
trol were absent in the difference spectrum indicating that this
compound does not bind Gai1 (Fig. S2).
3.4. Structure–activity relationship survey of 0990CL
0990CL interacted with purified G
a
i1 in its GDP-bound state which
After binding confirmation by NMR, compound 0990CL was
selected for further validation. Six 0990CL congener compounds
were subsequently purchased to explore substituents on the
aniline and phenyl rings of a quinazoline scaffold (Supp. Table 1).
we used in these experiments (Fig. 5). Interestingly, our NMR anal-
ysis revealed that compound 0990CL converted to the tautomer
state shown in Figure 5A in aqueous solution (H2O/DMSO-d6;
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K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
Table 1
Primary screening for GDIs through chemical diversity sampling
% Inhibition
CAS #
ID
G
ai
Gaq
864389-67-3
4630
38
<10
N+
O
N
S
H3C
907989-49-5
511514-03-7
849019-55-2
9827
0990
1217
35
25
30
<10
<10
<10
OH
N
HO
H3C
H3C
CH3
HN
N
N
NH
N
O
N+
NH
SH
O
O
CH3
O
301353-19-5
749837-97-6
7136
7507
13
<10
<10
NH
Me
N
O
O
<10
CH3
O
NH
N
N
N
S
NH
O
1070463-59-0
8005
<10
27
NH
N
N
N
H
C
3
CH
3
(continued on next page)
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8
K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1 (continued)
% Inhibition
CAS #
ID
Gai
Gaq
NH2
CH3
NH
284029-69-2
1254
<10
<10
—
—
O
S
H2N
NH
1020944-29-9
1174
H3C
CH3
H3C
H3C
CH3
N
N
N
1184184-00-6
2967
34
50
N
NH
H3C
CH3
O
CF3
NO2
O
1244948-00-2
6715
38
35
O-
CH3
CH3
CN
CN
H
N
H3C
CN
O
312609-02-2
1026
18
40
CH3
O-
N
O
N+
C-
N
111015-95-3
712346-46-8
8770
4799
<10
<10
32
25
N
O
NH
H3C
N
CF3
NH
Compound 9585, with a methyl group at positions 6 and 50 and
compound 9586, with a methyl at positions 6 and 30, both
need for the phenyl substituent at position 4 for exchange inhibi-
tion. Compound 6784, with a hydroxyl at the 50 and a phenyl and
methyl at positions 4 and 6, respectively, also showed no activity
towards nucleotide exchange inhibition. These data indicate the
requirement for hydrophobic substituents on the aniline ring and
the phenyl ring at position 4.
Our data thus far confirmed the quinazoline scaffold as amend-
able to GDI activity. We went further and synthesized and tested
ten additional congeners or fragments based on compound
0990CL (Supp. Table 1). Compound 2397, with a chloride at the
30 position and a phenyl at position 4, and compound 2395, with
displayed >50% inhibition of nucleotide exchange with
Gai.
Compound 9587, with a chloride at 50 and a methyl at position 6,
displayed only weak inhibition of nucleotide exchange on Gai
(ꢁ16%). These results suggest that hydrophobic interactions on
the aniline ring enhance exchange inhibition. Compound 9253,
with a carbonyl at the position 20, a methyl at 40, and methyl groups
at positions 4 and 6, proved ineffective at influencing nucleotide
exchange. Also ineffective was compound 8358 with methyl
groups at the 4 and 40 position. These two results emphasize the
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K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
Figure 4. Primary validation of inhibitors by Gai versus Gaq real-time fluorescence nucleotide exchange assay. (A and B) For each experiment 600 nM G
a
i or Gaq was pre-
incubated with vehicle (grey and red) or 300
nucleotide exchange was monitored via fluorescence for 60 min. Background signals were determined using BODIPY FL GTP
GTP S introduced to control reactions after 20 min (red) resulted in a decrease in fluorescence through time and demonstrates the nucleotide exchange viability of G-protein.
lM (green), 100
lM (purple), or 30
lM (olive) test compound 0990CL for 30 min prior to the addition of BODIPY FL GTP
cS, and
c
S alone and is represented in black. Unlabeled
c
(C and D) Endpoint results (60 min) of the kinetic assays depicted in A and B for G
from triplicate determinations from three independent experiments.
ai and Gaq for compounds 4630, 8005, and 0990CL on Gai and Gaq. Data in C–F was derived
a phenyl at position 4 both resulted in >30% inhibition of nucleo-
tide exchange on G i. Compounds 2353 and compound 2369
3.5. Molecular docking study of 0990CL
a
resulted in 23% and 18% inhibition of Gai nucleotide exchange,
respectively, suggesting that the quinazoline scaffold enhances
exchange inhibition. Compound 2410, with only a single nitrogen
on the aniline ring and phenyl at position 4; and 2437, with methyl
groups at positions 30 and 50 and a phenyl at position 4 were both
inactive toward nucleotide exchange. These results suggest that
both nitrogens are likely required for compound interaction.
Compound 2471, with carbonyl linker connecting the quinazoline
and the aniline ring, was inactive indicating that the phenyl-
quinazoline-aniline core structure enhances compound interac-
tion. Compound 2621, with a methoxy group at position 30 and a
phenyl at position 4, was inactive. Other inactive non-quinazoline
type compounds included 2355, with just a portion the nitrogen
trio of the quinazoline-aniline structure; and 8910, exploring this
same feature, emphasize the need for the basic phenyl-quinazo-
line-aniline root structure.
To further analyze the potential properties of the inhibitor/
G-protein interaction, we performed molecular docking simula-
tions on compound 0990CL. The top pose of interest for 0990CL
interacted with both Arg178 and Val199 (Fig. S4). Energy minimiza-
tion of the system which allowed the G-protein and nucleotide to
flex had no effect on the final pose. Other high probability interac-
tions were the central secondary amine of 0990CL donating a pro-
ton to Glu43 or Gln79 and H-arene interactions with Gln79 or Lys180
.
As we had hoped, these interactions mimic much of the biochem-
istry of the GPR/Goloco interactions with a mix of hydrophobic and
ionic interactions. These poses also indicate that the unmodified
phenyl is not planer with the rest of the molecule. Also the central
secondary amine is a flexible hinge allowing the overall molecule
to be convex. These features help to create a molecule with three
dimensional topology to present both hydrophobic and proton
donating features.
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10
K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 5. Saturation transfer difference (STD) NMR of G
a
i1 and 0990CL. (A) Chemical graph depicting inhibitor compound 0990CL, proton numbering scheme for STD NMR
i. Increases in STD signals are observed with
i inhibitor 0990 docked with G i GDP. T1-compensated STD
sat = 3 s were normalized to H30 which was set to 100%. (D) Enlargement of the aromatic region of the reference and STD NMR spectrum of 0990CL
i. Increases in STD signals are observed with saturation times ( sat) of 1 s (blue) and 3 s (red) and 0990CL and H8(GDP)-proton assignments are indicated.
and T1-relaxation times (italic). (B) Enlargement of the aliphatic region of the reference and STD NMR spectrum of 0990CL with G
saturation times ( sat) of 1 s (blue) and 3 s (red) and 0990CL proton assignments are indicated. (C) Tautomer G
effects observed using
with G
a
s
a
a
s
a
s
Through our synthetic, informatics, and structural characteriza-
tion we observed tautomerization of 0990CL. Specifically, the 30
carbon (Fig. 5A and C and Fig. S4) on the terminal pyrimidine is
capable of switching between alternate sp2 or sp3 hybridization.
Tautomer T1 was appealing as our earlier pose predictions indi-
cated the potential for the Arg178 guanidinium group donating a
hydrogen bond. However, our STD NMR experiments indicated
the sp3 tautomer was the primary interacting form in our experi-
ments. To further understand the consequences of tautomerization
buried while T2 had the phenyl portion of the quinazoline buried
(Fig. S5C). While both tautomers were able to form effective bonds
with Val179 in the simulations, T2 was able to form additional bonds
with GDP. Lastly, we took the top poses for each tautomer and
calculated the potential energy for the complex and individual
components (Fig. S5D). The poses predicted from the docking sim-
ulations gave the lowest energies while swapping the tautomers
gave poorer energies. Interestingly, T1 in the T2 pose was very unfa-
vorable in terms of energy, indicating T2 in its native pose is the
highest affinity interaction possible.
of 0990CL for interaction with Gai, we tested the two tautomers in
molecular simulations (Fig. S5). Using the same GDP site as used in
the primary virtual screening, both 0990CL tautomers were docked
3.6. Effect of GDIs on cellular cAMP
to Gai. Docking scores for the top 25 poses between the two were
nearly identical. Further investigating the poses, we recognized that
the poses were flipped with respect to each other and the GDP
binding site. Tautomer T1 consistently had the trimethylpyrmidine
We also assessed the cellular effects of small molecule G
on cAMP levels using the 2AR as a cellular model (Fig. 6 and
Fig. S3). 0990CL was able to partially restore cAMP levels following
ai GDIs
a
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K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
UK14304 treatment. Restoration was greatest at the lowest con-
centration of 0990CL (100 nM), which was able to restore ꢁ31%
of the reduction of cAMP elicited by Fsk and UK14304. At higher
GDP-bound confirmation of Gai and therefore directly measured
nucleotide exchange. The standard nucleotide exchange assay
was modified by optimizing the emission bandpass to avoid fluo-
rescence interference from test compounds. Fifteen compounds
were tested in the first phase of validation and eight were active
concentrations of 0990CL (10 lM), cytotoxic effects of the com-
pound began to negatively impact cell viability. The negative con-
trol compound 2355 showed no effect upon cAMP levels following
treatment with Fsk and UK14304 which demonstrates our inhibi-
tors likely do not interfere with the ability of UK14304 to activate
for G
exchange on G
ai inhibitors showed varying degrees of efficacy to increase
ai, although four were promiscuous and inhibited nucleotide
a
i and G q. 0990CL, 8770 and 2967 small molecular
a
G
the
signaling though
Compounds 8770 and 2967 demonstrated a lack of toxicity at
10 M indicated by measured max efficacy of Fsk in the presence
of UK14304. The fact 8770 did not inhibit G i1 in our nucleotide
exchange assay yet shows in vitro efficacy highlights the necessity
to conduct multiple assays to fully characterize compounds as
inhibitors in any drug screening platform.
a
2AR. Also of note, 8770 and 2967 were able to suppress cAMP
cAMP. From these data, a quinazoline scaffold emerged as tractable
for Gai inhibition. The structure–activity relationship (SAR) of the
seven quinazoline derivatives indicated the critical need of both
hydrophobic interactions provided by compound methyl groups
and hydrogen bond donors provided by compound amine groups.
We confirmed compound interaction by STD NMR. Data from the
exchange assay, SAR, NMR, and docking indicate that the 40-Me
moiety, and to a lesser degree, the 20-Me moiety, from 0990CL
are involved in specific hydrophobic interactions. There are also
significant compound interactions in the aromatic region and
implicate the pyrimidine secondary amine participates in hydro-
gen bonding. The compound 0990CL SAR did not indicate an obvi-
ous rout to substantial improvement, and future characterization
of hits such as 9595 and 9586 might provide better compounds
in the long run. However, these compounds do demonstrate the
a2AR in a concentration-dependent manner.
l
a
4. Discussion
Heterotrimeric G-proteins are central regulators of cellular
communication, and potent and selective inhibitors can serve mul-
tiple purposes ranging from being a pharmacologic probe to exper-
imental therapeutics. As G-proteins are signaling amplifiers, they
are an ideal target for therapeutic intervention and diagnosis. Here
we presented the rationale, techniques, and preliminary com-
feasibility of small molecule heterotrimeric G
GDIs are well suited to pursue receptor pharmacology of Gai
versus Gb signaling and their specific roles in cell signaling and
disease.69 A unique aspect of G
–GDIs is their ability to selectively
inhibit G with the potential to stimulate Gb signaling. This facet,
along with Gb directed pharmacologic tools and PTX, will allow
addressing the contribution of G , Gb , and G signaling in
living cells without alterations in protein stoichiometry. Several
pharmacologic regulators of Gb have been developed including
the SIGK peptide which dissociates the heterotrimer creating free
a GDIs.
c
pounds aimed at developing selective GDIs of Ga subunits.
a
The TAT-GPR peptide and our small molecule GDIs were
designed to stabilize the GDP bound confirmation of Gai by
blocking nucleotide release and also interacting G-protein isoform
specific residues around the nucleotide binding pocket. We deter-
mined that appending a TAT leader sequence was able to make the
GPR peptide cell permeable without suppressing nucleotide
exchange activity. Tagging with FGF was able to permeabilize the
GPR peptide, but inhibited nucleotide exchange activity. We then
determined that compared to PTX, TAT-GPR was very potent at
preventing UK14304 stimulated decreases in cAMP.
a
c
c
a
c
abc
c
G
G
a
a
and Gbc 70,71
.
The phosducin peptide inhibits Gbc but allows
to signal.72 With these tools, pharmacologic regulation of both
receptor-dependent and -independent signaling of individual
heterotrimeric subunits can provide a unique window into this
Using computational docking we probed the G
using 280,000 diverse compounds. We also used G
q–GDP as computational counter-screens. From these simula-
tions, we determined 210 potential i–GDP interacting
compounds. We desired to find compounds that stabilized the
a
i–GDP binding
major signaling axis. Therapeutically, G
a subunit inhibition by
ai–GTP and
GDI has the advantage of circumventing the need to directly
address the upstream component of GPCR-related signaling in
cases of mutations, polymorphisms, and expression-related defects
often seen in disease.
Ga
G
a
Author Contributions
All authors participated in research design. All authors
conducted experiments. Synthetic chemistry was designed and
performed by Lindsey. All authors performed data analysis. Lind-
sey, Hennig, and Peterson contributed new reagents or analytical
tools. All authors contributed to writing of the manuscript.
Acknowledgments
We thank Heidi Hamm, Anita Preininger, and Ali Kaya for
thoughtful discussions, purified G-protein, and recombinant
expression system. The G
aq baculovirus was a gift from Stephen
Graber. The 2AR plasmid was a gift from Stephen Lanier. We
a
thank John Hildebrandt, John Oatis, Joe Blumer, and Will Robesh-
aux at MUSC for support and thoughtful discussions. The initial
work with the cell-permeable GPR peptides was conducted while
YKP was a graduate student in the laboratory of Dr. Stephen M.
Lanier in the Department of Pharmacology at Louisiana Health
Sciences Center in New Orleans and was supported by the National
Institutes of Health Grant NS24821 awarded to S.M.L. K.J.B.
received an American Society of Pharmacology & Experimental
Figure 6. Effect of small molecule G
suppression. cAMP levels were measured in HEK293 GloSensor cell line was
transfected with 0.2 g/mL 2AR receptor. Cells were pre-treated prior to the
experiment with 100 ng/mL PTX (24 h), 0.1, and 1 M 0990CL (20 min) or 0.1, 1,
and 10 M 8770, 2967, and 2355 (20 min). Cells were subsequently treated with
250 nM Fsk or Fsk and 10 M of the 2AR agonist UK14304. Data was derived from
triplicate determinates from three independent experiments.
a inhibitors on a2AR/Gai mediated cAMP
l
a
l
l
l
a
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K. M. Appleton et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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by pilot research funding from an American Cancer Society Institu-
tional Research Grant awarded to the Hollings Cancer Center at
MUSC (YKP), and by the National Science Foundation (1126230
to M.H.).
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Please cite this article in press as: Appleton, K. M.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.035