The molecular pharmacology of AMD11070: An orally bioavailable CXCR4 HIV
Renee M. Mosia, Virginia Anastassovaa, Jennifer Coxa, Marilyn C. Darkesa, Stefan R. Idzana,
Jean Labrecquea, Gloria Lauc, Kim L. Nelsona, Ketan Patela, Zefferino Santuccia, Rebecca S.Y. Wonga,
Renato T. Skerljc, Gary J. Bridgera, Dana Huskensb, Dominique Scholsb, Simon P. Frickerc,*
aFormally of AnorMED Inc., #200 – 20353 64th Avenue, Langley, BC V2Y 1N5, Canada
bRega Institute for Medical Research, Leuven, Belgium
cGenzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA
The introduction of highly active antiretroviral therapy
(HAART) has dramatically changed the outcome for HIV-1 infected
persons with improvements in morbidity and mortality. However,
there are challenges associated with chronic use of reverse
transcriptase inhibitors and protease inhibitors, the components
of HAART, including emergence of resistance and chronic toxicities
which limit patient compliance such as diarrhea, anemia and
lipodystrophy . There is therefore a need for new agents with
different molecular targets. The inhibition of viral entry has
emerged as a validated target for HIV infection and the first
approved entry inhibitor was enfuvirtide which acts by blocking
gp41-mediated fusion . The discovery that HIV requires both
host CD4 and a chemokine receptor as a co-receptor for cell entry
stimulated research into chemokine receptor inhibitors as a new
class of HIV drugs [3,4]. Chemokines are 8–10 kDa proteins
defined by the number and relative spacing of cysteine residues at
the N-terminal end of the protein. The two major families are CC
and CXC in which there are two cysteine residues that are either
adjacent (CC) or separated by one amino acid residue (CXC). They
are mediators of hematopoiesis and inflammation, regulating
lymphocyte development, homing and trafficking. Their receptors
belong to the class A family of seven transmembrane (7 TM) G-
protein coupled receptors (GPCR) [5,6].
HIV uses two chemokine receptors, CCR5 and CXCR4, proven
targets for HIV drugs [3,7]. CCR5 is the principle co-receptor for
viral transmission in the early, clinically latent stage of the disease,
whereas CXCR4 usage emerges in the later stages and is associated
with a decrease in CD4 cell count and accelerated disease
progression . Virus that uses CCR5 as a co-receptor is
characterized as M (macrophage)-tropic, CCR5-using or R5, and
virus that uses CXCR4 as T (T lymphocyte)-tropic, CXCR4-using or
X4. The CCR5 inhibitor Selzentry1(maraviroc) was approved by
the FDA for use in treatment experienced patients in August 2007
[8–10], and in treatment naı ¨ve patients in November 2009 [11,12].
The bicyclam Mozobil1(plerixafor) is a selective antagonist of
CXCR4 [13,14] and is an inhibitor of T-tropic, X4 virus [15–17], and
was demonstrated to reduce the X4 viral load in HIV-1 infected
subjects . Inhibition of CXCR4 by plerixafor also leads to
leukocytosis and mobilization of hematopoietic stem cells (HSC)
from the bone marrow in both healthy volunteers and in patients
Biochemical Pharmacology 83 (2012) 472–479
A R T I C L E
I N F O
Received 19 August 2011
Accepted 21 November 2011
Available online 28 November 2011
A B S T R A C T
In order to enter and infect human cells HIV must bind to CD4 in addition to either the CXCR4 or the CCR5
chemokine receptor. AMD11070 was the first orally available small molecule antagonist of CXCR4 to
enter the clinic. Herein we report the molecular pharmacology of AMD11070 which is a potent inhibitor
of X4 HIV-1 replication and the gp120/CXCR4 interaction. Using the CCRF-CEM T cell line that
endogenously expresses CXCR4 we have demonstrated that AMD11070 is an antagonist of SDF-1a
ligand binding (IC50= 12.5 ? 1.3 nM), inhibits SDF-1 mediated calcium flux (IC50= 9.0 ? 2.0 nM) and SDF-
1a mediated activation of the CXCR4 receptor as measured by a Eu-GTP binding assay (IC50= 39.8 ? 2.5 nM)
or a [35S]-GTPgS binding assay (IC50= 19.0 ? 4.1 nM), and inhibits SDF-1a stimulated chemotaxis
(IC50= 19.0 ? 4.0 nM). AMD11070 does not inhibit calcium flux of cells expressing CXCR3, CCR1, CCR2b,
CCR4, CCR5 or CCR7, or ligand binding to CXCR7 and BLT1, demonstrating selectivity for CXCR4. In addition
AMD11070 is able to inhibit the SDF-1b isoform interactions with CXCR4; and N-terminal truncated variants
of CXCR4 with equal potency to wild type receptor. Further mechanistic studies indicate that AMD11070 is
an allosteric inhibitor of CXCR4.
? 2011 Elsevier Inc. All rights reserved.
* Corresponding author. Tel.: +1 508 271 4598; fax: +1 508 661 8791.
E-mail address: email@example.com (S.P. Fricker).
Contents lists available at SciVerse ScienceDirect
jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m
0006-2952/$ – see front matter ? 2011 Elsevier Inc. All rights reserved.
with non-Hodgkin’s lymphoma (NHL) and multiple myeloma
(MM) [19,20] and improves mobilization when combined with the
cytokine G-CSF [21–23]. Plerixafor was evaluated in two successful
randomized Phase III clinical trials and was subsequently approved
by the U.S.F.D.A. in 2008 in combination with G-CSF to mobilize
hematopoietic stem cells to the peripheral blood for collection and
subsequent autologous transplantation in patients with NHL and
MM. The EMA approved Mozobil in 2009 [24–26].
Plerixafor is administered subcutaneously for HSC mobilization
whereas an orally bioavailable drug is preferred for HIV treatment.
This prompted the development of an orally available CXCR4
antagonist resulting in the discovery of the non-cyclam molecule
AMD11070 (Fig. 1) . AMD11070 is a potent inhibitor of CXCR4,
and inhibits T-tropic viral infectivity. In clinical trials in healthy
volunteers AMD11070 was found to be well tolerated and orally
bioavailable . In a proof of concept clinical trial in HIV infected
patients harboring X4 virus 4/9 patients had a greater than 1log10
reduction in X4 viral level .
In this paper we present in detail the molecular pharmacology
and mechanism of CXCR4 inhibition by AMD11070. We demonstrate
that AMD11070 is a potent inhibitor of X4 HIV replication.
Confirmation that AMD11070 inhibits viral infection via CXCR4
blockade is provided by the inhibition of cell/cell fusion in an assay
that mimics the binding of gp120 to CXCR4. Using a T cell line that
naturally expresses CXCR4 we show that AMD11070 inhibits the
binding of CXCL12 (SDF-1), the cognate ligand for CXCR4, and
inhibits subsequent receptor activation and downstream signaling.
In addition AMD11070 was shown to have no activity against other
chemokine receptors demonstrating selectivity for CXCR4. Mecha-
nistic studies further probing the molecular interactions of
AMD11070 with CXCR4 demonstrate the allosteric nature of
2. Materials and methods
2.1. Cell lines
All parent cell lines were obtained from the ATCC (Manassas, VA).
CCRF-CEM cells, which naturally express CXCR4, CCR4 and CCR7,
were cultured in RPMI 1640 containing 1 mM sodium pyruvate,
2 mM L-glutamine and 10% fetal bovine serum. HEK293F cells were
transfected to express CCR1, CCR2b, CXCR3, CCR4, or CCR5. Cells
expressing CXCR3 and CCR4 were also co-transfected with a
chimeric Gaqi5 protein to improve signaling. CHO-S cells were
transfected to express the LTB4receptor BLT1. Canine thymus Cf2Th
cells transfected with CXCR7 were maintained in DMEM containing
1 mM sodium pyruvate, 4 mM L-glutamine and 20% FBS. All other
cells were cultured in DMEM containing 1 mM sodium pyruvate,
4 mM L-glutamine, 0.1 mM non-essential amino acids and 10% fetal
bovine serum and 800 mg/mL geneticin. Hygromycin B was added to
cultures expressing the chimeric G-protein.
Canine thymus Cf2Th cells were transiently transfected with
the CXCR4 C-terminus truncated variants R334X and E343X. The
CXCR4 variants, R334X and E343X, were constructed by site
directed mutagenesis of the cloned wild type CXCR4 using the
Strategene Quikchange mutagenesis kit (Agilent Technologies,
Cedar Creek, TX, USA). The primers used for the mutagenesis were
50-caagatcctctccaaaggaaagtgaggtggacattcatctg-30(for R334X) and
50-gtggacattcatctgtttccacttagtctgagtcttcaag-30for (E343X), with
the respective reverse complement primers. After the mutagene-
sis, DNA sequencing was performed on the mutants to confirm that
the target sequence was obtained. An endotoxin-free preparation
of the mutated DNA was prepared by the Qiagen endofree plasmid
kit (Qiagen, Mississauga, Ontario). The canine thymus cell line,
Cf2Th, was transiently transfected with either R334X or E343X
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). For
control experiments Cf2Th cells were transiently transfected with
wild type CXCR4.
2.2. Compounds and chemokines
AMD11070 was synthesized as previously described . All
chemokines were provided by the late I. Clark-Lewis (University of
British Columbia, Vancouver, BC).
All chemicals and buffer salts were obtained from Calbiochem
(now EMD Biosciences, Rockland, MA) unless otherwise stated. Cell
culture media was obtained from Hyclone Inc., Logan, UT. Other
chemicals, including Eu-GTP and [35S]-GTPgS, were purchased
from Perkin-Elmer Life Sciences, USA.
2.3. Anti-HIV activity
Inhibition of HIV-1 replication in MT-4 cells (CD4+CXCR4+
lymphocytic cell line) was performed as previously described.
Anti-HIV-1 activity was determined by assaying the viability of
HIV-1 NL4.3 X4 virus infected MT-4 cells with increasing
concentrations of AMD11070. The IC50 was defined as the
concentration of AMD11070 required to inhibit viral cytopathicity
by 50% . PBMCs were isolated from healthy donors by density
gradient centrifugation and stimulated with PHA at 1 mg/mL
(Sigma Chemical Co., Bornem, Belgium) for 3 days at 37 8C. The
activated cells (PHA-stimulated blasts) were washed three times
with PBS, and infected with HIV-1 NL4.3 X4 virus. HIV-infected or
mock-infected PHA-stimulated blasts were cultured in the
presence of 25 U/mL of IL-2 and varying concentrations of
AMD11070. Supernatant was collected at day 10, and HIV-1 core
antigen in the culture supernatant was analyzed by the p24 viral
Ag ELISA kit (Perkin-Elmer, Zaventem, Belgium) [15,17].
2.4. CXCR4-mediated cell fusion
P4-R5 MAGI cells express endogenous CD4 and CXCR4 on the
cell surface and have also been stably transformed with b-
galactosidase that is under the control of HIV-LTR, which is
transactivated by HIV or SIV Tat protein. CHO-K1 cells (>95%
confluent) were transiently transfected with an endotoxin free
preparation of the plasmid pDOLHIVenv using Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA) accordingly to supplier’s protocol.
Plasmid pDOLHIVenv contains a 3108 bp SalI–XhoI fragment
encompassing the open reading frames of the env, tat, rev coding
regions from pNL4-3. Twenty-four hours after the transfection,
cells were lifted and resuspended at 4 ? 105cells/mL and mixed
with P4-R5 MAGI cells (1:1 ratio) in white 96-well plates in the
presence of AMD11070. Cells were incubated for 16–20 h at 37 8C,
5% CO2. Interaction of CXCR4 expressed on P4-R5 MAGI cells and
gp120 on the transiently-transfected CHO-K1 cells induced fusion
of the two cell types and resulted in the transactivation of the b-
galactosidase gene. Fusion level was monitored using the
Galscreen substrate (Applied Biosystems, Carlsbad, CA, USA), with
luminescence measured by Victor 2 spectrophotometer (Perkin-
Elmer, USA) .
Fig. 1. Structure of AMD11070.
R.M. Mosi et al. / Biochemical Pharmacology 83 (2012) 472–479
2.5. Receptor binding assays
The SDF-1 ligand binding assay against CXCR4 was performed
using the CCRF-CEM cells, and for CXCR7 using Cf2Th cells
transfected to express CXCR7 as previously described [13,32].
Briefly 500,000 cells were incubated for 3 h at 4 8C in binding buffer
(PBS containing 5 mM MgCl2, 1 mM CaCl2, 0.25% BSA, pH 7.4) with
100 pM [125I]-SDF-1a (Perkin-Elmer, 2200 Ci/mmol) on Millipore
AMD11070 was included in the incubation mix at appropriate
concentrations as required. Unbound [125I]-SDF-1a was removed
by washing with cold 50 mM HEPES, 0.5 M NaCl pH 7.4. The
competition binding assay against BLT1 was performed on
membranes from CHO-S cells expressing recombinant BLT1. The
membranes were prepared by mechanical cell lysis followed by
high speed centrifugation, re-suspended in 50 mM HEPES, 5 mM
MgCl2buffer and flash frozen. The membrane preparation was
incubated with AMD11070 for 1 h at room temperature in an assay
mixture containing 50 mM Tris, pH 7.4, 10 mM MgCl2, 10 mM
CaCl2, 4 nM LTB4 mixed with 1 nM [3H]-LTB4 (195.0 Ci/mmol,
Perkin-Elmer Life Sciences) and 8 mg membrane. The unbound
[3H]-LTB4was separated by filtration on Millipore Type GF-C filter
plates. The bound radioactivity was counted using a LKB Rackbeta
1209 Liquid Scintillation Counter.
2.6. Calcium flux assays
Chemokine-stimulated calcium flux was assayed on the
following cells: CCRF-CEM for CXCR4 and CCR7, HEK293F cells
expressing CCR1, CCR2b, CXCR3, CCR4 or CCR5, and Cf2Th cells
expressing the CXCR4 C-terminal truncated variants R334X and
E343X. Cells were loaded with the calcium-indicator Fluo-4-AM
(Molecular Probes Inc.). The loaded cells were then washed and
resuspended in HBSS containing 20 mM HEPES, 0.2% BSA, 2.5 mM
probenecid, pH 7.4. Before the assay, cells were pre-incubated for
15 min at 37 8C with AMD11070 at appropriate concentrations.
Changes in intracellular calcium concentration upon addition of
Eem= 525 nM, using a FLEXstation fluorescent plate reader
(Molecular Devices, CA, USA). Chemokine ligand concentrations
used were 2.5 or 10 nM SDF-1a (CXCL12) for inhibition studies,
and a concentration range of 0.2 nM–1 mM for mechanistic studies,
and 10 nM SDF-1b for CXCR4, 10 nM MIP-1a (CCL3) for CCR1,
13.3 nM MCP-1 (CCL2) for CCR2b, 7 nM TARC (CCL17) for CCR4,
30 nM RANTES (CCL5) for CCR5, 200 nM MIP-3b (CCL19) for CCR7,
and 40 nM IP10 (CXCL10) for CXCR3 [13,32]. Results were
normalized with respect to a control without AMD11070.
Eex= 485 nM,
2.7. GTP binding assays
For the GTPgS binding studies, a concentration range of
AMD11070 was incubated for 1 h at 30 8C in an assay mixture
containing 5 mM GDP, 30 mM NaCl, 10 nM SDF-1a, 1 nM [35S]-
GTPgS and 8 mg CCRF-CEM membrane. The unbound [35S]-GTPgS
was separated by filtration on Millipore Type GF-C filter plates and
the data analyzed as described above .
The CXCR4 Eu-GTP assay was performed as previously
described . A concentration range of AMD11070 was incubated
for 1 h at 30 8C on Acrowell plates (Pall-Gelman, East Hills, NY,
USA) in the presence of 10 mg/well CCRF-CEM membrane, 5 nM
SDF-1a in 50 mM HEPES, pH 7.4, 5 mM GDP, 5 mM MgCl2, 10 mM
NaCl, 0.1 mg/mL saponin, and 5 nM Eu-GTP (Perkin-Elmer), a
nonhydrolyzable, europium-labeled analogue of GTP. Unbound
Eu-GTP was separated by filtration and the bound was counted by
time-resolved fluorescence, Eex= 340 nM, Eem= 615 nM, using a
Victor 2 fluorescent plate reader (Perkin-Elmer).
2.8. Chemotaxis assay
For the chemotaxis studies, cells were loaded with 5 mM
calcein-AM (Molecular Probes, InVitrogen, Carlsbad, CA, USA). Cells
were then washed and resuspended in media (RPMI containing
10 mg/mL BSA). Cells were preincubated for 10 min at 37 8C with a
concentration range of AMD11070. In a Corning Transwell plate
(5 mm pore 24-well plate), AMD11070 and 10 nM SDF-1a were
added to the lower well and the cell-AMD11070 mixture was
added to the upper well. Plates were incubated for 3–5 h at 37 8C,
5% CO2. Migration of the cells towards the SDF-1a containing lower
chamber was quantified by fluorescence reading Eex= 485 nM,
Eem= 525 nM using a FLEXstation (Molecular Devices) [13,32].
2.9. Data analysis
Ligand binding and concentration/response curves were ana-
lyzed by nonlinear regression using PRISM 3.0 (GraphPAD
Software, San Diego, CA) or XLFit add-in for Microsoft Excel.
Results are expressed as mean ? S.E.
3.1. AMD11070 is a potent inhibitor of X4 HIV-1 replication
AMD11070 is an inhibitor of X4-tropic HIV, with antiviral
activity demonstrated against HIV-1 NL4.3 in both MT-4 cells and
PBMC with IC50values of 9.6 ? 0.3 nM and 10.1 ? 0.3 nM respec-
tively (Table 1). The CC50for AMD11070 was around 30 mM in MT-4
cells, giving a selectivity index (SI) of more than 3000. In addition
AMD11070 potently inhibited cell fusion between a CHO-K1 cell line
expressing gp120 and the P4-R5 MAGI cells which express endoge-
nous CD4 and CXCR4 with an IC50 of 1.5 ? 0.3 nM (Fig. 2a). No
antiviral activity was observed with AMD11070 against HIV-1 R5
(BaL) in PBMC (IC50> 10 mM).
3.2. AMD11070 inhibits SDF-1a binding to cells expressing CXCR4
The CCRF-CEM T-lymphoblastoid cell line has been reported to
naturally express CXCR4 . CXCR4 expression was confirmed by
flow cytometry as described previously . This cell line was
therefore chosen and used throughout to demonstrate the
interactions of AMD11070 with the CXCR4 receptor. AMD11070
was shown to inhibit [125I]-SDF-1a ligand binding to CCRF-CEM
cells in a heterologous competition binding assay. A typical result
Inhibition of CXCR4-mediated biological responses by AMD11070.
gp120/CXCR4 cell fusiona
9.6 ? 0.3
aResults are IC50concentration (nM) giving 50% inhibition expressed as mean ? S.E.
bConcentration (mM) causing 50% cytotoxicity of MT-4 cells expressed as mean ? S.E.
cSelectivity index: ratio of CC50for MT-4 cells/IC50NL4.3 HIV activity in MT-4 cells.
dNL4.3 HIV activity in PBMCs.
27.5 ? 2.8
10.1 ? 0.3
1.5 ? 0.3
12.5 ? 1.3
39.8 ? 2.5
19.0 ? 4.1
9.0 ? 2.0
19.0 ? 4.0
R.M. Mosi et al. / Biochemical Pharmacology 83 (2012) 472–479
is shown in Fig. 2b. The data was fitted to a single site binding
model and gave an IC50of 12.5 ? 1.3 nM (Table 1).
3.3. AMD11070 inhibits SDF-1-mediated cell signaling
Chemokine receptors are G-protein coupled receptors [5,6], i.e.
the mechanism of receptor activation is dependent upon coupling
to an intracellular heterotrimeric G-protein composed of the Ga,
Gb and Gg subunits, which in its basal state binds the guanine
nucleotide GDP. Upon activation by ligand binding GDP is released
and replaced by GTP. This leads to subunit dissociation into a bg
dimer and the a monomer to which the GTP is bound. The GTP is
rapidly hydrolysed to GDP resulting in re-association of the
receptor and the trimeric G protein complex. This process is
assayed using a nonhydrolysable analogue of GTP such as the
fluorescently labeled Eu-GTP or radio labeled [35S]-GTPgS, thus
trapping the formation of the GTP/G-protein. Typical results are
shown in Fig. 3a and b. AMD11070 inhibited CXCR4 activation with
IC50values of 39.8 ? 2.5 and 19.0 ? 4.1 nM in the Eu-GTP-binding
and [35S]-GTPgS assays respectively.
Upon the activation of the G-protein coupled receptor,
intracellular signaling pathways are triggered resulting in the
release of calcium from intracellular stores. This calcium flux can
be assayed using a calcium-chelating molecule, Fluo-4, which
fluoresces upon binding calcium. AMD11070 was able to inhibit
SDF-1a (2.5 nM SDF-1a) mediated calcium flux in CCRF-CEM cells
with an IC50of 9.0 ? 2.0 nM. A typical result is shown in Fig. 3c.
A key property of all chemokines is that they induce a
chemotactic response to a chemokine concentration gradient.
AMD11070 was able to inhibit SDF-1a mediated chemotaxis of
CCRF-CEM cells with an IC50of 19.0 ? 4.0 nM as shown in Fig. 3d. All
results are summarized in Table 1.
-10 -9-8 -7-6-5
Avg IC50 = 39.8 ± 2.5 nM n=25
log [AMD070] M
-10 -9-8 -7-6 -5
Avg IC50=19.0 ± 4.1 nM n=6
Log [AMD070] (M)
% Total Response
Log [AMD070] (M)
Avg IC50 =9 ± 2 nM n=10
% Maximal Response
-10 -9-8 -7 -6-5
=19.0 ± 4 nM n=6
Log [AMD070] (M)
% Maximal Chemotaxis
Fig. 3. Inhibition of SDF-1a mediated signaling by AMD11070: (a) inhibition of SDF-1a stimulated Eu-GTP binding; (b) inhibition of SDF-1stimulated [35S]-GTPgS binding; (c)
inhibition of SDF-1a-induced calcium flux and (d) inhibition of SDF-1a stimulated CCRF-CEM chemotaxis.
-10-8 -6 -4
Log M [AMD070]
%of Maximum Response
IC50 = 1.5 ± ± 0.3 nM (n=3)
Log [AMD070] (M)
-9-8 -7 -6-5
IC50=12.5 ± 1.3 nM n=23
125ISDF-1α α Bound (CPM)
Fig. 2. Inhibition of CXCR4/ligand interactions where the ligands are HIV gp120 and the cognate ligand SDF-1. (a) Inhibition of cell fusion between a CHO-K1 cell line
expressing gp120 and the P4-R5 MAGI cells which express endogenous CD4 and CXCR4. (b) AMD11070 inhibition of [125I]-SDF-1a binding to CXCR4+CEM-CCRF cells.
R.M. Mosi et al. / Biochemical Pharmacology 83 (2012) 472–479
3.4. AMD11070 activity is selective for CXCR4
In order to demonstrate the specificity of AMD11070 for CXCR4
it was tested in calcium signaling assays against a panel of
chemokine receptors, and in ligand binding assays for BLT1, the
receptor for leukotriene B4 (LTB4), and CXCR7. LTB4is a potent
chemoattractant and its receptor is a GPCR. The results in Table 2
show that the IC50of AMD11070 against CCR1, CCR2b, CCR4, CCR5,
CCR7, CXCR3, and LTB4was >50 mM in all cases. AMD11070 did
not inhibit SDF-1a binding to CXCR7 at a concentration of 10 mM,
the maximum concentration tested in this assay. Together these
data show that AMD11070 is a selective inhibitor of CXCR4.
3.5. AMD11070 inhibits SDF-1 signaling of C-terminal variants of
From an HIV therapeutic perspective it is important that CXCR4
antagonists can act on CXCR4 variants. C-terminal truncated
variants of CXCR4 have been reported associated with WHIM
syndrome; nonsense mutations resulting in a 19 amino acid
truncation (R334X), and a 10 amino acid truncation (E343X) and a
frameshift mutation resulting in a 13 amino acid truncation
(S339fs342X) [35,36]. In order to address this we cloned the R334X
and E343X CXCR4 variants and transiently expressed them in the
canine thymus cell line Cf2Th. This cell line was chosen due to its
lack of expression of CXCR4 . Wild type CXCR4 was similarly
sub-cloned into this cell line for control studies. In control studies
it was demonstrated that both these C-terminal truncated variants
were able to respond to SDF-1a in the calcium flux assay with a
similar potency to wild type CXCR4. The EC50values for SDF-1a
were 13.6, 11.3 and 15.3 nM against the wild type, R334X and
E343X variants of CXCR4 respectively (Fig. 4a). The inhibitory
effect of AMD11070 on SDF-1a-mediated calcium flux was
assessed for the two CXCR4 variants. Both variants were inhibited
to a similar extent as the wild type CXCR4 with IC50values of 3.1,
8.5 and 4.6 nM for the wild type, R334X and E343X variants
respectively (Fig. 4b).
3.6. AMD11070 is an allosteric inhibitor of CXCR4
In order to further analyze the mechanism of AMD11070
inhibition of the SDF-1/CXCR4 interaction we investigated the
effect of increasing concentrations of inhibitor on the SDF-1a dose/
response in the calcium flux assay. The data in Fig. 5a shows a
concentration dependent depression of the SDF-1a dose/response
curves as high concentrations of the ligand are unable to overcome
the inhibitory response of high concentrations of AMD11070. A
more detailed analysis of these data provides further insight into
the mechanism of inhibition. Whereas a linear correlation of a
logarithmic plot of SDF-1a EC50versus AMD11070 concentration
would be expected for a competitive inhibitor the plot clearly
shows a non-linear relationship which is indicative of a non-
competitive, or mixed inhibitor (Fig. 5b). Assuming EC50is directly
related to ligand KDit is apparent that ligand binding affinity is
modified by increasing inhibitor concentration. Furthermore in
Fig. 5c the maximal SDF-1a-induced calcium response (Emax) is
decreased by increasing concentrations of inhibitor. The effect on
both EC50and Emaxindicates that AMD11070 inhibits the SDF-1/
CXCR4 response with a mixed-type inhibition mechanism,
indicative of allosteric inhibition.
Several splice variants of SDF-1 have been reported [37–40].
The two most studied are SDF-1a and SDF-1b, with SDF-1a being
the predominant isoform. The gene for SDF-1 encodes an 89 amino
acid protein with a 21 amino acid signal peptide cleavage site
giving rise to a 68 amino acid protein which is SDF-1a. The splice
variant SDF-1b is a 72 amino acid protein with an additional 4
residues at the C-terminus . As shown in Table 3 both isoforms
were able to stimulate a calcium flux in CCRF-CEM cells with EC50
values of 10 ? 0.5 nM for SDF-1a and of 2.9 ? 0.7 nM for SDF-1b. A
concentration of 10 nM SDF-1a or b was used for subsequent
inhibition studies with AMD11070. The data in Table 3 show that
AMD11070 was able to inhibit CXCR4-mediated calcium flux initiated
by both isoforms of SDF-1 with IC50values of 18.2 ? 2.0 nM for SDF-
1a, and 45.8 ? 2.5 nM for SDF-1b. If we hypothesize that AMD11070
acts via a mixed-inhibition mechanism as indicated above then the
IC50is a good approximation of the Ki. As the Kivalues are
different for the two ligand isoforms this further supports the
hypothesis that AMD11070 is acting as an allosteric inhibitor of the
AMD11070 is an orally bioavailable CXCR4 antagonist with
potent activity against T-tropic X4 HIV [27,29]. AMD11070 was
-12-11 -10 -9-8 -7-6 -5
-12-11-10 -9 -8-7 -6-5 -4
Fig. 4. AMD11070 inhibition of CXCR4 N-terminus truncated variants. (a) SDF1-a stimulation of calcium flux in wild type and R334X and E343X CXCR4 variants, and (b)
inhibition of SDF-1a stimulated calcium flux in wild type and R334X and E343X CXCR4 variants.
AMD11070 is a selective inhibitor of CXCR4. Data is shown for calcium flux response
for CCR1, CCR2b, CXCR3, CCR4, CCR5 and CCR7; and ligand binding for CXCR7 and
R.M. Mosi et al. / Biochemical Pharmacology 83 (2012) 472–479
able to inhibit replication of the HIV-1 T-tropic NL4.3 strain in the
low nanomolar range in vitro. Confirmation that this was due to the
inhibition of CXCR4 was provided by the demonstration of
inhibition of the viral envelope/CXCR4 interaction in a cell/cell
fusion assay using a cell line expressing viral envelope from the X4
virus pNL4.3. In this paper we further investigate the molecular
pharmacology of CXCR4 inhibition by AMD11070.
Using the CCRF-CEM cell line, which naturally expresses CXCR4
[13,32] we have shown that AMD11070 inhibits SDF-1a ligand
binding to CXCR4 with an IC50of 12.5 ? 1.3 nM. AMD11070 also
inhibits CXCR4 activation and signaling as shown by inhibition of
SDF-1a mediated G-protein activation of the CXCR4 receptor in two
assays using either the fluorescent Eu-GTP or the radiolabeled [35S]-
GTPgS binding assays with IC50values of IC50of 39.8 ? 2.5 nM and
19.0 ? 4.1 nM respectively, and inhibition of SDF-1a mediated
calcium flux with an IC50of 9.0 ? 2.0 nM. AMD11070 also inhibited
SDF-1a-mediated chemotaxis, a CXCR4-mediated physiological
response, with an IC50 of 19.0 ? 4.0 nM. In addition, AMD11070
had little or no inhibitory effect on either MIP1a, MCP-1, TARC,
RANTES, MIP-3b, or IP10 mediated calcium flux, ligands for CCR1,
CCR2b, CCR4, CCR5, CCR7 and CXCR3 respectively, or SDF-1a binding
to CXCR7, or LTB4binding to BLT1, an alternative GPCR that mediates
chemotaxis. These data indicate that AMD11070 is a selective
inhibitor of CXCR4 over these chemokine receptors.
Given the relative size of the cognate ligand for CXCR4 with a
molecular weight of approximately 10 kDa, and that of AMD11070
with a molecular weight of 350 Da, it is unlikely that the small
molecule inhibitor would act as a direct competitive antagonist of
CXCR4. In fact it has been reported that other small molecule
antagonists of chemokine receptors such as the CXCR1/2 inhibitors
repertaxin and Sch527123 [42,43], and the CCR5 inhibitors TAK-
779, SCH-C and 873140 [7,44–46], are allosteric inhibitors of their
respective chemokine receptor. We therefore decided to ascertain
if AMD11070 was an allosteric inhibitor using classical pharma-
cology. We compared the dose/response of SDF-1a in the calcium
flux assay in the presence of increasing concentrations of inhibitor.
The dose/response curves were depressed with increasing
AMD11070 could not be overcome by high ligand concentrations.
A similar observation was reported for the allosteric inhibitor of
CXCR2, Sch527123 . These data show that AMD11070 is not a
competitive inhibitor of the SDF-1a/CXCR4 interaction, but is most
likely an allosteric inhibitor. Interestingly the SDF-1a EC50was not
significantly affected at low inhibitor concentrations suggesting
that the affinity of the agonist for the receptor was not changed by
the inhibitor at these concentrations. At higher AMD11070
concentrations there was an apparent increase in the EC50
suggesting a change in agonist affinity. Additionally the maximum
response, Emax, was depressed by increasing concentrations of
AMD11070. The effects on both EC50and Emaxare indicative of a
mixed-type mechanism of inhibition and are typical of an allosteric
modulator . Further support for allosteric inhibition comes
from a comparison of the effect of different SDF-1 isoforms on
CXCR4-mediated calcium flux, and inhibition by AMD11070. SDF-
1a and SDF-1b stimulate calcium flux with different EC50values,
10 ? 0.5 nM and of 2.9 ? 0.7 nM respectively, and AMD11070
inhibits the respective response with IC50values of 18.2 ? 2.0 nM
and 45.8 ? 2.5 nM. These differential effects suggest that different
isoforms of the ligand may influence binding of the inhibitor to an
In previously reported studies we have mapped the binding
site of AMD11070 on the CXCR4 receptor to the extracellular
-13 -12 -11 -10
0 nM 07
0.3 nM 07
3 nM 07
15 nM 07
75 nM 07
150 nM 07
13 ± 2 nM
15 ± 3 nM
8.7 ± 1.3 nM
14 ± 3 nM
35 ± 12
91 ± 36
Log [SDF-1] (M)
Ca2+ RFU Change
5.0× ×10- 8
1.0× ×10- 7
1.5× ×10- 7
2.0× ×10- 7
Fig. 5. Allosteric inhibition of SDF-1a-induced calcium flux in CCRF-CEM cells. (a) The effect of increasing concentrations of AMD11070 on the SDF-1a dose/response curve,
(b) the effect of increasing concentrations of AMD11070 on the SDF-1a EC50, and (c) the effect of increasing concentrations of AMD11070 on the maximum SDF-1a response
A comparison of the inhibitory effects of AMD11070 on CXCR4-mediated calcium
flux stimulated by different isoforms of SDF-1.
10 ? 0.5
18.2 ? 2.0
2.9 ? 0.7
45.8 ? 2.5
R.M. Mosi et al. / Biochemical Pharmacology 83 (2012) 472–479
transmembrane region of the receptor . CXCR4 mutagenesis
and molecular modeling studies have shown that binding of the
bicyclam CXCR4 antagonist plerixafor to the CXCR4 receptor is
dependent upon three positively charged amino acids in
transmembrane regions TMIV and TMVI, Asp171, Asp262and
Glu288[48,49]. Similar studies have shown that these interac-
tions are important for AMD11070 binding. However, significant
additional interactions with Tyr45(TMI), Trp94and Asp97(TMII)
were also identified for AMD11070. As one single binding mode
could not explain all these interactions we proposed three
alternative binding modes for AMD11070 . Though the
precise binding mode of SDF-1 has not been elucidated it is
thought that binding is a two-stage process with initial binding
to the N-terminus of CXCR4 with subsequent presentation of the
ligand to the extracellular loops and the transmembrane region
. Key amino acids involved in receptor signaling have been
identified in ECL2 and 3, and Asp97and Glu288have been
identified as important interaction sites in the transmembrane
region [50–52]. AMD11070 therefore can potentially interact
with residues involved in SDF-1 interactions, but can also
occupy unique binding sites which supports an allosteric,
mixed-inhibition model. Interestingly Asp97is required for
HIV gp120 interaction with CXCR4 [50,53], thus indicating that
binding to this residue can contribute to the anti-HIV activity of
We have extended this mutational analysis. It has been
reported that mutations of the intracellular regions of CXCR4,
including the intracellular loops and the C terminal cytoplasmic
tail, have no effect on HIV infectivity [4,50]. Mutations in CXCR4
resulting in truncation of the intracellular C-terminus of the
receptor have been linked to the rare autoimmune disease, WHIM
syndrome [35,36]. We cloned and expressed two of these CXCR4
variants and demonstrated that AMD11070 was able to inhibit
CXCL12-mediated calcium flux in these C-terminal truncated
variants. These data further indicate that AMD11070 acts via
interaction with the extracellular region of CXCR4. Furthermore it
is significant from the perspective of AMD11070 as a potential
therapeutic option for HIV-1 that it can inhibit multiple variants of
In conclusion we have shown that AMD11070 is a selective
inhibitor of the chemokine receptor CXCR4. It can inhibit binding of
different isoforms of SDF-1 to CXCR4, and inhibit SDF-1 stimula-
tion of different variants of CXCR4. Furthermore, we present
evidence that AMD11070 is an allosteric inhibitor of the SDF-1/
CXCR4 interaction, though further studies are required to fully
define the precise mechanism of inhibition.
A proof-of-concept clinical trial has shown that AMD11070 can
reduce the viral load of X4 HIV . Together these data add
further support to the potential beneficial role of an orally
bioavailable CXCR4 inhibitor as a therapeutic option for HIV/AIDS.
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