JOURNAL OF VIROLOGY, Aug. 2004, p. 8654–8662
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 16
Spirodiketopiperazine-Based CCR5 Inhibitor Which Preserves
CC-Chemokine/CCR5 Interactions and Exerts Potent Activity
against R5 Human Immunodeficiency Virus
Type 1 In Vitro
Kenji Maeda,1,2Hirotomo Nakata,1,2Yasuhiro Koh,1,2Toshikazu Miyakawa,2
Hiromi Ogata,1,2Yoshikazu Takaoka,3Shiro Shibayama,3Kenji Sagawa,3
Daikichi Fukushima,3Joseph Moravek,4Yoshio Koyanagi,5
and Hiroaki Mitsuya1,2,6*
Department of Hematology1and Department of Infectious Diseases,2Kumamoto University School of Medicine, Kumamoto 860-
8556, Minase Research Institute, Ono Pharmaceutical Co. Ltd., Osaka 618-8585,3and Department of Virology, Tohoku
University Graduate School of Medicine, Sendai 980-8575,5Japan; Moravek Biochemicals, Inc., Brea, California
928214; and Experimental Retrovirology Section, HIV and AIDS Malignancy Branch,
National Cancer Institute, Bethesda, Maryland 208926
Received 6 January 2004/Accepted 31 March 2004
We identified a novel spirodiketopiperazine (SDP) derivative, AK602/ONO4128/GW873140, which specifi-
cally blocked the binding of macrophage inflammatory protein 1? (MIP-1?) to CCR5 with a high affinity (Kd
of ?3 nM), potently blocked human immunodeficiency virus type 1 (HIV-1) gp120/CCR5 binding and exerted
potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates, including multidrug-
resistant HIV-1 (HIV-1MDR) (50% inhibitory concentration values of 0.1 to 0.6 nM) in vitro. AK602 compet-
itively blocked the binding to CCR5 expressed on Chinese hamster ovary cells of two monoclonal antibodies,
45523, directed against multidomain epitopes of CCR5, and 45531, specific against the C-terminal half of the
second extracellular loop (ECL2B) of CCR5. AK602, despite its much greater anti-HIV-1 activity than other
previously published CCR5 inhibitors, including TAK-779 and SCH-C, preserved RANTES (regulated on
activation normal T-cell expressed and secreted) and MIP-1? binding to CCR5?cells and their functions,
including CC-chemokine-induced chemotaxis and CCR5 internalization, while TAK-779 and SCH-C fully
blocked the CC-chemokine/CCR5 interactions. Pharmacokinetic studies revealed favorable oral bioavailability
in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.
Highly active antiretroviral therapy has had a major impact
on the AIDS epidemic in industrially advanced nations (5, 20);
however, eradication of human immunodeficiency virus type 1
(HIV 1) appears to be currently impossible, in part due to the
viral reservoirs remaining in blood and infected tissues (6, 27).
The limitation of antiviral therapy of AIDS is exacerbated by
complicated regimens, the development of drug-resistant
HIV-1 variants (11), and a number of inherent adverse effects.
Successful antiviral drugs, in theory, exert their virus-specific
effects by interacting with viral receptors, virally encoded en-
zymes, viral structural components, viral genes, or their tran-
scripts without disturbing cellular metabolism or function (20).
However, at present, no antiretroviral drugs or agents are
likely to be completely specific for HIV-1 or to be devoid of
toxicity or side effects in the therapy of AIDS, which has been
a critical issue because patients with AIDS and its related
diseases will have to receive antiretroviral therapy for a long
period of time, perhaps for the rest of their lives (6, 27). Thus,
the identification of new antiretroviral drugs which have
unique mechanisms of action and produce no or minimal side
effects remains an important therapeutic objective (20). In this
respect, it has been thought that certain chemokine receptor
inhibitors might produce no or minimal toxicity.
In the present study, we designed, synthesized, and identi-
fied a novel small nonpeptidic CCR5 inhibitor, AK602/
ONO4128/GW873140, and related compounds which showed
high binding affinity to CCR5, potently inhibited CCR5 gp120
interactions, and had potent HIV-1-specific antiviral activity
against laboratory and clinical strains of HIV-1, including highly
drug-resistant HIV-1 variants. We describe here the pharmaco-
logical characteristics of AK602/ONO4128/GW873140 and its
unique feature that, despite the compound’s much greater
anti-HIV-1 activity compared to previously published CCR5
inhibitors, AK602/ONO4128/GW873140 preserves RANTES
and MIP-1? binding to CCR5?cells and their functions.
MATERIALS AND METHODS
Reagents. Two newly designed and synthesized spirodiketopiperazine (SDP)
derivatives, AK530 [(3S)-1-but-2-yn-1-yl-3-[(1S)-cyclohexylhydroxymethyl]-9
2,5-dione dihydrochloride] and AK602 [4-[4-[(3R)-1-butyl-3-[(1R) cyclohexylhy-
acid hydrochloride], are discussed in the present report. The methods for their
synthesis and physicochemical profiles will be described elsewhere. The struc-
tures of these two compounds are shown in Fig. 1. A previously reported pro-
totypic SDP derivative, E913 (17), was used as a reference compound. E921 and
* Corresponding author. Mailing address: Department of Hematol-
ogy, Kumamoto University School of Medicine, 1-1-1 Honjo, Kum-
amoto 860-8556, Japan. Phone: 81-96-373-5156. Fax: 81-96-363-5265.
AK671, which have the same structures as CCR5 inhibitors TAK-779 and SCH-
351125 (SCH-C), respectively, were synthesized as previously described by others
Zidovudine was purchased from Sigma (St. Louis, Mo.). Nelfinavir and sa-
quinavir were provided by Japan Energy (Tokyo, Japan) and Roche Products
(Welwyn Garden City, United Kingdom), respectively.
125I-labeled chemokines macrophage inflammatory protein-1? (MIP-1?),
macrophage inflammatory protein-1? (MIP-1?), and RANTES were purchased
from Amersham Pharmacia Biotech (Little Chalfont, United Kingdom) and
PerkinElmer Life Sciences, Inc. (Boston, Mass.), and three corresponding unla-
beled chemokines (MIP-1?, MIP-1?, and RANTES) were purchased from Pep-
roTech Inc. (Rocky Hill, N.J.). Recombinant HIV-1YU2gp120 (rgp120) and
human soluble CD4 (sCD4) were purchased from Immuno Diagnostics, Inc.
Cells, viruses, and anti-HIV-1 assay. Chinese hamster ovary (CHO) cells
expressing CCR5 (17) were maintained in Ham’s F-12 medium (Gibco-BRL,
Rockville, Md.) supplemented with 10% fetal calf serum (JRH Biosciences,
Lenaxa, Kans.), 50 U of penicillin per ml, and 50 ?g of streptomycin per ml in the
presence of 5 ?g of blasticidin S hydrochloride per ml. Peripheral blood mono-
nuclear cells were isolated from buffy coats of HIV-1-seronegative individuals
with Ficoll-Hypaque density gradient centrifugation and cultured at a concen-
tration of 106cells/ml in RPMI 1640-based culture medium supplemented with
10% fetal calf serum and antibiotics with 10 ?g of phytohemagglutinin per ml for
3 days prior to use (phytohemagglutinin-peripheral blood mononuclear cells).
Cell line CCR5?MOLT4 (18) was a kind gift from Yosuke Maeda, Kumamoto
A panel of HIV-1 strains was employed for drug susceptibility assays: HIV
1Ba-L(8), HIV-1JR-FL(13) HIV-1NL4-3(34), a wild-type HIV-1MOKWisolated
from a drug-naive AIDS patient (17), and two multidrug-resistant (HIV-1MDR)
primary HIV-1 strains (HIV-1JSLand HIV-1MM) (36). All primary HIV-1 strains
were passaged once or twice in phytohemagglutinin-peripheral blood mononu-
clear cell cultures, and the culture supernatants were stored at ?80°C until use.
Antiviral assays with phytohemagglutinin-peripheral blood mononuclear cells
were conducted as previously reported (12, 17, 26).
HIV-1 gp120 binding inhibition assays. CCR5?CHO cells were incubated
with rgp120 (5 ?g/ml) and sCD4 at 5 ?g/ml, biotinylated with EZ-link sulfo-
NHS-SS-biotin (Pierce, Rockford, Ill.) in the presence of the indicated concen-
trations of a CCR5 inhibitor for 1 h at 37°C. Cells were washed, and the binding
of the rgp120-sCD4 complex to CCR5?CHO cells was determined with phyco-
erythrin-conjugated streptavidin (BD PharMingen, San Diego, Calif.). Nonspe-
cific binding was determined based on the mean florescence intensity of phyco-
concentrations that brought about 50% inhibition (IC50) of mean fluorescence
intensity were then determined.
Generation of3H-labeled CCR5 inhibitors. Five CCR5 inhibitors, AK530,
AK602, E913, E921/TAK-779, and AK671/SCH-C, were tritiated by reductive
amination with sodium triacetoxyborotritide (10), methylation with [3H]methyli-
odide, and heterogeneous catalytic exchange with tritium gas (4). Detailed de-
scription of the radiosynthesis of the inhibitors will be presented by J.M. else-
where. In brief, [3H]E913, [3H]AK530, and [3H]AK602 were prepared by
reductive amination of the corresponding aldehyde with piperidine-containing
components of each inhibitor with an excess of sodium triacetoxyborotritide, and
the tritium label was positioned selectively into the methylene group connecting
the two components, generating inhibitors with specific activities of 10.2 Ci/
mmol, 17.5 Ci/mmol, and 8.3 Ci/mmol, respectively. [3H]E921/TAK-779 was
prepared by methylating the N-methyl precursor of E921/TAK-779 with [3H]m-
ethyliodide, generating [3H]E921/TAK-779, with a specific activity of 6.1 Ci/
mmol. For the preparation of [3H]AK671/SCH-C, methyl-2,4-dimethylpyridine-
3-carboxylate was tritiated by an exchange with tritium gas, catalyzed by
palladium on carbon in ethanol and triethylamine. Its conversion to N-oxide and
alkaline hydrolysis of the resulting ester provided [3H]2,4-dimethyl-pyridine-3-
carboxylic acid. Its condensation with N-tert-butoxycarbonyl precursor provided
[3H]AK671/SCH-C, with a specific activity of 5 Ci/mmol.
Saturation binding assay. CCR5?CHO cells (1.5 ? 105cells/well) were plated
onto 48-well, flat-bottomed culture plates, incubated for 24 h, rinsed with Ham’s
F-12 medium containing 20 mM HEPES and 0.5% bovine serum albumin
(Sigma), exposed to various concentrations of each3H-labeled CCR5 inhibitor,
washed thoroughly with cold phosphate-buffered saline, and lysed with 0.5 ml of
1 N NaOH, and the radioactivity in the lysates was measured. The nonspecific
binding of a radiolabeled compound was determined based on the radioactivity
detected in the CCR5?CHO cell-plated wells containing the same amount of
the3H-labled CCR5 inhibitor and a 200-fold greater amount of the correspond-
ing non radiolabeled compound. The Kd(dissociation) values of CCR5 inhibitors
and the maximal binding values (Bmax? number of CCR5/cell) were calculated
based on their specific radioactivity with Graphpad Prism software (Intuitive
Software for Science, San Diego, Calif.). All assays were performed in duplicate,
and the values shown in this report are the arithmetic means (?1 standard
deviation) of 3 to 10 independently conducted assays.
Chemokine binding inhibition and chemotaxis inhibition assays. CCR5?
CHO cells (1.5 ? 105) were plated onto 48-well microculture plates, incubated
for 24 h, rinsed, exposed to 3 nM [125I]MIP-1?, [125I]MIP-1?, or [125I]RANTES
in the presence of various concentrations of a CCR5 inhibitor at room temper-
ature for 1 h, thoroughly washed with phosphate-buffered saline, and lysed with
0.5 ml of 1 N NaOH, and their radioactivity was counted. The nonspecific
binding of the labeled chemokine to the cells was determined based on the
radioactivity detected in the wells plated with the same number of CCR5-
negative CHO (CHO-K1) cells exposed to each radiolabeled chemokine (3 nM).
Chemotaxis inhibition assays were conducted with CCR5?MOLT4 cells and
the ChemTx System (Neuro Probe, Inc., Gaithersburg, Md.). In brief, CCR5?
MOLT4 cells were exposed to various concentrations of each CCR5 inhibitor for
30 min, thoroughly rinsed, plated onto the upper chamber of the ChemTx
System, exposed to 0.5 nM RANTES contained in the lower chamber, and
incubated for 4 h at 37°C, and the number of the cells which migrated from the
upper chamber to the lower chamber was determined. Percent chemotaxis was
determined with the formula 100 ? [(number of CCR5 inhibitor-exposed cells
which migrated to the lower chamber in the presence of RANTES) ? (number
of CCR5 inhibitor-unexposed cells which migrated to the lower chamber in the
absence of RANTES)]/[(number of CCR5 inhibitor-unexposed cells which mi-
grated to the lower chamber in the presence of RANTES) ? (number of CCR5
inhibitor-unexposed cells which migrated to the lower chamber in the absence of
FACS analysis. Fluorescence-activated cell sorting (FACS) analysis was per-
formed as previously described (17) with minor modifications. Briefly, CCR5?
CHO cells (3 ? 105) were stained with a phycoerythrin- or fluorescein isothio-
cyanate-conjugated anti-CCR5 monoclonal antibody 2D7 (BD PharMingen, San
Diego, Calif.) or 45523 or 45531 (R&D Systems, Minneapolis, Minn.), with or
without a test CCR5 inhibitor, washed, and examined with an Epics XL (Beck-
man Coulter, Fullerton, Calif.).
FIG. 1. Structures of AK602 and AK530.
VOL. 78, 2004CCR5 INHIBITOR VERSUS R5 HIV-18655
Potent activity of AK602 against R5 wild-type and multi-
drug-resistant R5 HIV-1. We have previously reported that a
prototypic SDP derivative, E913, was active against R5 HIV-1
in vitro, with IC50values of 30 to 60 nM as tested in target
phytohemagglutinin-treated peripheral blood mononuclear
cells (17). Following optimization for increased potency
against R5 HIV-1 and favorable pharmacokinetic features, we
identified AK602 as the most potent agent among newly de-
signed and synthesized SDP derivatives. AK602 exerted potent
activity against three wild-type R5 HIV-1 strains (HIV-1Ba-L,
HIV 1JR-FLand HIV-1MOKW) with IC50values of 0.1 to 0.4 nM
(Table 1). It was of note that AK602 was substantially more
potent than two previously published CCR5 inhibitors, E921/
TAK-779 and AK671/SCH-C (1, 28).
During the extended study of the antiviral activity of the
prototypic E913, we noted that its activity against R5 HIV-
1Ba-Lin vitro varied substantially; the range of IC50values
spanned from 14 to 650 nM (Fig. 2). When we tested the
activity of E921/TAK-779 in phytohemagglutinin-treated pe-
ripheral blood mononuclear cells from multiple seronegative
donors, its variability was also substantial: its IC50values varied
from 2 to 200 nM. However, when we tested AK602, the
variability of AK602’s anti-HIV-1 activity was limited and sim-
ilar to that seen for zidovudine. The difference in the range of
the CCR5 inhibitor’s IC50values seems to correlate with the
potency of the inhibitor examined. Indeed, we have seen a
greater variability in the antiviral activity of the prototypic
E913 (Fig. 2). Moreover, AK602 suppressed the infectivity and
replication of two HIV-1MDRvariants, HIV-1MMand HIV-
1JSL(36), at extremely low concentrations (IC50values of 0.4 to
0.6 nM), while these two R5 HIV-1 variants were less suscep-
tible to zidovudine, nelfinavir, and saquinavir (IC50values were
greater by factors of 10 to 36, ?83, and 27 to 32, respectively,
compared to those against HIV-1Ba-L). As expected, none of
these CCR5 inhibitors suppressed the infectivity and replica-
tion of X4 HIV-1NL4-3in vitro. Although certain CC-chemo-
kines reportedly enhance the replication of X4 HIV-1 (19, 22),
no such enhancement of X4 HIV-1 replication was seen with
the CCR5 inhibitors examined in this study at concentrations
of up to 1 ?M (data not shown).
CCR5 binding properties of SDP derivatives. We deter-
mined the CCR5 binding profiles of SDP derivatives and com-
pared them with those of previously published CCR5 inhibitors
in saturation binding assays employing3H-labeled compounds.
Figure 3A depicts the CCR5 binding profile of AK602, show-
ing that it binds with high affinity to CCR5. The Kdvalues thus
determined for AK602, E913, E921/TAK-779, and AK671/
SCH-C were 2.9 ? 1.0 (Fig. 3A), 111.7 ? 3.5, 32.2 ? 9.6, and
16.0 ? 1.5 nM (data not shown), respectively.
We also asked whether the SDP derivatives blocked the
binding to CCR5 of rgp120 following exposure to sCD4. As
shown in Fig. 3B, AK602 potently blocked rgp120/sCD4 bind-
ing to CCR5 with an IC50value of 2.7 nM, followed by E921/
TAK-779 and AK-671/SCH-C, with IC50values of 12.0 and
16.5 nM, respectively. When we asked whether AK602 blocked
the intracellular Ca2?mobilization induced by MIP-1?, MDC,
SDF-1?, and MCP-1, whose primary receptors are CCR5,
CCR4, CXCR4, and CCR2, respectively, with the method we
TABLE 1. Anti-HIV-1 activity of SDP derivatives
Mean IC50(IC90) ? SD in p24 assay (nM)
0.4 ? 0.3 (12 ? 10)
0.1 ? 0.1 (4 ? 2)
0.2 ? 0.1 (5 ? 3)
0.6 ? 0.2 (11 ? 2)
0.4 ? 0.3 (7 ? 2)
32 ? 27 (324 ? 120)
13 ? 4 (144 ? 60)
82 ? 58 (709 ? 256)
81 ? 46 (?1,000)
51 ? 14 (941 ? 201)
61 ? 28 (?1,000)
64 ? 30 (713 ? 405)
28 ? 32 (256 ? 169)
5 ? 1 (237 ? 25)
11 ? 7 (194 ? 168)
14 ? 8 (352 ? 180)
7 ? 4 (316 ? 151)
4 ? 2 (79 ? 52)
2 ? 0.5 (56 ? 57)
2 ? 1 (54 ? 20)
3 ? 0.5 (138 ? 25)
2 ? 0.3 (84 ? 18)
7 ? 4 (48 ? 21)
10 ? 9 (157 ? 72)
6 ? 5 (47 ? 20)
250 ? 98 (?1,000)
70 ? 64 (?1,000)
11 ? 5 (181 ? 90)
12 ? 8 (105 ? 48)
14 ? 8 (82 ? 56)
20 ? 7 (75 ? 52)
11 ? 5 (60 ? 21)
5 ? 2 (49 ? 40)
300 ? 65 (?1,000)
350 ? 105 (?1,000)
10 ? 4 (48 ? 2)
aCytotoxic concentrations of a compound that reduces the number of cells by 50% (CC50) were determined as previously reported (17).
bHIV-1MOKWwas isolated from a drug-naive AIDS patient (17), while HIV-1MMand HIV-1JSLwere from patients who received antiretroviral therapy for a long period and whose virus acquired a number of mutations
in the RT- and PR-encoding HIV-1 genes (36).
cND, not determined.
8656MAEDA ET AL.J. VIROL.
published previously (17), AK602 completely blocked MIP-1?-
induced Ca2?mobilization at 0.1 ?M and beyond; however, it
failed to block Ca2?mobilization induced with MDC, SDF-1?,
and MCP-1 (data not shown).
We also attempted to illustrate where AK602 binds on the
CCR5 molecule by employing several monoclonal antibodies
known to bind to different domains of CCR5. FACS analyses
revealed that there was no AK602 inhibition of the binding of
monoclonal antibody 2D7, known to bind to the N-terminal
half (or domain A) of the second extracellular loop of CCR5
(14) (Fig. 4). In contrast, AK602 competitively blocked the
binding of two different monoclonal antibodies, 45523, report-
edly directed against multidomain epitopes of CCR5, and
45531, which is known to be specific against the C-terminal half
(or domain B) of the second extracellular loop (ECL2B) of
CCR5 (14), as examined with CCR5?CHO cells (Fig. 4).
These data suggest that the potent activity of AK602 against
R5 HIV-1 stems from its binding to ECL2B and/or its vicinity
with high affinity, resulting in inhibition of gp120/CD4 binding
to CCR5. It was of note, however, that another SDP derivative,
AK530, whose antiviral activity was moderate (the IC50value
against HIV-1Ba-Lwas 32 nM; Table 1), whose rgp120/sCD4
binding inhibition was the lowest among the inhibitors exam-
ined (IC50, 280 nM; Fig. 3B), and had only a moderate effect
on the binding of monoclonal antibody 45531 to CCR5?cells
(data not shown), had the highest binding affinity to CCR5 (Kd
value, 0.4 nM; data not shown) among the SDP derivatives,
suggesting that the binding pocket (or subsite) of certain SDP
derivatives (such as AK530) does not quite overlap that of
SDP derivatives bind to CCR5 but permit RANTES and
MIP-1? to bind to CCR5. We asked whether SDP derivatives
blocked the binding of CC-chemokines to CCR5 expressed on
the surface of CHO cells with [125I]RANTES, [125I]MIP-1?,
and [125I]MIP-1? and CCR5 inhibitors AK602, AK530, E921/
TAK-779, and AK671/SCH-C. As shown in Fig. 5A, the con-
centrations of E921/TAK-779 and AK671/SCH-C which
FIG. 2. Variability of anti-HIV-1 activity of AK602 in phytohem-
agglutinin-peripheral blood mononuclear cells. The range of IC50val-
ues of E913 and E921/TAK-779 against HIV-1Ba-Lvaried substantially
when examined in multiple phytohemagglutinin-peripheral blood
mononuclear cells as target cells, 14 to 650 nM (n ? 11) and 2 to 200
nM (n ? 15), respectively, while that of AK602 was relatively narrow,
0.1 to 1 nM (n ? 15), similar to that of zidovudine (ZDV), 1 to 9 nM
(n ? 14).
FIG. 3. CCR5 binding profiles and rgp120 binding blocking of various CCR5 inhibitors. (A) Binding affinity of AK602 to CCR5. CCR5?CHO
cells were incubated with the3H-labeled CCR5 inhibitors AK530, AK602, E913, E921/TAK-779, and AK671/SCH-C for 1 h. Following thorough
washing, cells were lysed, the radioactivity in the lysates was determined, and Bmaxand Kdvalues were calculated. The Kdvalues thus obtained were
0.4 ? 0.4, 2.9 ? 1.0, 111.7 ? 3.5, 32.2 ? 9.6, and 16.0 ? 1.5 nM, respectively. All assays were independently performed 3 to 10 times, and the values
represent the arithmetic means ? 1 standard deviation. (B) AK602 potently blocks the binding of rgp120/sCD4 to CCR5. CCR5?CHO cells were
incubated with rgp120 (5 ?g/ml) and sCD4 (5 ?g/ml) in the presence or absence of the indicated concentrations of CCR5 inhibitors, and the
binding of rgp120/sCD4 complex to CCR5?CHO cells was determined. The 50% binding inhibition (EC50) value was determined based on the
mean fluorescence intensity values obtained with or without CCR5 inhibitors. EC50values for AK602, AK530, E921/TAK-779, and AK671/SCH-C
were 2.7, 280, 12.0, and 16.5 nM, respectively.
VOL. 78, 2004CCR5 INHIBITOR VERSUS R5 HIV-1 8657
blocked RANTES binding to CCR5 by 50% (IC50) were 110
and 40 nM, respectively, and RANTES binding was completely
blocked in the presence of ?10 ?M E921/TAK-779 or AK671/
SCH-C. In contrast, AK602 only partially blocked RANTES
binding to CCR5 by 40% even at 10 ?M (Fig. 5A). The binding
of MIP-1? to CCR5 was also completely blocked by E921/
TAK-779 and AK671/SCH-C; however, AK602 failed to com-
pletely block MIP-1? binding (Fig. 5B). The MIP-1? binding
value in the presence of 10 ?M AK602 was 10%, and no
further blockade occurred at higher concentrations up to 40
?M (data not shown). AK530 also failed to completely block
the binding of RANTES and MIP-1? to CCR5.
These data suggest that the binding pockets (or subsites) of
CCR5 for SDP derivatives only partially overlap the CC-che-
mokine binding sites of CCR5 or that the conformational
changes ensuing the binding of SDP derivatives to CCR5 have
only moderate effects on the binding of RANTES and MIP-1?.
In the initial search for CCR5 inhibitors, lead compounds were
sought as those inhibiting MIP-1? binding to CCR5 and MIP-
1?-driven cytosolic Ca2?flux, and thus, as expected, AK602
blocked MIP-1? binding to CCR5 although AK530 was sub-
stantially less potent in blocking MIP-1? binding (Fig. 5C).
E921/TAK-779 and AK671/SCH-C were also found to com-
pletely block MIP-1? binding to CCR5 (Fig. 5C).
AK602 and RANTES bind simultaneously to CCR5. As de-
scribed above, AK602 and AK530 only partially inhibited
RANTES binding to CCR5?CHO cells; however, it was not
clear whether those SDP derivatives and RANTES bound si-
multaneously to CCR5. Therefore, competitive binding assays
employing3H-labeled and unlabeled AK602 and125I-labeled and
unlabeled RANTES were conducted. As shown in Fig. 6A, the
binding of [3H]AK602 (10 nM) to CCR5 was only partially inhib-
ited by ?4 nM RANTES. Also, the binding of [125I]RANTES
at 8 nM was only inhibited by up to 20% in the presence of 10
nM AK602 (Fig. 6B).
The interpretation that AK602 and RANTES bind simulta-
neously to CCR5 was corroborated by another experiment in
which a lower concentration of [3H]AK602 and much higher
concentrations of RANTES were used (Fig. 6A, inset). The
radioactivity counted for [3H]AK602 (5 nM) bound to CCR5?
CHO cells was only moderately blocked in the presence of 100
and 1,000 nM RANTES, by 32 and 46%, respectively (Fig. 6A,
inset). These data suggest that the SDP derivatives, in partic-
ular AK602, and RANTES bind simultaneously to CCR5, al-
FIG. 4. AK602 binds to the second extracellular loop of CCR5. AK602 at 100 nM almost completely inhibited the binding of two monoclonal
antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, recognizing ECL2B of CCR5. In contrast, E921/TAK-779 and
AK671/SCH-C moderately blocked the binding of 45523 and 45531. Note that there was no AK602 inhibition of the binding of a monoclonal
antibody 2D7, which is known to bind to domain A of ECL2 of CCR5.
FIG. 5. Inhibition of CC-chemokine binding to CCR5 by various CCR5 inhibitors. CCR5?CHO cells were incubated with 3 nM [125I]RANTES
(A), [125I]MIP 1 ? (B), or [125I]MIP-1 ? (Pnel C) in the presence and absence of various concentrations of CCR5 inhibitors. Note that while
AK671/SCH-C and E921/TAK-779 completely inhibited the binding of [125I]RANTES, [125I]MIP-1?, and [125I]MIP-1? to CCR5, SDP derivatives
partially blocked RANTES (A) and MIP-1? (B) binding, although they completely blocked MIP-1? binding (C).
8658 MAEDA ET AL.J. VIROL.
though conformational changes potentially caused by either of
the two might have occurred. Indeed, 15 to 25% inhibition was
seen at nearly equimolar concentrations of AK602 and RAN-
TES, which may reflect the involvement of the conformational
changes caused by either of the two agents or an overlap in
their binding sites (or domains).
AK602 permits RANTES-induced chemotaxis and CCR5 in-
ternalization at anti-HIV-1 activity-exerting concentrations.
We next asked whether AK602 allowed RANTES-induced
chemotaxis and CCR5 internalization with CCR5?MOLT4
cells and CCR5?CHO cells at its anti-HIV-1 activity-exerting
concentrations. As shown in Fig. 7A, AK671/SCH-C most po-
tently blocked chemotaxis, followed by E921/TAK-779. The
chemotaxis values at the IC50s against R5 HIV-1Ba-Lof
AK671/SCH-C and E921/TAK-779 (4 and 24 nM, respectively:
Table 1) were low, 18 and 8%, respectively, suggesting that
these two inhibitors considerably blocked chemotaxis at their
anti-HIV-1 IC50concentrations as determined in peripheral
blood mononuclear cells. In contrast, the chemotaxis seen at
the IC50level of AK602, 0.4 nM (see Table 1), was consider-
able, with 70% retained (Fig. 7A), while that seen AK530 was
much less (30%).
In order to corroborate the modest chemotaxis inhibition
seen with AK602, the inhibition of RANTES-induced CCR5
internalization was also examined. In the absence of CCR5
inhibitors, ?50% of CCR5 molecules were internalized from
the surface of CCR5?CHO cells incubated for 1 h at 37°C in
the presence of 10 nM RANTES; however, AK671/SCH-C and
E921/TAK-779 at 100 nM considerably blocked internaliza-
tion, and only 19 and 6%, respectively, of CCR5 molecules
were internalized. In the presence of higher concentrations of
AK671/SCH-C and E921/TAK-779, 300 and 1,000 nM, virtu-
ally no CCR5 internalization occurred (Fig. 7B). In contrast,
AK530 and AK602 at 100 nM allowed RANTES-induced
CCR5 internalization of 46 and 30%, respectively, and even at
300 and 1,000 nM, 10 to 34% CCR5 internalization occurred
A novel SDP derivative, AK602/ONO4128/GW873140, ex-
hibited high affinity to CCR5, blocked rgp120/sCD4 complex
binding to CCR5, and exerted potent activity against a wide
spectrum of laboratory and primary R5 HIV-1 isolates, includ-
ing HIV-1MDR. We recently examined AK602 against several
non-clade B R5 HIV strains and found that in general AK602
is comparably active against such non-clade B strains (data not
shown). It is of note that several small-molecule CCR5 inhib-
itors have been reported in the literature, including SCH-D
(D. Schurmann et al., Abstr. 11th Conf. Retroviruses Oppor-
tunistic Infections, 2004, abstr. 140LB), UK427,857 (A. L.
Pozniak et al. Abstr. 43rd Intersci. Conf. Antimicrob. Agents
Chemother., 2003, abstr. H-443), CMPD167 (32), and TAK-
220 (Y. Iizawa et al., Abstr. 10th Conf. Retroviruses Opportu-
nistic Infections, 2003, abstr. 11).
In the present study, we also demonstrated that AK602
potently blocked rgp120/sCD4 complex binding to CCR5.
With respect to gp120/CD4 binding to CCR5, Olson et al.
previously reported no correlation between fusion with and
entry into the target cell of HIV-1 and inhibition of rgp120/
sCD4 complex binding to CCR5, based on data with various
anti-CCR5 monoclonal antibodies (24). However, with all
small-molecule SDP derivatives examined in the present study,
inhibition of HIV-1 infectivity and replication generally corre-
lated with inhibition of the rgp120/sCD4 complex binding to
CCR5, strongly suggesting that the anti-HIV-1 activity of SDP
FIG. 6. AK602 and RANTES bind simultaneously to CCR5. (A) CCR5?CHO cells were exposed to 10 nM [3H]AK602 and various
concentrations of unlabeled RANTES. After 1 h of incubation, the cells were washed, and the [3H]AK602 bound to the cells was measured. Note
that 100% radioactivity on the ordinate denotes the radioactivity of cell-bound [3H]AK602 without RANTES and that ?90% of CCR5 molecules
are bound to AK602 at 10 nM (Fig. 3A). (B) CCR5?CHO cells were exposed to 10 nM unlabeled AK602 and various concentrations of
[125I]RANTES. After 1 h of incubation, the cells were washed, and the [125I]RANTES bound to the cells was measured. The binding profile of
[125I]RANTES alone is illustrated by open circles. Note that 100% radioactivity is equated to the radioactivity of cell-bound [125I]RANTES at 10
nM. The Kdvalues of RANTES in the presence and absence of 10 nM AK602 were 4.5 and 0.6 nM, respectively.
VOL. 78, 2004CCR5 INHIBITOR VERSUS R5 HIV-18659
derivatives stems from their inhibition of gp120 binding to
CCR5, as reported for other CCR5 inhibitors such as TAK-779
(3), although the binding pocket (or subsite) of CCR5 for
certain SDP derivatives (such as AK530) apparently does not
quite overlap the rgp120/sCD4 complex binding site of CCR5
(Fig. 3B). It is also possible that the conformational changes
ensuing upon AK602’s binding to CCR5 could differ from that
ensuing upon AK530’s binding to CCR5, thereby producing
differences in gp120/sCD4 binding and anti-HIV activity.
It is generally noted that although the determination of any
binding sites with antibodies provides “indirect” evidence, in
many cases it gives good insights (14). Indeed, SCH-C has been
reported to induce conformational changes in CCR5 and bind
to its transmembrane (TM) domain, thereby blocking HIV-
gp120 binding to CCR5. In our data, SCH-C completely
blocked the binding of the “multidomain”-reactive monoclonal
antibody 45523, which reportedly causes conformational
changes in CCR5, while it only moderately blocked the binding
of the ECL2B-specific monoclonal antibody 45531 (Fig. 4). In
contrast, AK602 completely blocked the binding of both 45523
and 45531. Considering that monoclonal antibody 45531’s
CCR5 binding is closely linked to amino acids 184 to 189 of
ECL2B, as shown by Lee and colleagues (14), it was thought
that the binding site of AK602 includes ECL2B or is vicinal to
it. Indeed, our recent analysis with the alanine-scanning algo-
rithm showed that AK602 totally failed to bind to a CCR5
mutant when a K191A substitution was introduced (Maeda et
al., unpublished data), corroborating and extending the idea
that AK602’s binding site involves the ECL2B domain.
It is noted that the IC50of AK602 against HIV-1 as deter-
mined in peripheral blood mononuclear cells (0.4, 0.1, and 0.2
nM against HIV-1Ba-L, HIV-1JR-FL, and HIV-1MOKW, respec-
tively: Table 1) are substantially lower than the Kdof AK602
(2.9 nM) and the IC50of AK602 for its inhibition of rgp120/
sCD4 complex binding to CCR5 (2.7 nM). The anti-HIV-1
IC50s of AK602 are also lower than the IC50s of AK602 for its
inhibition of MIP-1?-induced Ca2?influx (39.8 nM; unpub-
lished data) and that for its inhibition of CCR5 internalization
(?300 nM; unpublished data).
One possible explanation for these inconsistencies is the
different cell lines employed for each assay. However, it is of
note that when we determined the IC50values against several
R5 HIV strains and Kdvalues of AK602 in MAGI/CCR5 cells
(18), AK602’s IC50s (?0.2 nM) were reproducibly lower than
AK602’s Kd(3.8 nM) (data not shown). Thus, one can postu-
late that for the inhibition of HIV-1 infection by CCR5 inhib-
itors, not all CCR5 molecules might have to be occupied. In
this regard, our studies with
target cells expressing CCR5, MAGI/CCR5 (18) and U373-
MAGI (34), have shown that less than 30% of HIV-1 infection
occurred when approximately 50% of CCR5 molecules were
bound by AK602, and at its anti-HIV-1 IC50concentration,
AK602 was found to bind to 5 to 20% of CCR5 molecules on
the target cells (Maeda et al., unpublished data). These data
suggest that when one of the multimerized CCR5 molecules is
bound or occupied by AK602, inhibition of the cell is likely to
be blocked, although further stoichiometric analyses need to
It has been thought that individuals carrying a gene encoding
a mutant form of CCR5 called Delta32 are resistant to HIV-1
3H-labeled AK602 and CD4?
FIG. 7. AK602 allows RANTES-induced chemotaxis and CCR5 internalization. (A) CCR5?MOLT4 cells were exposed to various concen-
trations of AK530, AK602, E921/TAK-779, or AK671/SCH-C, thoroughly washed, plated onto the upper chamber of the ChemTx System, exposed
to 0.5 nM RANTES contained in the lower chamber, and incubated for 4 h; the number of the cells which migrated to the lower chamber was
determined, and chemotaxis was calculated. (B) CCR5?CHO cells were exposed to 10 nM RANTES in the presence or absence of various
concentrations of each CCR5 inhibitor and washed with acidic solution for removal of the cell-bound RANTES (21). The amount of cell surface
CCR5 was subsequently determined with monoclonal antibody 3A9 (BD PharMingen), which recognizes the N terminus of CCR5 and competes
with none of the CCR5 inhibitors tested. In panel A, the level of chemotaxis suppression by TAK-779 and SCH C was greater than that by AK530
and AK602 at four concentrations examined, although complete suppression was seen only at the highest concentration of the AK compounds,
1 ?M. However, in panel B, the level of CCR5 internalization suppression by TAK-779 and SCH-C was greater than that of the AK compounds
at all three concentrations examined.
8660MAEDA ET AL.J. VIROL.
infection and apparently do not have significant health prob-
lems (2, 15, 23, 25). One can assume that individuals with
homozygous CCR5-Delta32 might inherently have certain de-
fenses which could compensate for the deficiency of CCR5. In
this regard, there has been a report that individuals carrying
homozygous CCR5-Delta32 have longer survival of renal
transplants than those with other genotypes, suggesting that
such individuals might have compromised graft rejection im-
munity (7). Moreover, Woitas et al. have reported that indi-
viduals with homozygous CCR5-Delta32 have significantly
higher levels of hepatitis C virus in blood than their counter-
parts who have wild-type CCR5, suggesting that the CCR5-
Delta32 mutation may be an adverse host factor in hepatitis C
virus infection (35), although others have recently argued
against a role of CCR5 in susceptibility to hepatitis C virus
infection or response to antiviral therapy (9). Thus, sustained,
long-term suppression of the effect of CC-chemokines/CCR5
interactions, in particular in those who carry wild-type CCR5
and might not have a possible compensatory mechanism for
the absence of CCR5, might produce adverse effects, and cau-
tion should be used in the development of chemokine receptor
antagonists as potential therapeutics for HIV-1 infection.
In this respect, SDP derivatives such as AK602 can preserve
CC-chemokine/CCR5 interactions at their anti-HIV activity-
exerting concentrations; they allow RANTES and MIP-1?
binding to CCR5?cells and their functions at anti-HIV-1 con-
centrations. In contrast, two previously published CCR5 inhib-
itors, TAK-779 and SCH-C, fully blocked CC-chemokine/
CCR5 interactions (Fig. 5 and 7). It is of note that AK602’s
complete inhibition of the binding of MIP-1? was not surpris-
ing because in the initial search of lead compounds, we sought
compounds that blocked the binding of125I-labeled MIP-1? to
CCR5?CHO cells and MIP-1?-elicited cellular Ca2?mobili-
zation, as described previously (17).
In support of the above observation, the results of compet-
itive biding assays with [3H]AK602 and [125I]RANTES and
their corresponding unlabeled agents clearly indicated that
AK602 and RANTES bind simultaneously to CCR5 (Fig. 6).
Moreover, AK602 allowed CCR5?MOLT4 cells to undergo
RANTES-elicited chemotaxis (Fig. 7A) and CCR5?CHO
cells to internalize CCR5 in response to RANTES (Fig. 7B) at
concentrations much greater than AK602’s anti-HIV-1 activi-
ty-exerting concentration in peripheral blood mononuclear
cells. However, it is worth noting that although AK602 blocked
the binding of [125I]RANTES to CCR5?CHO cells only by
?40% at micromolar concentrations (Fig. 5A), it virtually
completely blocked the RANTES-induced chemotaxis at mi-
cromolar concentrations, as examined in CCR5?MOLT4 cells
(Fig. 7A). This apparent inconsistency could be explained by
the different cell lines employed for each assay and the fact
that the number of CCR5 molecules in CCR5?CHO cells (?5
? 105/cell) is substantially different from that of CCR5?
MOLT4 cells (?1 ? 105/cell), and thus, AK602 could more
efficiently block the chemotaxis of MOLT4 cells. It is also
possible that AK602 may more effectively block CCR5 mul-
timerization, which is reportedly important for the functional-
ity of the G protein-coupled receptor (29), rather than the
RANTES binding block to CCR5 per se. However, it is not
clear yet whether AK602’s unique profile that AK602 partially
allows RANTES and MIP-1? to bind to CCR5 despite
AK602’s tight binding to CCR5 brings about a clinical advan-
tage. This can be examined only in the setting of clinical trials
and careful clinical investigation in long-term treatment with
such an agent.
Several HIV-1 variants which acquired resistance to CC-
chemokines, including MIP-1? and CCR5 inhibitors, have
been reported. Trkola et al. described that when HIV-1 was
passaged in the presence of increasing concentrations of a
CCR5-specific, structurally SCH-C-related CCR5 inhibitor,
AD101, an escape mutant which contained 22 amino acid sub-
stitutions in the gp120 subunits emerged as early as after 19
passages (31). This escape mutant showed a ?20,000-fold re-
sistance to AD101 and was similarly resistant to SCH-C com-
pared with wild-type HIV-1, suggesting that HIV-1 can acquire
the capability of using CCR5 bound to certain classes of CCR5
inhibitors for its entry into the target cell (31). Maeda et al.
reported that HIV-1JR-FL, following in vitro selection against
MIP-1? over 3 months, acquired amino acid substitutions in
the V2 and V3 regions of HIV-1 gp120 and became four- to
sixfold more resistant to MIP-1?, MIP-1?, and RANTES (18).
In this regard, as of this writing, we have passaged HIV-1Ba-L
in CD4?CCR5?PM1 cells (16) in the presence of moderately
increasing concentrations of AK602 in one selection experi-
ment and aggressively increasing concentrations of AK602 in
another selection experiment over 22 months (45 passages);
however, the virus has acquired no detectable resistance to
AK602 and no significant amino acid substitutions (Nakata et
al., unpublished data).
It is worth noting that the anti-HIV-1 activity of AK602 is
virtually unaffected by the presence of human serum proteins.
For instance, the IC50of AK602 against HIV-1Ba-Lin the
presence of 10% fetal calf serum in culture medium was 0.4 ?
0.3 nM, while those of AK602 with 10 ?M ?1-acid glycoprotein
and 45% human serum added to the culture medium were 0.8
? 0.3 and 0.7 ? 0.7 nM, respectively. AK602 failed to induce
Ca2?flux, chemotaxis, or CCR5 internalization in CCR5?
cells (Maeda et al., unpublished data). As far as the sensitivi-
ties of our methods used in the present work, AK602 is to be
categorized as a nonagonist or antagonist. The phase 1 clinical
trial of AK602 in HIV-1-seronegative individuals has recently
been concluded, and no significant adverse effects have been
documented. Considering that AK602 potently inhibited the
replication of HIV-1 in vitro and in a nonobese diabetic-SCID
mouse model (Nakata et al., unpublished data) and that
AK602 has a favorable oral bioavailability in rodents, averag-
ing 20 to 30% (unpublished data), the present data strongly
suggest that AK602 is a promising CCR5 inhibitor as a poten-
tial therapeutic for HIV-1 infection.
We thank Steve LaFon, Larry Boone, Jim Demarest, Eddy Arnold,
Shigeyoshi Harada, Kazuhisa Yoshimura, and Yosuke Maeda for help-
ful discussion and critical reading of the manuscript.
This work was supported in part by a grant from the Research for
the Future Program (JSPS-RFTF 97L00705) of the Japan Society for
the Promotion of Science, a Grant-in-Aid for Scientific Research (Pri-
ority Areas) from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan (Monbu-Kagakusho), and a Grant for the
Promotion of AIDS Research from the Ministry of Health, Welfare,
and Labor of Japan (Kosei-Rohdosho).
VOL. 78, 2004CCR5 INHIBITOR VERSUS R5 HIV-18661
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