Inhibition of DC-SIGN-mediated HIV infection by a linear trimannoside mimic in a tetravalent presentation

Article abstract of DOI:10.1021/cb900216e

HIV infection is pandemic in humans and is responsible for millions of deaths every year. The discovery of new cellular targets that can be used to prevent the infection process represents a new opportunity for developing more effective antiviral drugs. In this context, dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a lectin expressed at the surface of immature dendritic cells and involved in the initial stages of HIV infection, is a promising therapeutic target. Herein we show the ability of a new tetravalent dendron containing four copies of a linear trimannoside mimic to inhibit the trans HIV infection process of CD4 T lymphocytes at low micromolar range. This compound presents a high solubility in physiological media, a neglectable cytotoxicity, and a long-lasting effect and is based on carbohydrate-mimic units. Notably, the HIV antiviral activity is independent of viral tropism (X4 or R5). The formulation of this compound as a gel could allow its use as topical microbicide.

Full text of DOI:10.1021/cb900216e

ARTICLE
Inhibition of DC-SIGN-Mediated HIV Infection
by a Linear Trimannoside Mimic in a
Tetravalent Presentation
Sara Sattin†, Anna Daghetti†, Michel The´paut‡,#, Angela BerziЌ, Macarena Sa´nchez-Navarro§,
Georges Tabarani‡,††, Javier Rojo§, Franck Fieschi‡,††, Mario Clericiʈ,¶, and Anna Bernardi†,*
†Dipartimento di Chimica Organica e Industriale and CISI, Universita` degli Studi di Milano, via Venezian 21, 20133 Milano,
Italy, ‡Laboratoire des prote´ines membranaires, CEA, DSV, Institut de Biologie Structurale, 41 rue Jules Horowitz, 38027
Grenoble, France, §Grupo de Carbohidratos, Instituto de Investigaciones Qu´ımicas, CSICϪUniversidad de Sevilla, Av.
Americo Vespucio 49, 41092 Seville, Spain, ʈDipartimento di Scienze e Tecnologie Biomediche, Universita` degli Studi di
Milano, via Flli Cervi 93, 20090 Segrate, Italy, ЌDipartimento di Scienze Precliniche, Universita` degli Studi di Milano, via
GB Grassi 74, 20157 Milano, Italy, ¶Don C. Gnocchi ONLUS Foundation IRCCS, Via Capecelatro 66, 20148 Milano, Italy,
#CNRS, UMR 5075, 38000 Grenoble, France, and ††Universite´ Joseph Fourier, 38000 Grenoble, France
uman immunodeficiency virus (HIV) remains
one of the main health problems of the 21st
century. Almost three decades have elapsed
ABSTRACT HIV infection is pandemic in humans and is responsible for millions
of deaths every year. The discovery of new cellular targets that can be used to pre-
vent the infection process represents a new opportunity for developing more effec-
tive antiviral drugs. In this context, dendritic cell-specific ICAM-3 grabbing non-
integrin (DC-SIGN), a lectin expressed at the surface of immature dendritic cells and
involved in the initial stages of HIV infection, is a promising therapeutic target.
Herein we show the ability of a new tetravalent dendron containing four copies of
a linear trimannoside mimic to inhibit the trans HIV infection process of CD4ϩ T
lymphocytes at low micromolar range. This compound presents a high solubility
in physiological media, a neglectable cytotoxicity, and a long-lasting effect and is
based on carbohydrate-mimic units. Notably, the HIV antiviral activity is indepen-
dent of viral tropism (X4 or R5). The formulation of this compound as a gel could al-
low its use as topical microbicide.
H
since the discovery of this virus, and despite the variety
of the available therapeutic arsenal, over 3 million
people die of AIDS every year. It is evident that more ef-
ficient drugs and strategies are needed. Unfortunately,
the hope to achieve a successful vaccine against HIV in
the near future remains elusive (1). New strategies have
been envisaged and evaluated, and HIV-entry inhibi-
tors appear one of the most promising alternatives (2).
Many efforts have been devoted to the search for novel
antagonists with the ability to target different HIV recep-
tors and coreceptors, such as CD4, CCR5, or CXCR4, at
the cell surface. In this context, a potential therapeutic
target was identified in dendritic cell-specific ICAM-3
grabbing non-integrin (DC-SIGN) a molecule involved in
the early stages of HIV infection (3). DC-SIGN is a tet-
rameric calcium-dependent (C-type) lectin, expressed
by dendritic cells (DC), which specifically recognizes
highly glycosylated structures displayed at the surface
of several pathogens such as viruses, bacteria, yeasts,
and parasites (4–7). Recognition by DC-SIGN plays a key
role in the infection process of some of these patho-
gens, most notably HIV, and it is considered an interest-
ing new target for the design of anti-infective agents
(8–12).
*Corresponding author,
anna.bernardi@unimi.it.
Received for review August 31, 2009
and accepted January 19, 2010.
Published online January 19, 2010
10.1021/cb900216e
DC-SIGNϩ immature DC, located in vaginal, cervical,
and rectal mucosae, are among the first cell types to en-
© 2010 American Chemical Society
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ment with the numerous glycans exposed by the viral
gp120 glycoprotein. Thus, the choice of DC-SIGN as a
therapeutic target implies a double challenge: first, the
development of a ligand unit featuring a good affinity for
the DC-SIGN CRD, and second, its functionalization on
a backbone allowing multiple ligand presentation to
compete against this natural multivalent-based
interaction.
The main carbohydrate ligand recognized by the car-
bohydrate recognition domain of DC-SIGN is the high
mannose glycan, (Man)9(GlcNAc)2, also known as Man9,
a branched oligosaccharide found in multiple copies
on several pathogen glycoproteins (gp120, GP1, etc.).
Therefore, structural analogues of Man9 terminal di- or
trisaccharides, able to compete with binding of gp120
to DC-SIGN, could be suitable for the development of
new anti-infective drugs. Indeed, recent data (20) have
shown that linear fragments of Man9 presented in high
density arrays bind with high affinity to DC-SIGN and
crystal structures of DC-SIGN complexes with linear oli-
gomannosides have been solved (21). We have re-
ported that the pseudo-dimannoside 1 (Figure 1),
mimicking the three-dimensional structure and confor-
mational behavior of the natural disaccharide Man␣1-
2Man, binds to DC-SIGN and exhibits moderate anti-
infective action in an Ebola virus infection model (8).
Additional results indicate that 2-C-substituted
branched D-mannose analogues bind to DC-SIGN with
affinity significantly greater than that of mannose (10).
Non-carbohydrate inhibitors with IC50 values in the low
micromolar range were identified by Kiessling and Bor-
rok via high-throughput screening of ϳ36,000 com-
pounds from commercial libraries (9). Multivalent
oligomannosides blocking DC-SIGN were described by
Wong et al. (22) Finally, Penade´s et al. have just reported
that gold nanoparticles displaying different multivalent
linear and branched mannosyl oligosaccharides behave
as potent inhibitors of HIV trans infection processes at
the cellular level (23). We have previously demonstrated
that commercially available hyperbranched dendritic
Boltorn polymers are good platforms for sugar multiva-
lent presentation (24, 25). Using a biosensor with a SPR
detection method, we proved that this mannosylated
glycodendritic polymer binds to DC-SIGN and inhibits in-
teraction to gp120-coated SPR chips (26). Also, using
an infection model based on pseudo-typed viral par-
ticles of Ebola virus, we have found that at least the third
generation of Boltorn polymer bearing 32 copies of man-
Figure 1. Mimics of linear mannose disaccharide 1 and trisaccha-
ride 2.
counter HIV during sexual transmission. DC-SIGN ex-
pressed by DC at mucosal tissues captures HIV at low
titer by binding the envelope glycoprotein gp120. DC-
SIGN acts as an attachment factor rather than an entry
receptor, binding and concentrating HIV on the cell sur-
face. Two different pathways are involved in the trans-
mission of DC-SIGN-bound HIV to T lymphocytes (13,
14). The first, responsible for short-term HIV transfer
(24 h after HIV exposure), involves virion internalization
into intracellular compartments where the virus is pro-
tected from degradation and retains a high infective ca-
pacity during DC migration to lymphoid tissues (15). The
second, involved in long-term HIV transfer (72 h after ex-
posure), follows DC infection in cis by transfer of DC-
SIGN-bound virus to canonical HIV entry receptors, CD4
and CCR5, resulting in infected DC with a continuous
production of virus for the T cells (14, 16). In both cases,
upon arrival at lymphoid tissues, DC efficiently transmit
HIV to CD4ϩ T lymphocytes, a process called infection in
trans. Hence, blocking the interaction between DC-
SIGN and HIV could be an efficient strategy to prevent
HIV transmission and infection of the host.
DC-SIGN efficiency in HIV capture is associated with
two main characteristics of this molecule. First, the car-
bohydrate binding domains (CRD) of DC-SIGN spike up
(320 Å) from the cell surface and are the first to encoun-
ter the virus (17). Second, the protein is organized as a
tetramer containing 4 CRDs and is distributed as clus-
tered patches at the cellular surface (18, 19). Altogether,
these features lead to attachment platforms of high
avidity for virus particles, allowing multipoint attach-
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nose at the surface was able to inhibit with an IC50 of
0.3 ␮M the cis infection of Jurkat cells via the interac-
tion with DC-SIGN (25).
SCHEME 1. Synthesis of linear trimannoside mimic 2b
The previous data from our groups convinced us to
explore the possibility of combining dendritic platforms
with glycomimetic compounds to obtain novel DC-SIGN
antagonists endowed with strong binding affinities and
able to block this lectin and to inhibit the HIV infection
process. The pseudo-trisaccharide 2a (R ϭ allyl) was re-
cently designed in one of our laboratories as a mimic
of the linear mannotrioside Man␣1-2 Man␣1-6 Man␣
by replacing the central mannose unit with a carbocy-
clic diol (Figure 1) (27). The results described here show
that a tetravalent construct presenting four copies of the
pseudo-trimannoside 2 on a Boltorn-type dendron in-
hibits DC-SIGN-mediated HIV trans infection of CD4ϩ T
lymphocytes at low micromolar concentration. Infection
inhibition data of this functionalized dendron were also
compared to the activity of the monovalent ligand 2b
and of the corresponding tetravalent construct present-
ing four copies of mannose as the active component.
Man-BSA containing 15 glycosylation sites displaying
the Man␣1-3[Man␣1-6]Man trisaccharide. The DC-SIGN
extracellular domain (ECD) exhibited good affinity (in the
micromolar range) for this surface. Inhibition studies
were then performed using DC-SIGN ECD, at a fixed con-
centration, injected alone or in the presence of an in-
creasing amount of the ligands. As a reference, a com-
petition experiment has been performed with mannose.
Sensograms are included in the Supporting Informa-
tion (Supplementary SPR Data).
The efficiency of inhibition as a function of a com-
pound’s concentration is directly related to the ligand af-
finity toward DC-SIGN ECD (Figure 2). An IC50 of 2.5 mM
was estimated for mannose. Using this assay, an IC50
of 1.8 mM has been obtained previously for mannose
(11), and a KI of 7.7 mM has been also reported using
a solid-phase competition assay (6). Indeed, within ex-
perimental deviations, the IC50 is in the range of previ-
ously reported experiments, thus validating the test.
From Figure 2, the IC50 values of 1 and 2b were deter-
mined to be 1.0 mM and 125 ␮M, respectively. While
the interaction properties of the pseudo-disaccharide 1
with DC-SIGN have already been demonstrated by NMR
(8), it is shown here for the first time that its affinity is
RESULTS AND DISCUSSION
Synthesis of Monovalent Units. The synthesis of 1
(Figure 1) was previously described (8). The synthesis
of 2b (Figure 1) was based on the reported synthesis of
2a (27), with appropriate modifications. Briefly, (2-
azidoethyl)-2,3,4-tri-O-acetyl-␣-D-mannopyranoside 3
was prepared from (2-chloroethyl)-2,3,4,6,-tetra-O-
acetyl-␣-D-mannopyranoside 4 (28) via the known azide
5 (27, 29) by standard steps (Scheme 1). Opening of
the epoxide 6 (30) with 3 was promoted by Cu(OTf)2 to
afford the pseudo-1,6-dimannoside 7 in 53% yield. This
was mannosylated in 85% yield using the tetra-O-
benzoyl trichloroacetimidate 8 (31) as donor and
TMSOTf (20%) as the promoter in methylene chloride
at Ϫ20 °C. Zemplen deprotection of the pseudo-
trisaccharide 9 afforded 2b quantitatively.
Evaluation of Monovalent Glycomimetics by Surface
Plasmon Resonance (SPR). Surface plasmon resonance
(SPR) was used to compare the DC-SIGN recognition
properties of the two monovalent ligands 1 and 2b
(Figure 1). As previously described (11), because of the
natural low affinity of monovalent compounds, evalua-
tion of their relative affinity cannot be obtained in a di-
rect interaction mode and can only be accessed through better than that of mannose, thus confirming a direct
a competition assay that we recently reported (11). For
contribution of the additional carbocyclic-diol in the
this assay, we used a CM4 SPR chip functionalized with binding to DC-SIGN CRD. In compound 2b, the addition
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Both mimics discussed here represent interesting li-
gands to pursue with multivalent versions. Considering
the highest affinity of the trimannoside mimic 2b, the
latter has been selected for further synthetic effort to-
ward multivalent presentation versions for biological
testing.
Synthesis of Multivalent Compounds. The functional-
ized monovalent pseudo-trimannoside 2b (Figure 1, R ϭ
CH2CH2N3) was used for the synthesis of the corre-
sponding tetravalent dendron. Hydrogenolytic reduc-
tion of azide 2b with Pd(C) catalyst afforded amine 10
in 99% yield, which was coupled to dendron 11 (32) us-
ing HATU (33, 34) as the condensing agent. LH-20 Seph-
adex chromatography of the reaction crude afforded
the tetrafunctionalized dendron 12 (92%), which was
fully characterized by NMR and MS analysis (Scheme 2).
The tetravalent mannosylated analogue 14 was pre-
pared using the same approach from mannosyl deriva-
tive 13 and the same dendritic core 11. Coupling condi-
tions and purification steps were as described for
glycodendron 12.
Figure 2. Comparison of inhibitory properties of different compounds on
DC-SIGN/BSA-mannotriose interaction. DC-SIGN ECD at 20 ␮M is
incubated with mannose (Œ), 1 (), or 2b (9) and injected on a BSA-
mannotriose functionalized surface (1208 RU immobilized). IC50 values
are 2515 ␮M for mannose, 1005 ␮M for compound 1, and 125 ␮M for
compound 2b.
of a second mannose unit mimicking an ␣1-6 link and
resulting in a linear trimannoside mimic improves the af-
finity by 1 order of magnitude relative to mannose.
SCHEME 2. Synthesis of dendrons 12 and 14
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Infection Studies. Synthesized compounds were
tested for the ability to inhibit HIV transmission in an
in vitro trans infection assay. B-THP-1/DC-SIGN cells are
derived from B-THP-1 human B cell line by transfection
with DC-SIGN expression vector in order to express high
levels of the DC-SIGN receptor. This cell line supports
efficient DC-SIGN-mediated HIV transmission and is a
widely used model system to mimic HIV capture and
transmission to T lymphocytes by dendritic cells (3). In
a first series of experiments, B-THP-1/DC-SIGN cells pre-
incubated for 30 min in the presence or in the absence
of the DC-SIGN inhibitors were subsequently exposed to
HIV (the R5 tropic laboratory-adapted strain HIV-1 BaL)
in the continued presence of inhibitors. Mannan is
known to inhibit DC-SIGN-mediated viral infection (4,
35, 36) and was used as positive control (0.25 mg
mLϪ1). Non-transfected B-THP-1 cells were used as a
negative control and, as expected, did not transmit in-
fection (Figure 3, panel a). After washing, the B-THP-1/
DC-SIGN cells were co-cultured with activated CD4ϩ T
lymphocytes from healthy volunteer donors. Viral infec-
tion of CD4ϩ T lymphocytes was assessed by measur-
ing the concentration of the HIV core protein p24 in the
co-culture supernatants by ELISA. p24, immunologically
distinct from the protein of most other retroviruses, is a
major structural core component of HIV-1and is esti-
mated to be present at 2000–4000 molecules in each
virion. The measurement of p24 levels is therefore a
commonly exploited method to verify the successful in-
fection by the virus. Each point was obtained in triplicate
using CD4ϩ T lymphocytes from at least three different
healthy donors. Results showed that the tetravalent
compound 12 at 50 ␮M reduced the trans infection of
CD4ϩ T lymphocytes by over 90%; moreover, at 100 ␮M
and at 250 ␮M inhibition was almost complete. Al-
though the uncertainties deriving from the involvement
of three different donors are very high in these experi-
ments, the data suggest that higher concentrations of
the monovalent compound 2b (1 mM and 0.5 mM) are
necessary to obtain a comparable inhibitory effect
(Figure 3, panel a).
Figure 3. Inhibition of HIV transmission after treatment with 2b and 12.
Data obtained in triplicate, from 3 different healthy donors. Values are
mean ؎ SD. a) After 30 min of preincubation with 2b and 12, B-THP-1/
DC-SIGN cells or B-THP-1 cells were incubated for 3 h with HIV-1 BaL in
the presence of the indicated concentrations of the DC-SIGN inhibi-
tors, then washed and co-cultured with CD4؉ T lymphocytes for
3 days. Viral infection was assessed by measuring the concentration of
p24 in the co-culture supernatants. Mannan (0.25 mg mL؊1) was used
as positive control. Non transfected B-THP-1 cells do not trasmit infec-
tion. b) Effect of cell washing after treatment with the inhibitors. B-THP-
1/DC-SIGN cells were washed after 30‘ min of incubation with the in-
hibitor, before exposure to HIV-1 BaL. Results obtained with or without
intermediate wash step are compared. Different concentrations of 2b
and 12 were assayed. Mannan (0.25 mg mL؊1) was included as a
control.
tration (data not shown), did not show anti-infective ac-
tivity. Although alternative explanations cannot be
discarded (see Discussion), these data suggest that
the tetravalent system possesses significant affinity to
DC-SIGN, which allows it to deploy its antiviral activity
and to prevent the interaction between HIV gp120 and
DC-SIGN even after a washing cycle. On the contrary, the
In a second set of experiments the B-THP-1/DC-SIGN
cells were first incubated with the inhibitors and then
washed with buffer prior to exposure to HIV (BaL). In this
case (Figure 3, panel b) the tetravalent system 12 dis-
played highly efficient anti-infective properties with
Ͼ94% inhibition at 100 ␮M. On the contrary, the mono-
valent compound 2b, even when used at 5 mM concen- affinity of 2b is not sufficient for the DC-SIGN/2b com-
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washing and co-culture with the CD4ϩ T lymphocytes,
viral infection was assessed using analysis of p24 con-
centration in the supernatants. At 100 ␮M the inhibition
of infection was complete for both donors and an IC50
of 5 ␮M could be estimated (Figure 4). Dose–response
data were also obtained for 2b using CD4ϩ T lympho-
cytes from a susceptible donor and allowed estimation
of an IC50 of 80 ␮M for the monovalent ligand (Supple-
mentary Figure 2).
Dendritic cells are permissive toward infection sup-
ported both by X4- and R5-tropic strains. Thus, we next
verified the capability of 12 to block transmission of
both IIIB (X4 tropic) and 89.6 (dual tropic, R5/X4) strains
of HIV. Results showed that transmission of virus to
CD4ϩ T lymphocytes was almost completely prevented
at 250 and 100 ␮M, and about 90% at 50 ␮M, when
B-THP-1/DC-SIGN cells were challenged with either viral
strains (Figure 5, panels a and b). In subsequent analy-
ses the primary HIV isolates V6 (R5 tropic) and V17 (X4
tropic) were utilized; also in this case results indicated
an inhibition of HIV infection with both types of virus
Ͼ99% at all concentration tested (Figure 5, panels c
and d).
To evaluate the duration of the inhibitory properties
of 12, B-THP-1/DC-SIGN cells were treated for either
30 min or 2 h with 12 at 250 ␮M. The compound was
subsequently removed, and cells were exposed to HIV-1
BaL either immediately thereafter or after 6 or 12 h. Sub-
sequently B-THP-1/DC-SIGN cells were co-cultured with
CD4ϩ T lymphocytes as described above. Virus trans-
mission to CD4ϩ T lymphocytes was almost completely
abrogated at 0 and 6 h post compound removal; nota-
bly, infection was still reduced by over 80% after 12 h.
Two hour pretreatment did not appear to prolong inhibi-
tory effect of 12 compared to 30 min pretreatment
(Figure 6).
Figure 4. Dose؊response results for 12. B-THP1/DC-SIGN cells were in-
cubated for 3 h with HIV-1 BaL in the presence of different concentra-
tions of 12. After washing, B-THP1/DC-SIGN cells were co-cultured for
3 days with CD4؉ lymphocytes of individuals genetically less (P1) or
more (P2) susceptible to HIV infection. Viral infection was assessed by
measuring the concentration of p24 in the supernatant. Each donor was
tested in duplicate. Values are mean ؎ SD.
plex to survive the washing step, and hence viral trans-
mission can occur when the cells are challenged with
BaL. A quantitative assessment of the relative potency
of 12 and 2b was obtained in dose–response single-
donor experiments that will be described below. The
protocol in which inhibitors were not removed by wash-
ing was applied in the following trans infection experi-
ments, unless otherwise specified.
The properties of 12 were further analyzed. Compari-
son with dendron 14, the corresponding tetravalent sys-
tem presenting four mannose units (Scheme 2) was per-
formed. Results showed that dendron 12 displayed a
stronger inhibitory activity at both concentrations tested
(100 and 10 ␮M). Notably, at 100 ␮M 12 abrogated al-
most totally HIV BaL transmission to CD4ϩ T lympho-
cytes, whereas the inhibition provided by 14 was only
partial (approximately 65%) (Supplementary Figure 1).
The results obtained are consistent with a 1 order of
magnitude difference in affinity between 12 and 14,
which corresponds to the affinity difference estimated
by SPR for the corresponding monovalent binding ele-
ments, the linear trimannoside mimic 2b in 12 and man-
nose in 14.
Evaluation of Toxicity of Compounds. 7-Amino-
actinomicin D (7-AAD) labeling of the cells after the incu-
bation period with 12 showed that the anti-infective
properties of the inhibitors are not an epiphenomenon
due to cell death. The data obtained for 12 indicate that
the percentage of 7-AAD positive cells (apoptotic cells)
was below 1% and did not change significantly in the
absence of the compound or in its presence up to
250 ␮M (Supplementary Figure 3).
Dose–response curves were obtained for 12 using
CD4ϩ T lymphocytes from healthy donors character-
ized as being endowed with the genetic markers associ-
ated with reduced susceptibility to HIV infection (P1) or
lack thereof (augmented susceptibility to infection, P2).
B-THP-1/DC-SIGN cells were challenged with HIV BaL in
the presence of increasing concentrations of 12. After
Effect of Compounds on DC-SIGN Expression. Com-
pounds 2b and 12 could exert their effect secondarily to
the suppression of DC-SIGN expression. To examine this
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with anti-DC-SIGN anti-
body, and both percent-
age of DC-SIGN positive
cells and mean fluores-
cence intensity (MFI) were
evaluated after 30 min or
3.5 h of incubation with dif-
ferent concentrations of 2b
and 12 or in the absence of
inhibitor. Results showed
that incubation of cells with
the compounds did not
modify percentages of DC-
SIGN-positive cells (data
not shown). A reduction of
MFI was observed after
treatment of the cells with
12 (Supplementary
Figure 4). The effect is
dose-dependent: ϳ20%
reduction of fluorescence
intensity was observed for
a 50 ␮M concentration of
12, up to a 52% reduction
for a 250 ␮M concentra-
tion of 12. The monovalent
ligand 2b was tested at
Figure 5. Inhibition of trans infection mediated by laboratory-adapted HIV strain (IIIB and
86.9) or by primary viral isolates (V6 and V17). B-THP1/DC-SIGN cells were incubated for
3 h with (a) HIV-1 strains IIIB, (b) 89.6 HIV, (c) V6 (primary isolate; R5- tropic), and (d) V17
(primary isolate; X4-tropic) in the presence of indicated concentration of 12. Virus trans-
mission was determined by measuring the concentration of p24 in the supernatants. Ex-
periments were performed on 3 healthy donors. Values are mean ؎ SD.
possibility, DC-SIGN expression was studied by flow cy-
tometry experiments. B-THP1/DC-SIGN cells were stained
higher concentrations (Supplementary Figure 4). A
change in MFI was observed only after 3.5 h of incuba-
tion and for ligand concentrations
Ն0.5 mM. The effects observed may be at
least partially due to increased internaliza-
tion of DC-SIGN receptors after binding of
the dendron. However, the concentration re-
quired to exert a noticeable effect in the
flow cytometry studies seems somewhat
higher than the infection inhibition
concentration.
Discussion. DC-SIGN recognizes highly
mannosylated glycoproteins at the surface
of a broad variety of pathogens. At least for
some of these agents, including human im-
munodeficiency virus (HIV) (3), Dengue virus
(37), Ebola virus (38), hepatitis C virus (39),
SARS (40), and Mycobacterium tuberculosis
(41), this interaction appears to be an impor-
tant part of the infection process, and as a
consequence, the initial stages of the infec-
Figure 6. Persistence of inhibitory effect of 12 after compound removal.
B-THP-1/DC-SIGN cells were incubated with 12 (0.25 mM) for 30 min
or 2 h. After extensive washing, cells were pulsed with HIV-1 BaL fol-
lowing 0, 6, or 12 h. Levels of infection (determined by p24 concentra-
tion in co-culture supernatants) 3 days post infection were shown. Ex-
periment was performed on two donors. Each condition was tested in
duplicate. Values are mean ؎ SE.
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tion could be prevented by inhibiting DC-SIGN mediated
pathogen recognition.
Therapies based on antimicrobial drugs that kill
pathogens apply a strong selective pressure to the mi-
the CCR5 receptor. Therefore GML action is dependent
on viral tropism, acting only on the R5 tropic strain, as
opposed to the molecules described here. Griffithsin
(GRFT) (49), like cyanovirin (CNV) (50), acts by binding
crobial population, which in turns favors the emergence glycoproteins of the HIV envelope and is capable of
of resistant strains: hence the never-ending search for
new, more powerful agents capable of fighting such
strains. For HIV, this is particularly exacerbated by the
rapid rate of mutations of the viral population. The
mechanism by which DC-SIGN antagonists work aims
at interfering with the initial steps in the infection pro-
cess without killing the virus. Preventing the adhesion
blocking cell-to-cell HIV transmission. So far, all clinical
trials involving microbicides designed to prevent muco-
sal transmission of HIV infection have failed (51).
A handful of compounds have been developed so
far in an effort to target DC-SIGN-mediated viral trans-
mission (Supplementary Tables 2 and 3 collect the avail-
able affinity information). Among them, some potent,
of HIV to its specific dendritic cell receptor, the lectin DC- highly mannosylated polyvalent compounds have been
SIGN, can block the infection while applying minimal se- described. The Wong group has reported oligomannose
lective pressure to the pathogen, which is simply made dendrons that display complex oligomannoses in high
non-harmful.
density and inhibit binding of gp120 to recombinant
dimeric DC-SIGN with IC50 in the nanomolar range (22).
These systems are excellent models of DC-SIGN inhibi-
tion, but their practical application as therapeutic agents
seems unlikely, given the complexity of the oligosaccha-
Current antiviral therapies are based on drugs
directed at different targets of the virus or of the micro-
bial infection cycle. Highly active antiretroviral therapy
(HAART) markedly reduces morbidity and mortality of
HIV-infected individuals but is not able to eradicate HIV rides used. More remarkably, Penade´s, Alcami, and
infection, and lifelong treatment is required. Moreover, their groups have reported that gold nanoparticles dis-
people living in developing countries often do not have playing various linear and branched mannosyl oligosac-
access to this therapy (42). Several efforts have been
made to develop a vaccine against HIV, but vaccine
candidates have so far met with limited success (43,
44).
charides (Manno-GNPs) are potent inhibitors of DC-
SIGN-mediated HIV trans infection of human activated
peripheral blood mononuclear cells (23). However,
in vivo use of GNPs raises some concerns, mostly be-
cause of the potential toxicity produced by gold
accumulation.
The development of glycomimetic drugs appears as
a new, expanding, and promising approach to circum-
vent chemical drawbacks associated with the use of car-
bohydrates for therapeutic purpose (12). The pseudo-
glycosylated dendron disclosed here is the first example
of a low-valency compound based on a sugar mimic
that, used in low micromolar concentrations, can com-
pletely block DC-SIGN-mediated trans infection of CD4ϩ
T lymphocytes. The anti-infective action is exerted up-
It is the general consensus that the development of
efficient, low-cost microbicides and entry inhibitor mol-
ecules that can be applied topically to prevent sexually
transmitted HIV infections should be given high priority
(45). Mannan is known to inhibit DC-SIGN-mediated
viral uptake, but it cannot be used in vivo because it is
highly toxic and mitogenic, which limits its therapeutic
applications in vivo (46, 47). The polyvalent ligand 12
described herein inhibits viral uptake by DC-SIGN-
expressing cells, thus avoiding viral dissemination. Ap-
plication of this new tetravalent pseudo-trimannoside
dendron as topical formulations may lead to topical anti- stream of cytokine involvement and therefore is inde-
infective agents with a protective action against HIV.
Some topical anti-infective agents against HIV have
been recently disclosed, all of which attempt to exploit
inhibitory pathways independent of the DC-SIGN recep-
tor. New microbicides were developed and studied in
cell, tissue, and animal models. Glycerol monolaurate
(48) (GML) blocks CCR5ϩ cell-attracting chemokines
and pro-inflammatory cytokines induced by SIV, thus
inhibiting the recruitment of SIV target cells expressing
pendent of viral tropism, as shown by inhibition of a
series of laboratory-adapted strains and primary iso-
lates with different tropism.
The distance spanned by two ligand units of 12 at
full extension of the dendron arm is 28–30 Å at best,
too short to allow simultaneous binding to two CRDs of
the same DC-SIGN tetramer (from the recently published
tetrameric model of DC-SIGN, derived from small-angle
X-ray scattering studies, the distance between two adja-
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ARTICLE
cent Ca2ϩ sites is close to 40 Å) (17). Depending on
protein density on the cell surface, simultaneous bind-
ing to two CRDs on different tetramers could occur. Most
probably, however, the ligand presentation on den-
drons allows achievement of a high local concentration
of recognition elements. With dendrimers this is ob-
tained without increasing the viscosity of the system
and thus is fully compatible with use in physiological
fluids.
The time-course studies (Figure 6) and the experi-
ments shown in Figure 3, panel b show that the anti-
viral effect of 12 persists for hours even after the cells
have been washed. The mechanism of this inhibition
after removal could be based on the persistency of the
multivalent ligand on the receptor binding site (slow off-
rate of the tetravalent compound from the protein). How-
ever, the dose-dependent reduction in MFI observed in
the flow cytometry studies (Supplementary Figure 4)
suggests that exposure to 12 may also alter somewhat
significantly the observed cell surface concentration of
DC-SIGN, possibly by induced endocytosis. Depletion of
receptor membrane concentration is an interesting fea-
ture of 12 and could at least partially account for its
antiviral activity. Complex mechanisms can be involved,
and for this reason further studies are in progress to
quantitatively understand the correlation between anti-
infectivity and modification of cell-surface receptor con-
centration in this system, as well as to establish the
underlying mechanisms.
ride mimic on a Boltorn-type dendron, allows efficient
synthesis, amenable to large-scale production. The com-
pound is highly soluble in physiological media and
shows negligible cytotoxicity. Its chemical stability in
aqueous buffer was evaluated at pH 5 and 7.4 using
MALDI-MS and 1H NMR spectroscopy. At pH 5, which is
relevant for topical vaginal application, 12 was found to
be stable for at least 1 week. In pH 7.4 aqueous buffer
the stability is reduced to 6–12 h (52). Although stabil-
ity to mannosidase has not been tested yet for 2 or 12,
the parent pseudo-disaccharide 1 was found to resist
mannosidase hydrolysis (30).
The anti-infective activity of 12 appears to depend
both on the good affinity for DC-SIGN of its pseudo-
trimannoside binding unit and on the tetravalent
presentation on the dendron scaffold. Indeed, the infec-
tion studies show that neither the monovalent pseudo-
trisaccharide 2b nor the tetravalent mannosylated den-
dron 14 perform as well as 12 as inhibitors of viral
transmission. Thus, the structure of this lead can be im-
proved by increasing the affinity of the monovalent bind-
ing unit, but it can also be tuned on the controlled level
of presentation depending on the dendrimer/dendron
used. Furthermore, functionalization of the dendron
focal point in 12 allows further structural manipula-
tions (i.e., introduction of a lypophilic tail), which will
help to modulate the pharmacokinetics of the molecule
without affecting the affinity for DC-SIGN. Further devel-
opments along these lines are currently actively pursued
in our laboratories.
The structural simplicity of ligand 12, which displays
only four copies of a simple linear mannose trisaccha-
25 mM Tris-HCl, pH 8, 150 mM NaCl, 4 mM CaCl2, and 0.005%
of P20 surfactant as running buffer. The original sensograms are
reported as Supporting Information.
METHODS
Surface Plasmon Resonance. DC-SIGN ECD protein (residues
66–404) has been overexpressed and purified as described pre-
viously (17). All experiments were performed on a BIAcore 3000
using functionalized CM4 chips and the corresponding reagents
from BIAcore. Two flow cells were activated as previously de-
scribed (53). Flow cell one was then blocked with 50 ␮L of 1 M
ethanolamine and served as the control surface. The second one
was treated with BSA-Man␣1-3[Man␣1-6]Man (BSA-Mannotriose,
Dextra) (60 ␮g mLϪ1) in 10 mM acetate buffer, pH 4. Remaining
activated groups were blocked with 50 ␮L of 1 M ethanolamine.
The final density immobilized on the surface of the second flow
cell was 1200 RU. The BSA-Mannotriose used to functionalize the
CM4 chip harbors 15 glycosylation sites according to manufac-
turer. The affinity of the various sugars and mimics was then esti-
mated through a DC-SIGN ECD binding inhibition assay. The ECD
of DC-SIGN was injected onto the BSA-Mannotriose surface, at a
20 ␮M alone or in presence of an increasing concentration of the
sugar derivatives. Injection were performed at 20 ␮L/min using
Infection Studies. Cell Culture and Virus. Human B cell lines
B-THP1 and B-THP1/DC-SIGN (contributed by Drs Li Wu and Vinet
N.Keval Ramani) were grown in RPMI 1640 (PBI International)
supplemented with 10% fetal bovine serum (FBS), penicillin and
streptomycin (P/S), and L-glutamine (L-Gln) (all from EuroClone).
The following laboratory-adapted HIV-1 strains were used
in the experiments: the R5 tropic HIV-1 BaL (contributed by
Drs. S. Gartner, M. Popovic, and R. Gallo), the R4 tropic HIV-1 IIIB
(contributed by Drs. M. Popovic and R. Gallo), and the dual tropic
strain HIV-1 89.6 (contributed by Dr. R Collman). All strains were
grown to high titer in peripheral blood mononuclear cells (PBMC)
from healthy donors. Primary isolates HIV-1 V6 (CCR5 tropic) and
V17 (CXCR4 tropic) were kindly provided by Dr. M. Andreoni
(University of Rome Tor Vergata, Italy).
CD4+ T Lymphocytes Preparation. Peripheral blood was col-
lected from healthy donors and PBMC were isolated by centrifu-
gation on a Ficoll discontinuous density gradient (Lympholyte-H,
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Cederlane Laboratories). CD4ϩ T lymphocytes were separated
from PBMC by direct magnetic labeling using the CD4ϩ microbeads
(Miltenyi Biotech) according to manufacturer’s protocol.
H6M=B, H6MB), 3.80–3.53 (m, 38H, D1, H6M=A, H6MA, H3M, H4M, H4M=,
H7, H5M, H5M=, CH2N3), 3.69 (s, 24H, COOCH3), 3.41 (bs, 8H, H8),
3.46–3.38 (m, 8H, D4, D5), 2.72–2.62 (m, 8H, CH2COO), 2.60–
2.49 (m, 8H, CH2CONH), 2.24–2.06 (m, 8H, D6eq, D3eq), 1.92–1.72
(m, 8H, D6ax, D3ax), 1.32 (s, 3H, CH3 a=), 1.25 (s, 6H, CH3 b=). 13C NMR
(100 MHz, D2O, ppm): ␦ 181.9 (COO); 178.2, 177.9 (COOCH3);
174.7, 174.66 (COOCH2 b, CONH); 174.3 (COOCH2 a); 100.5 (C1M);
99.2 (C1M=); 75.1 (C1D); 71.5 (C2D); 74.0, 72.5, 71.2, 71.1 (C3M,
C3M=, C4M, C4M=); 71.1 (C2M=); 70.6 (C2M); 68.5 (C6M); 67.5, 67.4
(C5M, C5M=); 66.8 (C7, OCH2 a, OCH2 b), 66.5 (OCH2CH2N3), 61.6
(C6M=), 53.2 (CH3O); 50.0 (CH2N3); 47.3, 47.0 (Cquat a==, b==);
39.6 (C4D, C5D); 39.5 (C8); 30.6 (CH2CONH); 29.8 (CH2COO);
27.6, 27.3 (C6D, C3D), 17.5 (CH3 a=, b=). MS (MALDI) calcd for
[C129H201N7O82Na]ϩ ϭ 3184.97, found ϭ 3184.60. ESI-HRMS calcd
for [C129H201N7O82] monoisotopic peak ϭ 3160.17735, found ϭ
3160.18380. Calculated and found isotopic distribution are re-
ported in Supporting Information (Supplementary Figure 5 and
Table 1)
CD4ϩ T cells (at density of 2 ϫ 106 cells mLϪ1) were activated
by culturing them in complete RPMI (RPMI 160 with 20% FBS, P/S,
and L-Gln), in presence of IL2 (15 ng mLϪ1; R&D Systems) and PHA
(7.5 ␮g mLϪ1; Sigma Aldrich) for two days.
Inhibition of HIV Infection in “trans”. Inhibitors were diluted to
desired concentration into culture medium (complete RPMI).
B-THP1/DC-SIGN cells or B-THP1 cells (106 cells mLϪ1) were pre-
incubated with different concentration of compounds 2b, 12, 14,
and mannan (Sigma) or culture medium alone (negative control) for
30 min prior to exposure to HIV-1 BaL, IIIB, 89.6, V6, or V17 (final
concentration of all HIV-1 strains ϭ 0.08 ng mLϪ1 of p24) in the con-
tinued presence of the inhibitors for 3 h at 37 °C and 5% CO2.
After extensive washing, to remove unbound virus and inhibi-
tors, B-THP1/DC-SIGN cells were co-cultured with activated CD4ϩ
T cells in complete RPMI with IL2 (15 ng mLϪ1) at 37 °C and 5% CO2
for 3 days. Cellular concentration of both B-THP1/DC-SIGN cells
and CD4ϩ T lymphocytes was 2 ϫ 106 cells mLϪ1, and the ratio of
B-THP1/DC-SIGN to CD4ϩ T lymphocytes was 1:4.
For some experiments B-THP-1/DC-SIGN cells were treated
30 min or 2 h with the inhibitor or medium alone and washed
with PBS to eliminate unbound compounds. After wash B-THP-1/
DC-SIGN cells were put in culture medium and then were exposed
for 3 h to HIV BaL (0.08 ng mLϪ1 of p24) immediately or after 6 or
12 h. After incubation with the virus the cells were the co-cultured
with CD4ϩ T lymphocytes, as previously described.
Acknowledgment: The reagents B-THP1, B-THP-1/DC-SIGN,
HIV BaL (courtesy of the NIH AIDS Research and Reference Re-
agent Program), HIV IIIB, and HIV 89.6 were provided through
the EU programme EVA centre for AIDS Reagents NIBSC, U.K.
(AVIP contract number LSHP-CT-2004-50-3487). This work was
made possible by the following grants: Azioni Integrate Italia-
Spagna (IT074ABCCM and HI2005-0212), the Ministry of Sci-
ence and Innovation (MICINN, CTQ2008-01694), the FIRB pro-
gram CHEM-PROFARMANET (RBPR05NWWC), and in part, Marie
Curie ITN FP7 project CARMUSYS (PITN-GA-2008-213592), Isti-
tuto Superiore di Sanita’ “Programma Nazionale di Ricerca sull’
AIDS”, the EMPRO and AVIP EC WP6 Projects, the nGIN EC WP7
Project, the Japan Health Science Foundation, 2008 Ricerca
Finalizzata [Italian Ministry of Health], 2008 Ricerca Corrente
[Italian Ministry of Health]. M.S.-N. thanks MEC for a FPU fellow-
ship. A.B. was supported by a fellowship of the Doctorate School
of Molecular Medicine, University of Milan. M.T. was supported
by a fellowship of Sidaction-Ensemble contre le Sida.
ELISA. Co-culture supernatants were collected at day 3, and
p24 concentration, as a measure of HIV infection, was assayed
using the Alliance HIV-1 p24 Antigen kit (Perkin-Elmer) following
manufacturer’s instruction.
Flow Cytometry. DC-SIGN expression in the cell line B-THP1/
DC-SIGN was checked by staining with the PE-labeled anti-
human DC-SIGN monoclonal antibody (clone AZND1, Beckman
Coulter). To evaluate the toxicity of 12 B-THP-1/DC-SIGN cells
were incubated with different concentrations of 12 for 3 h and
30 min. The apoptosis was monitored evaluating the number of
dead cells according to forward and side scatters of flow cytom-
etry analysis and the staining with DNA incorporating the dye
7-aminoactinomicin D (7-AAD, Beckman Coulter). All flow cyto-
metric analyses were performed using a CYTOMICS FC-500 flow
cytometer interfaced with CXP 21 software (Beckman Coulter).
Synthesis. General information is reported in Supporting In-
formation. The following compounds are known and have been
previously described: 4 (28), 5 (28, 29), 6 (30), 8 (8), and 13
(29). Full synthetic procedures, compound characterization for
2b, 10, and 14, and numbering used in spectral assignment and
isotopic distribution of dendron 12 are reported as Supporting
Information.
Tetravalent Dendron of the ␣(1,2)␣(1,6)Pseudomannotriose 12.
To a solution of the scaffold dendron 11 (32) (4.5 mg, 5.39 ␮mol,
1 equiv) in 100 ␮L of dry N,N-dimethylacetamide (DMA) under nitro-
gen atmosphere were added HATU (17 mg, 43.36 ␮mol, 8 equiv)
and diisopropylethylamine (DIPEA) (15 ␮L, 86.7 ␮mol, 16 equiv).
After 15 min a solution of 10 (26 mg, 43.36 ␮mol, 8 equiv) in dry
DMA (170 ␮L) was added. The reaction was stirred at RT for 3 days.
MALDI mass analysis of a reaction aliquot showed completion of
the reaction. The reaction mixture was diluted in methanol and
charged directly onto a LH-20 Sephadex column equilibrated in
methanol. Slow elution led to the purification of product 12, which
was isolated in good yield (15.6 mg, 92% yield). [␣]20D ϭ ϩ55.2 (c
0.7, CH3OH). 1H NMR (400 MHz, D2O, ppm): ␦ 5.00 (s, 4H, H1M= as-
signed on the basis of interglycosidic NOE signal in ROESY experi-
ment between H1M= and D1 and D3eq), 4.84 (s, 4H, H1M), 4.33 (bs, 6H,
OCH2CH2N3, CH2O a), 4.24 (bs, 8H, CH2O b), 4.03 (bs, 1H, D2),
Supporting Information Available: This material is available
free of charge via the Internet at http://pubs.acs.org.
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