In silico-aided design of a glycan ligand of sialoadhesin for in vivo targeting of macrophages

Article abstract of DOI:10.1021/ja307501e

Cell-specific delivery of therapeutic agents using ligand targeting is gaining interest because of its potential for increased efficacy and reduced side effects. The challenge is to develop a suitable ligand for a cell-surface receptor that is selectively

Full text of DOI:10.1021/ja307501e

Communication
pubs.acs.org/JACS
In Silico-Aided Design of a Glycan Ligand of Sialoadhesin for in Vivo
Targeting of Macrophages
Corwin M. Nycholat,† Christoph Rademacher,†,‡ Norihito Kawasaki, and James C. Paulson*
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, United States
S
* Supporting Information
wound healing,11,12 the restricted expression and endocytic
properties of Sn make it an ideal receptor for the development
of a macrophage-targeted delivery system for therapeutic
intervention.
ABSTRACT: Cell-specific delivery of therapeutic agents
using ligand targeting is gaining interest because of its
potential for increased efficacy and reduced side effects.
The challenge is to develop a suitable ligand for a cell-
surface receptor that is selectively expressed on the desired
cell. Sialoadhesin (Sn, Siglec-1, CD169), a sialic acid-
binding immunoglobulin-like lectin (Siglec) expressed on
subsets of resident and inflammatory macrophages, is an
attractive target for the development of a ligand-targeted
delivery system. Here we report the development of a
high-affinity and selective ligand for Sn that is an analogue
of the natural ligand and is capable of targeting liposomal
nanoparticles to Sn-expressing cells in vivo. An efficient in
silico screen of a library of ∼8400 carboxylic acids was the
key to identifying novel 9-N-acyl-substituted N-acetylneur-
amic acid (Neu5Ac) substituents as potential lead
compounds. A small panel of targets were selected from
the screen and synthesized to evaluate their affinities and
selectivities. The most potent of these Sn ligands, 9-N-
(4H-thieno[3,2-c]chromene-2-carbamoyl)-Neu5Acα2−
3Galβ1−4GlcNAc (TCCNeu5Ac), was conjugated to lipids
for display on a liposomal nanoparticle for evaluation of
targeted delivery to cells. The TCCNeu5Ac liposomes were
found to target liposomes selectively to cells expressing
either murine or human Sn in vitro, and when
administered to mice, they exhibited in vivo targeting to
Sn-positive macrophages.
In general, Siglecs bind with low intrinsic affinity (0.1−1
mM) to their natural sialoside ligands.13 Several reports have
demonstrated the importance of sialic acid substituents (e.g., at
C9 of Neu5Ac) for increasing the affinity and selectivity of
ligand binding to Siglecs.8,14−17 In this regard, an exemplary
ligand for CD22 (Siglec-2) on B cells, 9-N-BPC-Neu5Acα2−
6Galβ1−4GlcNAc (6′-BPCNeu5Ac; BPC = biphenylcarbamoyl)
was found to support targeting of chemotherapeutic-loaded
liposomes and prolong life in a murine model of human B cell
lymphoma.18 More recently, we showed that liposomes
decorated with a related ligand, 9-N-BPC-Neu5Acα2−
3Galβ1−4GlcNAc (1, 3′-BPCNeu5Ac), are capable of targeting
Sn-expressing cells in vitro. However, this ligand was not
sufficiently selective for in vivo targeting.19 Here we describe
the in silico-aided design approach that we employed to
develop a high-affinity ligand for Sn that can be used for in vivo
targeting of macrophages.
To develop ligands with higher selectivity and affinity for Sn,
we adopted a strategy that takes advantage of the existing
crystal structure to identify novel 9-N-acyl-Neu5Ac substituents
that could favorably bind to Sn in the hydrophobic pocket
occupied by the previously described biphenyl substituent. To
this end, we conducted a virtual screen of a library of carboxylic
acids guided by the cocrystal structure of murine Sn (mSn) and
BPCNeu5Ac-OMe.8 As an alternative to solution-based screen-
ing methods, which would require extensive synthetic effort, the
in silico approach has the advantage of rapidly screening large
compound libraries to identify lead structures.20,21 Figure 1
outlines the in silico screening approach for the representative
carboxylic acid shown in Figure 1A. Initially, up to 250
conformers were calculated for each of ∼8400 carboxylic acids
from a commercial building-block library. The resulting
conformations were treated as unique acid structures and
virtually coupled to the amino group of 9-NH2-Neu5Ac fixed
within the binding pocket. An aromatic ring pharmacophore
was implemented using the coordinates of the first benzene ring
of the biphenyl substituent in BPCNeu5Ac-OMe. The tethered
docking of the acid conformers was scored on the basis of
London dispersion energy using Molecular Operating Environ-
ment (MOE). Four representative solutions from this tethered
docking approach for the acid in Figure 1A are shown in Figure
igand-targeted liposomal nanoparticles offer promising
Lapplications in human medicine for selective delivery of
therapeutic agents to the desired cells, resulting in increased
efficacy and decreased side effects.1−5 The challenge is to
identify cell-surface receptors that are selectively expressed on
the targeted cell and to develop ligands that target and bind
those receptors with high selectivity. Siglecs, a family of sialic
acid-binding immunoglobulin-like lectins with restricted ex-
pression on one or a few immune cell types, represent attractive
targets for cell-directed therapies.6,7 Among them, sialoadhesin
(variously denoted as Sn, Siglec-1, or CD169) is an endocytic
surface receptor that is expressed on subsets of resident and
inflammatory macrophages and has a preference to bind glycan
ligands with the Neu5Acα2−3Galβ1−4GlcNAc sequence
(Neu5Ac = N-acetylneuraminic acid; Gal = galactose; GlcNAc
= N-acetylglucosamine).8−10 Because macrophages have both
protective and pathological activities, including antitumor
immune response, allergy and asthma, atherosclerosis, and
Received: July 30, 2012
Published: September 11, 2012
© 2012 American Chemical Society
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Scheme 1. Chemo-enzymatic Synthesis of C-9 N-Acyl-
a
Modified Sialoside Analogues
a
Reagents and conditions: (a) CMP-9-NH2-Neu5Ac, PmST1; (b)
RC(O)NHS, THF, H2O (see Table 1 for R).
Table 1. Inhibitory Potencies of Sialoside Analogues 1−9
a
against Murine Sn
a
All of the titrations were performed in triplicate, and standard
deviations (SDs) of three independent measurements are given.
1B−E. From this preliminary evaluation, the top 3000 poses
were selected for further inspection using AutoDock 4.2.22
Figure 1F depicts a tethered AutoDock solution for the
representative carboxylic acid. The final nontethered docking
solutions resembled the canonical sialic acid binding pose and
provided a ranking of the acids based on calculated binding
energies. From this ranking, a small panel of six target
structures (2−7) were selected from the top-ranked 100 for
their structural diversity based on the computed two-dimen-
sional molecular fingerprints of the corresponding acids; their
structures are shown in Table 1. In addition, as “nonranked”
controls, we selected two additional sialosides (8 and 9) with
N-acyl substituents that were eliminated from the screen at an
early stage.
Figure 1. Representative structures from the in silico screening
strategy used to identify high-affinity ligands for sialoadhesin (Sn). (A)
Representative carboxylic acid from a commercial building-block
library whose members were screened as potential substituents of 9-
NH2-Neu5Ac. (B−E) Four representative solutions for the acid from
(A) obtained using the tethered docking approach, highlighting
varying docking poses of the tethered acid substituent. Conformations
of the respective substituents were calculated and conjugated via in
silico amide coupling to 9-NH2-Neu5Ac, which was fixed within the
binding site. The top 3000 hits from this initial docking evaluation
were further inspected using AutoDock. (F) Representative AutoDock
solution for the selected carboxylic acid coupled to 9-NH2-Neu5Ac. O,
C, and N atoms are highlighted in red, white, and blue, respectively.
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Figure 2. Inhibitory potencies of selected sialoside analogues in a mSn
competitive bead-binding assay. Each compound was analyzed in
triplicate for inhibition of the binding of mSn-Fc chimera to
Neu5Acα2−3Galβ1−4GlcNAc-coated magnetic beads as described
in the Supporting Information. See Table 1 for glycans 1, 2, and 4.
Figure 3. Fluorescence-assisted cell sorting (FACS) analysis of in vitro
binding of targeted TCCNeu5Ac or nontargeted “naked” liposomes to
cells expressing murine and human Siglecs. Binding is shown as mean
fluorescence intensity (MFI) SD (n = 3).
Figure 4. TCCNeu5Ac liposomes target Sn-positive macrophages in
vivo. (A) Wild-type mice were injected subcutaneously with
fluorescently labeled naked or TCCNeu5Ac liposomes, and the
neighboring lymph nodes were harvested after 2.5 h. Cells were
stained with various antibodies as described in the Supporting
Information and analyzed by flow cytometry. (B) Wild-type and Sn
knockout mice (n = 3 in each group) were injected with the liposomes.
T h e b i n d i n g o f l i p o s o m e s t o m a c r o p h a g e s
(TCRb−NK1.1−CD19−CD11b+) was analyzed by flow cytometry.
The plots show the geometric mean of fluorescent intensity (MFI).
All of the targets were synthesized chemo-enzymatically
(Scheme 1). Briefly, Galβ1−4GlcNAc-ethyl azide (10) was
reacted with CMP-9-NH2-Neu5Ac (CMP = cytidine mono-
phosphate) using Pasteurella multocida α2−3-sialyltransferase 1
(PmST1)23 to afford the trisaccharide scaffold 11. Divergent
reaction of 11 with the panel of NHS-activated carboxylic acids
afforded the final targets 2−9. The reference ligand 1
substituted with BPC was also prepared.
The inhibitory potencies (IC50) of the glycan derivatives 1−9
were evaluated in a flow cytometry assay based on the
competitive binding of mSn-Fc chimera to beads decorated
with the natural ligand Neu5Acα2−3Galβ1−4GlcNAc. The
IC50 values required to displace the bound mSn-Fc were
determined with serial dilutions of the competitors (Table 1
and Figure 2).
A wide range of binding affinities was observed for sialosides
2−7 containing the top-ranked substituents. Relative to the
reference compound 1 (IC50 = 4.8 μM), two were weak
inhibitors (6 and 7; IC50 > 200 μM), three were of comparable
affinity (3−5; IC50 of 7.3−26 μM), and one had ∼13-fold
higher affinity (2; IC50 = 0.38 μM). This result is particularly
notable in view of the affinity of the natural nonsubstituted
ligand, Neu5Acα2−3Galβ1−4GlcNAc, which had an IC50 of
1300 μM in a comparable assay.13 Analysis of 2 in complex with
Sn suggested that the bioisostere replacement of the first benzyl
group in 1 provides improved shape complementarity and
additional contacts to the protein (data not shown). Of the two
unranked substituents, one was weakly active (9; IC50 > 500
investigation to validate its potential and general utility, it
provided a significant lead with minimal investment in synthetic
resources. Accordingly, we proceeded to evaluate further the
high-affinity lead structure, 9-N-(4H-thieno[3,2-c]chromene-2-
carbamoyl)-Neu5Acα 2−3Galβ1−4GlcNAc (2, TCCNeu5Ac).
We next tested the selectivity of TCCNeu5Ac (2) for Sn when
incorporated into targeted liposomes. To accomplish this,
fluorescent targeted liposomes were formulated to include high-
affinity glycan ligand 2 coupled to PEGylated lipid (see the
Supporting Information), and these liposomes were then tested
for binding to a panel of cell lines expressing human and
murine Siglecs.18,19 Cells expressing Siglecs were incubated
with fluorescently labeled targeted liposomes, washed, and
analyzed by flow cytometry. The results revealed that the
TCCNeu5Ac ligand is highly selective for Sn (Figure 3). Only
cells expressing murine or human Sn bound the targeted
liposomes, while nontargeted “naked” liposomes did not bind
any of the cells.
To evaluate the specificity of the TCCNeu5Ac ligand in an in
vivo setting, we sought to determine whether TCCNeu5Ac-
decorated liposomes could target Sn-expressing macrophages in
peripheral lymph nodes. Accordingly, wild-type (WT) or Sn
knockout (KO) mice were subcutaneously injected in the flank
with Alexa Fluor 647-labeled TCCNeu5Ac liposomes. After 2.5
h, immune cells from neighboring lymph nodes on the same
μM) and the other showed relatively high affinity (8; IC50
=
26.5 μM). Thus, while our approach of using the in silico
screen to identify target substituents requires further
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Populations of T, B, and NK lymphocytes and myeloid cells
were identified using specific antibodies (Figure 4). Further-
more, Sn-positive myeloid cells (macrophages) were identified
using an anti-Sn antibody. The gated cell populations revealed
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effective in vivo targeting of Sn-positive macrophages. We
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for Sn will provide a powerful means of active in vivo delivery
of antigens or therapeutic agents to macrophages.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Supplementary synthetic scheme, experimental protocols,
synthetic methods, and compound characterization. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
jpaulson@scripps.edu
■
Present Address
‡Max Planck Institute of Colloids and Interfaces, Department
of Biomolecular Systems, 14424 Potsdam, Germany.
Author Contributions
†C.M.N. and C.R. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.R. thanks EMBO for a long-term fellowship. This work was
supported by the National Institutes of Health (P01HL107151
and R01AI05143).
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