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CellFy—A Cell-Based Fragment Screen against C-Type Lectins Jessica Schulze, Hannes Baukmann, Robert Wawrzinek, Felix Fuchsberger, Edgar Specker, Jonas Aretz, Marc Nazaré, and Christoph Rademacher ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00875 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on November 28, 2018
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ACS Chemical Biology
CellFy — A Cell-Based Fragment Screen against C-Type Lectins Jessica Schulze1,2, Hannes Baukmann (0000-0003-3560-6777)1,2, Robert Wawrzinek (00000002-1186-7625)1,2, Felix Fuchsberger (0000-0002-9379-9792)1,2, Edgar Specker3, Jonas Aretz (0000-0002-4623-5820)1,2, Marc Nazaré3, and Christoph Rademacher (0000-0001-70827239)1,2*. 1
Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Potsdam, Germany
2
Freie Universität Berlin, Department of Biology, Chemistry and Pharmacy, Berlin, Germany
3
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
Supporting Information ABSTRACT: Fragment-based drug discovery is a powerful
screening against multiple CLRs allowing a selectivity
complement to conventional high-throughput screening,
counterscreening. Overall, this sensitive cell-based fragment
especially for difficult targets. Screening low molecular
screening assay provides a powerful tool for rapid
weight
identification of bioactive fragments, even for difficult targets.
fragments
usually
requires
highly
sensitive
biophysical methods due to the generally low affinity of the identified ligands. Here, we developed a cell-based fragment screening assay (cellFy) that allows sensitive identification of
INTRODUCTION
fragment hits in a physiologically more relevant environment
Fragment-based drug discovery (FBDD) is a promising
in contrast to isolated target screenings in solution. For this a
way for the development of novel drugs, especially for difficult
fluorescently labeled multivalent reporter was employed,
targets.1 Due to the generally low binding affinities of
enabling direct measurement of displacement by low
fragments, higher compound concentrations and more
molecular weight fragments without requiring enzymatic
sensitive
reactions or receptor activation. We applied this technique to
resonance (NMR) spectroscopy is recognized as a highly
identify hits against two challenging targets of the C-type
powerful technique in FBDD.1 The approach to discover and
lectin
evolve
receptor
(CLR)
family:
Dendritic
Cell-Specific
assays
fragments
are
to
necessary.
high-affinity
Nuclear
drugs
magnetic
by
1H-15N-
Intercellular adhesion molecule-3-Grabbing Non-integrin
heteronuclear single quantum coherence (HSQC) NMR was
(DC-SIGN) and Langerin. Both receptors are involved in
introduced two decades ago,2 and hit validation by HSQC is
pathogen recognition and initiation of an immune response,
generally considered the “gold standard” for hit validation3.
which renders them attractive targets for immune modulation.
Although being very sensitive and well-established, NMR
Due to their shallow and hydrophilic primary binding site, hit
experiments and other commonly used techniques in FBDD
identification for CLRs is challenging and drug-like ligands for
rely on purified protein.
CLRs are sparse. Screening of a fragment library followed by hit validation identified several promising candidates for further fragment evolution for DC-SIGN. Additionally, a multiplexed assay format was developed for simultaneous
In contrast, cell-based systems take into account cellular localization of target molecules, interaction partners, posttranslational
ACS Paragon Plus Environment
modifications,
and
formation
of
active
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metabolites. Also the cell metabolism and the localization
quinoxalinone-based
within a cell can change the overall ligand activity.4 Hence,
throughput
developing a highly sensitive cell-based fragment assays
affinities.22,23
screening
DC-SIGN exhibit
inhibitors low
from
high-
micromolar
range
presents a cornerstone with the potential to identify specific,
Langerin is a trimeric CLR, mainly expressed on human
bioactive small molecule ligands. Yet, only a handful of cell-
Langerhans cells and has been shown to interact with self-
based fragment assays have been reported, indicating the
and non-self-glycan epitopes, e.g. from HIV or Candida
complexity of these screening approaches.5–8 One reason
albicans.24–28 Since Langerin is an efficient endocytic
might be the difficulty to find sensitive readout methods.
recycling receptor, it is an attractive target for the
Moreover, high compound concentrations, which are
development of anti-infectives and for new skin vaccine
necessary in fragment-based applications, have a higher
therapies to systemically modulate the immune system.29,30
tendency to unspecifically interfere with cellular processes
However, while glycomimetic-based ligands have been
complicating the search for specific small molecules that are
described, drug-like small molecule ligands are lacking.31
active in living cells. Nevertheless, just recently a phenotypic-
The Ca2+-dependent binding of monosaccharides by CLRs
based screening approach gained attention by identifying
is characterized by weak interactions with dissociation
new leads from fragment-based libraries that were modified
constants in the millimolar range due to their shallow and
with a photoreactive group and an alkyne handle.9,10 The
highly solvent-exposed binding site.32 Consequently, natural
combined technology provided new opportunities for the
ligands possess low affinity with high promiscuity. Most CLR
identification of yet untargeted proteins, however, fragment
ligands lack drug-like properties such as high affinity,
functionalization is tedious and can also unfavorably impact
bioavailability and adequate blood circulation time. These
the protein-ligand interaction. Hence, a combined cellular
characteristics and past efforts using classical approaches
target-based fragment screening approach may open new
render CLRs as challenging targets for drug discovery.
opportunities even for challenging targets.
Here, we present the development of an innovative cell-
C-type lectin receptors (CLRs) have evolved as relevant
based fragment screening assay (cellFy) using the example
pharmaceutical targets as several members of this large
of two highly attractive, but challenging targets: the CLRs
family of glycan-binding proteins are often involved in
DC-SIGN and Langerin. Screening our fragment library
pathogen recognition and immune homeostasis.11 CLRs bind
resulted in rapid identification of potential DC-SIGN ligands,
to glycans that mediate many essential functions in mammals
which display novel therapeutic agents when further
such as cell-cell interactions, cell proliferation, and immune
optimized. Additionally, the multiplexed cellFy allows for
responses.12 Particularly, DC-SIGN (Dendritic Cell-Specific
sensitive simultaneous identification of candidates for
Intercellular adhesion molecule-3-Grabbing Non-integrin)
pursuing fragment evolution, therefore providing a powerful
and Langerin have been well studied due to their involvement in
pathogen
recognition
and
subsequent
tool for rapid identification of bioactive fragments, even for
antigen
difficult targets.
presentation to the immune system. DC-SIGN is a tetrameric CLR expressed on myeloid
RESULTS and DISCUSSION
dendritic cells and macrophages.13–15 It is involved in the recognition
of
a
including
Assay development. Crucial for developing the highly
Mycobacterium tuberculosis, HIV, Ebola virus, and Candida
sensitive cell-based fragment assay (cellFy) was the
albicans.16 In particular, since DC-SIGN promotes HIV trans-
identification of a suitable reporter molecule as well as a fast
infection of T cells, this CLR has drawn much attention as a
and sensitive read-out method to detect weak fragment-
target for anti-viral
plethora
therapy.17
of
pathogens
As of today, carbohydrate-
target interactions in a cellular environment to recombinantly
derived inhibitors have been developed showing affinities in the medium to high micromolar
range,18–21
expressed target receptors on model cell lines.
while drug-like,
2
ACS Paragon Plus Environment
We selected high-throughput flow cytometry that allows
A
several simultaneous and sensitive measurements to
B R2 = 0.97 IC50 = 4.5 mM Z` = 0.82
1300
level. A set of FITC-labeled reporter molecules were tested
500
800
R2 = 0.91 IC50 = 369 µM Z` = 0.56
400
MFI
MFI
analyze binding of fluorescent molecules on a single-cell
300
for effective binding to human Langerin-expressing Raji cells,
200
300 0 1 2 log [mannose] (mM)
-1
C
binding was detected with FITC-derivatized forms of
SSC-A
molecular weights of 500 kDa and 2000 kDa (Figure S1). For the detection of at least 10% fluorescent cells (or a two-fold MFI over background), high ligand concentrations were
Fixed cells 93.3%
0
necessary for zymosan (>0.1 mg ml-1) and mannosylated
0
BSA (>0.05 mg ml-1). Conversely, a two-fold MFI increase
FSC-A
D
0.025 mg ml-1. Hence, we proceeded with FITC-dextrans as potential reporter molecules. By competing the dextran-based reporter with increasing concentrations of mannose, a natural ligand of Langerin, the reporter construct with a molecular weight of 500 kDa showed excellent assay parameters (Z’-factor = 0.72, R2
0 0
1M
FSC-A
1M
E
1.5 0.5
1
FITCdextran 10 mM mannose Bkgrd
0 3 1 2 log [mannose] (mM)
80 60 40 20 0
0
0 100
FITC
10 20 30 40 DMSO (%)
106
FITCdextran 10 mM mannose Background 2 mM Fragments
F
100
1.0 0
Single cells 98.2%
4 h, Z` = 0.71 1 h, Z`= 0.77 0.5 h, Z`= 0.76
2.5 2.0
-1 0 log [ligand] (mM)
500
Viability (%)
with dextran ligands was observed below concentrations of
-2
1M
FSC-H
1M
zymosan, mannosylated BSA and two dextrans with
3
count
which have been described before.33 Dose-dependent ligand
MFI (x1000)
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ACS Chemical Biology
MFI Screen1(x1000)
Page 3 of 11
4.0 3.0 2.0 1.0 0
2.0 1.0 0 MFI Screen2 (x1000)
Figure 1. Validating and optimizing the cellFy. (A) The IC50
value = 0.94) and a high sensitivity reflected by an IC50 value
of mannose was measured with a reduced reaction volume
of 8.7 mM (Figure S2). Moreover, dextran binding was
of 25 µl. FITC-dextran (0.025 mg/ml) was incubated together
calcium dependent (Figure S2D). Next, to minimize signal
with different concentrations of mannose for 1 h at 4°C. After
fluctuations arising from cell activity during data acquisition,
incubation,
cells were fixed with paraformaldehyde. These fixed cells
cells
were
washed
and
fixed
with
paraformaldehyde for 20 min before measuring by flow
were tested and compared to cells that were fixed after the
cytometry. (B) IC50 determination of a human Langerin-
ligand incubation step. The latter showed a large signal range
targeting ligand. Here, viable cells were directly analyzed by
and an improved Z’-factor (Figure S3). Thus, unless stated
flow cytometry. (C) Cells analyzed in the cellFy were gated in
otherwise, all further experiments were conducted under
two steps before analyzing the FITC fluorescent intensity of
these conditions applying the 500 kDa FITC-dextran reporter.
single, fixed cells plotted in a histogram. (D) The ligand
In a next step, the fragment screening assay was further
incubation period was varied; all other steps were mentioned
optimized for a lower sample volume of 25 µl without
as before. (E) DMSO toxicity was analyzed by incubating
impairing the quality or sensitivity of the assay (Figure 1A). In
viable cells with increasing concentrations of DMSO for 1 h
addition, we evaluated a ligand for human Langerin with this
at 4°C. (F) 72 fragments were compiled and screened in the
assay.34 The IC50 value of the glycomimetic ligand was
cellFy together with 8 repeats of negative controls (DMSO
determined to be 369 µM, which is in the same range as the
only), 8 repeats of positive controls (10 mM mannose) and 8
affinity constant previously measured via a 19F NMR reporter
wells with cells only for measuring the background signal.
displacement assay and twelve-fold better than mannose
Two separate plates were prepared and measured on
(Figure 1B).31
different dates to analyze the assay robustness (p
