CellFy: A Cell-Based Fragment Screen against C-Type Lectins - ACS

Shuker, S. B., Hajduk, P. J., Meadows, R. P., and Fesik, S. W. (1996) High-Affinity Ligands for Proteins: SAR by NMR. Science (Washington, DC, U. S.) ...
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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

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modifications,

and

formation

of

active

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Page 2 of 11

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

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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