Development and evaluation of a non-peptidic ligand for the molecular imaging of inflammatory processes using S100A9 (MRP14) as a novel target

Article abstract of DOI:10.1039/c5cc07019h

The establishment of novel molecular imaging tools to monitor the local activity of inflammation remains an interdisciplinary challenge. Our target, the alarmin S100A9, one subunit of the heterodimer S100A8/S100A9 (calprotectin), is locally secreted in hi

Full text of DOI:10.1039/c5cc07019h

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Development and evaluation of a non-peptidic
ligand for the molecular imaging of inflammatory
processes using S100A9 (MRP14) as a novel
target†
Cite this:DOI: 10.1039/c5cc07019h
Received 20th August 2015,
Accepted 9th September 2015
ab
c
a
abde
bce
abe
A. Faust,‡* T. Voller,‡ F. Busch, M. Schafers,
T. Vogl
J. Roth,
S. Hermann
and
DOI: 10.1039/c5cc07019h
¨
¨
bce
www.rsc.org/chemcomm
The establishment of novel molecular imaging tools to monitor the both, the extracellular matrix and Toll-like receptor 4 (TLR4) or
local activity of inflammation remains an interdisciplinary challenge. RAGE expressing immune cells, S100A8/S100A9 exhibits a high
Our target, the alarmin S100A9, one subunit of the heterodimer local concentration gradient amplifying local inflammatory reac-
S100A8/S100A9 (calprotectin), is locally secreted in high concen- tions. Recently, S100A8/S100A9 has been established as a biomarker
1a,5
trations from immigrated and activated phagocytes at local sites of in many inflammatory diseases.
S100A8/S100A9 serum levels
inflammation. Calprotectin is already a well established serum could for example be characterized as an accurate predictor for
biomarker for many inflammatory disorders. Here we show the the risk of cardiovascular events in patients. Moreover, the knockout
À/À
development and first evaluation of the novel S100A9 specific of S100A9 significantly reduced disease severity in ApoE
molecular imaging probe Cy5.5-CES271 for optical imaging of local mice, a mouse model for atherosclerosis.
6
inflammatory activity in vivo.
Previously, we reported the use of antibody-based optical
probes such as Cy5.5t-labelled antibodies targeting the S100A9
subunit (anti-S100A9-Cy5.5) of the S100A8/S100A9 heterodimer
1
Inflammatory reactions like autoimmune and infectious diseases
2
as well as cardiovascular diseases such as atherosclerosis or in vivo. We could observe excellent correlations between signal
3
myocardial ischemia characteristically feature local immune intensity and local S100A9 expression. Furthermore, we could
cell activation and inflammation. These inflammatory reactions predict the individual disease course of a single mouse in different
7
that initially serve the host by avoiding local tissue damage and mouse models for inflammatory and infectious diseases. However,
by clearing infections, can in fact harm the patient during translation of this imaging strategy into patients is challenging due
overwhelming immune responses or under chronic inflamma- to known limitations of antibody-based approaches. Therefore, we
tory conditions such as rheumatoid arthritis. The disease activity developed the first S100A9 affine optical imaging tracer based on a
and progression, e.g. in atherosclerosis correlate well with the small non-peptidic structure, which can also be used for many
1b,4
number of immigrated immune cells.
Activated phagocytes other imaging modalities such as SPECT or PET when radio-
and epithelial cells express and locally secrete high levels of the labelled. We chose linomide 1 as lead compound, a synthetic
S100 protein complex S100A8/S100A9, which acts as a so called immuno-modulator based on 3-quinolinecarboxamides which
8
alarmin or Danger Associated Molecular Pattern (DAMP) mole- is firstly described in prostate 1996. A modified substance of 1,
1a,4
cule with potent pro-inflammatory capacities.
By binding to laquinimod 2 was already selected for clinical studies in man
and its efficacy has been demonstrated in animal models of
9
a
several autoimmune diseases, including multiple sclerosis. The
European Institute for Molecular Imaging (EIMI), University of M u¨ nster,
Waldeyerstr. 15, 48149 M u¨ nster, Germany. E-mail: faustan@uni-muenster.de
Cells-in-Motion Cluster of Excellence (EXC 1003–CiM), University of M u¨ nster,
1
0
drug was granted a fast track review by the FDA in 2009.
b
c
Multivariate analytical tools were used to derive the SAR for
the binding activity of a series of laquinimod analogues towards
S100A9 with the assumption that similar analogs bind to the
same binding site in a similar binding mode.
Recently, we could show that the most potent ligands are based
on 3-quinolinecarboxamides with an ethyl group in 5-position
4
8149 M u¨ nster, Germany
Institute of Immunology, University Hospital of M u¨ nster, R o¨ ntgenstr. 21,
8149 M u¨ nster, Germany
4
d
e
Department of Nuclear Medicine, University Hospital of M u¨ nster,
Albert-Schweitzer-Campus 1, 48149 M u¨ nster, Germany
Interdisciplinary Center for Clinical Research (IZKF M u¨ nster),
University of M u¨ nster, Domagkstr. 3, 48149 M u¨ nster, Germany
Electronic supplementary information (ESI) available: Experimental procedures
11
instead of the chloride (Fig. 1). We propose that the b-hydroxy-
†
carboxamide unit is responsible for binding to the protein. In
comparison the quinoline moiety, where slight variations are
possible, the N-phenyl or N-methylamine used for forming the
and analytical data of all compounds, details on ELISA, binding studies, mice and
biodistribution. See DOI: 10.1039/c5cc07019h
‡
Authors contributed equally.
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15
amination of the mesyl-PEG-azide 4 with aniline gave low yields
the mesylate was converted into the corresponding bromide and
we got the secondary amine in satisfactory amounts. The key
intermediate 3 was then coupled to the secondary amine and the
resulting carboxamide was then reduced to give the free amine as
precursor for coupling with the cyanine dye. An activated NHS-
TM
ester derivative of Cy5.5
was added under basic conditions
16
yielding Cy5.5-CES271 suitable for optical imaging of S100A9.
For verification the HPLC-purified tracer was analyzed by high
resolution mass spectrometry showing the fourfold anionic
species (Scheme 1B and C).
Fig. 1 Lead compound (top) and the established synthetic immuno-
modulators linomide 1 and laquinimod 2.
Studies were then conducted to verify strong binding affinity
amide bond is free for attaching different linker systems. In of Cy5.5-CES271 for S100A9 (Fig. 2A). In this regard, we modi-
addition a connection of a quinolinecarboxamide on a gold fied our specific S100-ELISA to analyse S100A9 binding to TLR4/
1
1
surface is possible without loss of inhibition.
MD2. Instead of the capturing anti-S100A9 antibody we coated
The synthesis of the target compound Cy5.5-CES271 for optical TLR4/MD2 (3146-TM-050/CF, R&D Systems) to the wells of a
7
imaging of S100A9 is outlined in Scheme 1. Starting from the 96-well plate which served as capturing molecule. After blocking
commercially available 3-nitrophthalic acid anhydride, diethyl of the unspecific binding sites by PBS/5% skim milk powder,
malonate was condensed and the resulting lactone was treated plates were washed three times. S100A9 protein was added at a
1
2
À1
with hydrochloric acid to give 2-acetyl-6-nitrobenzoic acid. The concentration of 1 mg ml in the presence or absence of 100 mM
anthranilic acid was synthesized in a two-step hydrogenation Cy5.5-CES271 and incubated for two hours at room temperature.
procedure and treated with phosgene to give the corresponding Unbound S100 protein was removed by washing the plates for
13
isatoic anhydride in good yield. After N-alkylation with iodo- three times, followed by the addition of a primary anti-S100A9-
methane the anhydride was opened with diethyl malonate under antibody (1 mg ml , polyclonal, rabbit). After a washing step, the
decarboxylation and reclosed yielding the corresponding secondary anti-rabbit-IgG-antibody coupled to HRP (1 mg ml
À1
À1
,
b-hydroxyester. Acidic cleavage yielded the 3-quinolinecarboxylic Cell Signalling) was added. TMB was used as substrate for HRP
acid 3 as key intermediate for functionalization for different to quantify binding efficiency by absorbance readings at 450 nm
14
labelling techniques. For the first S100A9 affine imaging probe in an ELISA reader (Anthos Mikrosysteme). Addition of the non-
TM
the cyanine dye Cy5.5
label was coupled to intermediate 3 peptidic S100A9 ligand (Cy5.5-CES271) markedly blocked bind-
through a PEG (polyethylene glycol) linker in order to avoid inter- ing of S100A9 to TLR4/MD2, as indicated by a decrease of signal
TM
actions between the Cy5.5
label and the protein. After the given by the TLR4-S100A9 ELISA. These results confirm that the
Scheme 1 (A) Synthesis of Cy5.5-CES271: (a) Ac
2
O, NEt
O, NaOH, 110 1C, 3 h then rt, 24 h, 90% (two steps); (d) diphosgene, THF, toluene, rt, 5 h, 86%; (e) NaH,
MeI, DMF, rt, 14 h; (f) NaH, diethyl malonate, DMF, 85 1C, 14 h, 53% (two steps); (g) HCl/HOAc 60 1C, 6 h, 50%; (h) LiBr, CH CN, reflux, 16 h, 80%; (i) aniline,
O, reflux, 16 h, 42%; ( j) NEt , SOCl , CH Cl , 0 1C, 4 h, 82%; (k) H , Pd/C, THF, rt, 16 h, 79%; (l) Cy5.5-NHS, NEt , DMF, rt, 16 h, 50%. (B) Analysis and
verification of Cy5.5-CES271 (here M species) by high resolution ESI-MS; (C) quality control (RP-HPLC) of Cy5.5-CES271.
3 2 2 2
, diethyl malonate, 40 1C, 4 h, 88%; (b) HCl, H O, toluene, 100 1C, 14 h, 80%; (c) 1. PtO , H O,
s
NaOH, 90 1C 3 h then rt, 14 h; 2. RANEY -Ni, H
2
3
H
2
3
2
4
2
2
2
3
À
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Fig. 2 (A) Analysis of the binding of Cy5.5-CES271 to S100A9. S100A9 binding to TLR4/MD2 was quantified by an ELISA-based assay. Specific binding of
CES271-Cy5.5 to S100A9 inhibits formation of the S100A9–TLR4/MD2 complex and thereby diminishes the ELISA signal (n = 5; p o 0.05). (B) Estimation
of the binding constant of Cy5.5-CES271 to human and murine S100A9. The constant was calculated using the one site saturation regression model.
Each dot represents the mean value of four independent experiments.
attachment of the dye Cy5.5 does not interfere with the binding
of Cy5.5-CES271 to S100A9. This blocking study also proves
target specificity of Cy5.5-CES271, and shows that binding of
S100A9 to TLR4/MD2 can be efficiently blocked in the presence
of Cy5.5-CES271 (resulting in low absorption).
Next, we determined the binding constants of Cy5.5-CES271
to murine and human S100A9 by fluorimetric measurements
À1
(
Fig. 2B). Briefly, 0,3788 mM S100A9 (5 mg ml of homodimer
S100A9) solved in 50 ml PBS was coated to the bottom of a 96-well
plate and served as capturing molecule. For each S100A9 coated
well a control well was used with 50 ml PBS alone. After a washing
step, unspecific binding sites were blocked by PBS/5% skim milk
powder. Cy5.5-CES271 was added at increasing concentrations.
After 1 h incubation at 4 1C, the supernatants were removed and
the fluorescence intensity was measured with a fluorimeter.
Non-linear regression analysis was performed with a one site
saturation model, to calculate the binding constant of Cy5.5-
Fig. 3 Biodistribution of Cy5.5-CES271 in healthy Balb/c mice. The tracer
was intravenously injected at a dose 2 nmol per mouse. The tracer accu-
mulation was measured 1 and 3 h post injection (n = 5 for each time point).
CES271 to either murine or human S100A9. The K -values of
d
determined as sufficient for both murine and human S100A9
for in vivo optical imaging techniques. Ongoing in vivo studies
in the inflamed tissue in models of ear inflammation, arthritis
or myocardial infarction will give us information about the
feasibility as optical probe for specific S100A9 targeted imaging.
The established synthesis and imaging strategy allows further
labelling methods (e.g. radionuclides) and is therefore suitable
for clinical translation.
2.66 mM (murine) and 2.06 mM (human, Fig. 2B) confirm that
Cy5.5-CES271 is eligible for imaging purposes. We observed a
strong binding affinity of the tracer to S100A9 that is not affected
by the attachment of Cy5.5 to CES271.
Finally, we analysed the biodistribution of Cy5.5-CES271
injected to healthy Balb/c mice by the measurement of fluores-
cence intensity in various organs. Fig. 3 shows the tracer accumu-
lation 1 and 3 h after injection. The tracer was injected at a dose of
We gratefully acknowledge financial support from the DFG
2
nmol per mouse. We could observe a good tissue availability of
(SFB656 A9 and Z5), the Interdisciplinary Centre of Clinical
Cy5.5-CES271 and an elimination that was mainly driven by renal
excretion, as indicated by the high renal uptake and the increasing
concentrations of the tracer in the urinary bladder urine in
comparison to the relatively low hepatic uptake. This kinetics,
that is different to our previously published antibody based tracer
Research (IZKF M u¨ nster, core unit PIX) and the Medical College
M u¨ nster.
Notes and references
7
anti-S100A9-Cy5.5 kinetics favors Cy5.5-CES271 for imaging of
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In conclusion, we developed the first optical imaging probe
Cy5.5-CES271 based on non-peptidic 3-quinolinecarboxamide
for specific imaging of extracellularly released S100A9 protein
indicating local phagocyte activity. The specificity was confirmed
by a modified S100-ELISA and the binding potency was
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4.2 mmol) was dissolved in 400 mL dry dimethylformamide provided
with 10 mL triethylamine. To this solution, Cy5.5-NHS ester (GE)
(1 mg, 0.9 mmol) was added. The reaction mixture was vortexed for
16 h at room temperature in the dark. Purification of Cy5.5-CES271
was performed by gradient-HPLC using a Knauer system with two
K-1800 pumps, an S-2500 UV detector and a RP-HPLC Nucleosil
100–5 C18 column (250 mm  4.6 mm). Eluent A: water (0.1% TFA).
Eluent B: Acetonitrile (0.1% TFA). Gradient from 95% A to 40% A
over 19 minutes, holding for 5 minutes and back to 95% in one
3
4
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(
b) M. G. Ionita, A. Vink, I. E. Dijke, J. D. Laman, W. Peeters,
À1
P. H. van der Kraak, F. L. Moll, J. P. de Vries, G. Pasterkamp and
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A. Faust, C. Geyer, B. Petersen, K. Roebrock, M. Sch ¨a fers, C. Bremer
and J. Roth, Nat. Commun., 2014, 5, 4593.
minute at a flow rate of 5.5 ml min , detection at l = 254 nm. The
appropriate fractions (t = 16.5 min) were collected, lyophilized,
R
5
6
redissolved in 1 mL water and finally stored at À20 1C. The average
À1
content of Cy5.5-CES271 was 0.45 Æ 0.02 mmol ml (E50%) as
determined by photometric measurements with labs = 678 nm and
À1
À1
À
3À
7
e678 = 250000 M cm . MS (ES ): m/z = 464.1 (100%), 464.5, 464.8 [M] ;
2
À
À
696.7, 697.2, 697.7 [M + H] . HRMS (ES ): m/z = 347.84493,
348.09560, 348.34570, 348.59556, 348.84580 [M]
347.84511, 348.09589, 348.34575, 348.59593, 348.84592 [M]
4À
calculated:
4À
8
I. B. Joseph and J. T. Isaacs, Prostate, 1996, 29, 183.
.
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