LDV peptidomimetics equipped with biotinylated spacer-arms: Synthesis and biological evaluation on CCRF-CEM cell line

Article abstract of DOI:10.1016/j.bmcl.2011.10.078

The tripeptide Leu-Asp-Val (LDV) is known to bind α₄β ₁ integrin in leukemia cells. Here we have synthesized a LDV peptidomimetic equipped with a biotin-conjugated spacer-arm. Compound 9 acts as an inhibitor of the α₄β₁ integrin in an adhesion assay using fluorescently labeled, α₄β ₁ integrin-expressing leukemia CCRF-CEM cells. Furthermore, when bound to neutravidin-coated plates, compound 9 could capture CCRF-CEM cells. Such biotin-conjugated LDV peptidomimetic may thus represent a novel tool for biotechnological applications using avidin interaction for leukapheresis or leukemia cell targeting.

Full text of DOI:10.1016/j.bmcl.2011.10.078

Bioorganic & Medicinal Chemistry Letters 22 (2012) 586–590
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
LDV peptidomimetics equipped with biotinylated spacer-arms: Synthesis
and biological evaluation on CCRF-CEM cell line
Estelle Gérard a, , Aline Meulle b, , Olivier Feron b, Jacqueline Marchand-Brynaert a,
⇑
a Université catholique de Louvain (UCL), Institut de la Matière Condensée et des Nanosciences, Bâtiment Lavoisier, Place L. Pasteur, L4.01.02, B-1348 Louvain-la-Neuve, Belgium
b Université catholique de Louvain (UCL), Pole of Pharmacology, FATH5349, Institute of Experimental and Clinical Research, Avenue E. Mounier n°52, B-1200 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The tripeptide Leu-Asp-Val (LDV) is known to bind a4b1 integrin in leukemia cells. Here we have synthe-
sized a LDV peptidomimetic equipped with a biotin-conjugated spacer-arm. Compound 9 acts as an
Received 8 September 2011
Revised 21 October 2011
Accepted 23 October 2011
Available online 31 October 2011
inhibitor of the 4b1 integrin in an adhesion assay using fluorescently labeled, 4b1 integrin-expressing
a
a
leukemia CCRF-CEM cells. Furthermore, when bound to neutravidin-coated plates, compound 9 could
capture CCRF-CEM cells. Such biotin-conjugated LDV peptidomimetic may thus represent a novel tool
for biotechnological applications using avidin interaction for leukapheresis or leukemia cell targeting.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
a4b1 Integrin antagonists
LDV peptidomimetics
Biotin-conjugated peptidomimetics
CCRF-CEM cell line
The cellular adhesion receptor
a4b1, also known as Very Late
leukemia cells expressing the a
4b1 integrin.12 Combined with the
Antigen-4 (VLA-4, or CD49d/CD29), is a member of the integrin
family and is expressed on the surface of activated leukocytes,
including leukemia cells.1,2 It mediates cellular adhesion and acti-
vation through a variety of cell–cell and cell–matrix interactions
that regulate leukocyte trafficking during inflammatory responses
or cancer progression.3,4 This receptor binds to two natural ligands,
the vascular cell adhesion molecule-1 (VCAM-1),5 expressed on
endothelial cells in response to inflammatory cytokines, and the
alternatively spliced portion of the type III connecting segment of
the extracellular matrix protein fibronectin (Fn), known as the
CS-1 region. The tripeptide Leu-Asp-Val (LDV) was identified as
the minimum sequence of the CS-1 region of fibronectin necessary
extraordinary affinity between biotin and avidin/streptavidin
(KD = 10À14 to 10À15 M),13 LDV peptidomimetics could indeed en-
able the removal of malignant white blood cells in people with
leukemia or hyperleukocytosis. Leukapheresis could also be useful
when debulking of circulating leukocytes is advisable before lytic
therapy. More generally, biotin-conjugated LDV peptidomimetics
have the potential to be exploited to image in vivo
a4b1 integrin-
expressing tumor cells or for the production of dedicated drug
targeting systems.
We decided to synthesize small molecules that mimic the LDV
motif recognized by the
a4b1 integrin. These molecules were
equipped with spacer-arms to introduce a minimum spacing be-
tween the biotin and the peptidomimetic core and to favor the
availability of the biotin/avidin binding without altering the pepti-
domimetic recognition by the integrin.14
to bind
medicinal chemistry for the design of small molecules or pepti-
a
4b1 integrin.6 This sequence was the starting point in
domimetics, antagonists of the
of the interaction between the
a
a
4b1 integrin.7,8 Indeed, inhibition
4b1 integrin and its ligands may
We began our study by the synthesis of peptides 1 and 2
prevent the recruitment and tissue infiltration of leukocytes and
may thus be a potential treatment for inflammatory, cancer and
autoimmune pathologies.9,10 This approach has been validated
with the approval of the humanized a4 monoclonal antibody
‘natalizumab’ for the treatment of multiple sclerosis and Crohn’s
disease.11
containing the active LDV sequence, recognized by the a4b1 inte-
grin, and a diaryl urea cap on the N-terminal function (Fig. 1).
Lin et al. have previously demonstrated the possibility to increase
the binding affinity towards this integrin by the introduction of a
similar motif.7 The C-terminal function was protected with a phen-
ethyl amide or with a hydrophobic spacer-arm. The molecules 1
and 2 were prepared according to the classical peptide synthesis
methods in solution (see Supplementary data).7,15
In this study, we were interested in the development of biotin-
conjugated LDV peptidomimetics for the detection/capture of
The activity of these two molecules towards the
a4b1 integrin
was evaluated in a standard in vitro cell adhesion assay, consisting
in the inhibition of leukemia cell adhesion on plates coated with
⇑
Corresponding author. Tel.: +32 10 47 27 46; fax: +32 10 47 41 68.
E-mail address: jacqueline.marchand@uclouvain.be (J. Marchand-Brynaert).
fibronectin, the natural ligand of the
a4b1 integrin. The human
These authors have equally contributed to the work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.078

E. Gérard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 586–590
587
3-(5-amino-2-hydroxyphenyl)propanoate template, such as the
peptidomimetic 3 (Scheme 1)15 structurally close to compounds
of the Biogen group.17 Starting from 3, we envisaged the synthesis
O
OH
O
O
O
O
H
N
H
N
of the two peptidomimetics 6 and 7 equipped with
a,x-diamino
N
H
N
H
O
O
spacer-arms. Our general synthetic approach to obtain the mole-
cules 6 and 7 is outlined in Scheme 1 (see also Supplementary
data). Two spacer-arms, featuring different hydrophilicities but
the same number of carbon atoms, were introduced on compound
3. An alkyl chain 4 and an oligoethylene glycol (OEG) 5 were cho-
sen to study the effect of the nature of the linker on the adhesion of
CCRF-CEM cells. Ethylene glycol oligomers are usually considered
due to their hydrophilic character, repulsive effect versus non-spe-
cific adsorption of proteins and to improve the solubility of mole-
cules.18 Moreover, previous studies have shown that the
introduction of a hexaethylene glycol chain (12 carbons) enabled
to maintain the activity of our lead peptidomimetics, antagonists
O
O
O
O
N
H
N
H
1
OH
H
N
H
N
N
H
NH2
n = 10
N
H
O
N
H
N
H
2
a
vb3 integrin.19 The synthesis of the two linkers 4 and 5,
(TFA salt)
of the
mono-protected at one end by a tert-butyl carbamate (Boc), were
described in Supplementary data.
Figure 1. LDV modified peptides 1 and 2.
20,21
Thetwodifferentspacer-armswerecoupledtothepeptidomime-
tic core 3 after activation with COMU (1-[(1-(cyano-2-ethoxy-2-
oxo-ethylideneaminooxy)-dimethylamino-morpholinomethylene)]
methanaminiumhexafluorophosphate)inthepresenceofN,N-diiso-
propylethylamine(DIPEA).22 Thiscouplingagent,chosentofacilitate
the work up and the purification, gave the desired products with
good to moderate yields (80% and 55% for compounds with the alkyl
and the OEG chains, respectively). Then, simultaneous deprotection
oftheBoccarbamateandthetert-butylesterwithtrifluoroaceticacid
(TFA) afforded the target compounds 6 and 7 quantitatively.
A reference molecule 11, featuring the diaryl urea motif solely
and the alkyl chain, was prepared by conventional chemistry, as
described in Scheme 2 (see also Supplementary data). The diaryl
urea molecule 10 was obtained by reaction of 2,6-dimethylphenyl
isocyanate with p-aminophenylacetic acid (see Supplementary
data).23 The coupling of 10 and the spacer-arm 4 was then per-
formed with PyBOP (benzotriazolyloxy-tris(pyrrolidino)-phospho-
nium hexafluorophosphate) as activation agent, and in presence
of triethylamine, with a yield of 89%.24 Lastly, reaction with triflu-
oroacetic acid gave quantitatively the compound 11.
T lymphoblast-like cell line CCRF-CEM expressing the a4b1 integrin
(derived from a child with acute lymphoblastic leukemia) was cho-
sen for this assay among different leukemia cells; in preliminary
experiments, attachment of this cell line to fibronectin was indeed
specifically inhibited by anti-
we also used quiescent human umbilical vein endothelial cells
(HUVEC) which do not express 4b1 integrin and the adhesion of
a4 or anti-b1 antibody. As a control,
a
which on fibronectin failed to be prevented by anti-
a4b1 integrin
antibodies (not shown).
The pre-labeling of CCRF-CEM cells with calcein allowed track-
ing by fluorescence measurements, the number of adherent cells
after 1 h incubation at 37 °C with increasing amounts of our mol-
ecules (see Supplementary data). The results showed an inhibition
activity of the LDV modified peptides 1 and 2 at the micromolar le-
vel (Table 1) with a prominent activity of compound 2 (equipped
with the spacer-arm) reaching an IC50 value of 2
this activity was obtained without addition of Mn2+ divalent cat-
ions, a treatment necessary to activate 4b1 integrin in normal
lymphocytes.16 This observation confirms the constitutive activa-
tion of 4b1 integrin in CCRF-CEM as observed in many leukemia
lM. Interestingly,
a
The inhibition activities of the peptidomimetics 6 and 7 on the
CCRF-CEM cells adhesion were also determined (Table 1). Com-
pound 6, equipped with the alkyl chain, was a better inhibitor than
compound 7 with an OEG chain (p <0.05), revealing an important
influence of the nature of the linker. Indeed, the reference com-
a
cells and thereby further supports the potential of LDV peptidom-
imetics to specifically interfere with leukemia cells.
From this encouraging result, that is, no loss of activity by the
incorporation of a spacer-arm, we decided to synthesize small mol-
ecules that mimic the LDV peptide 2. Indeed, peptidomimetics are
usually preferred because of their better stability towards enzy-
matic hydrolysis and their easiness of synthesis, purification and
characterization.
pound 11 was also slightly more active than compound
7
(although not statistically significant, p = 0.07) and deletion of
the spacer-arm in 11 resulted in a loss of activity (see compound
10). The activity of compound 11 seems to result of the hydropho-
bic character of the linker. Moreover, peptidomimetic 6 was able to
detach the CCRF-CEM cells which had previously adhered on
fibronectin during 24 h, contrary to 7 (vs the inhibition of the
We have previously reported the synthesis of a series of good
antagonists of the
a4b1 integrin, based on the methyl
Table 1
Half-maximal inhibitory concentrations (IC50) of the different compounds on the adhesion of
a4b1-expressing CCRF-CEM cells
a
Compound
Description
IC50 (lM)
1
2
Peptide LDV with a phenethyl chain
Peptide LDV with an alkyl chain
36.3
2.6
3
Peptidomimetic LDV
68.4
6
7
9
10
11
12
Peptidomimetic LDV with an alkyl chain
Peptidomimetic LDV with an OEG chain
Biotinylated peptidomimetic LDV with an alkyl chain
Diaryl urea cap
Diaryl urea cap with an alkyl chain
Biotinylated diaryl urea cap with an alkyl chain
123.0
169.8
14.1
>1000
151.4
97.7
a
Inhibition of adhesion of CCRF-CEM leukemia cells to fibronectin (10
of the listed compounds (1–500 M) (see examples in Fig. S1).
lg/mL) was measured after 1h incubation at 37 °C in the presence or the absence of increasing doses
l

588
E. Gérard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 586–590
H
N
O
O
O
O
N
H
N
H
O
O
3
H2N
N
H
n = 10
O
O
OH
a,b
H
N
4
O
O
H
N
O
n = 4
H2N
a,b
O
O
O
5
OH
O
O
N
H
N
H
O
H
N
O
6
O
NH
(TFA salt)
OH
O
O
N
N
H
H
n = 10
O
S
7
NH2
O
NH
(TFA salt)
HN
H
NH
H
c
O
O
O
N
O
O
O
8
n = 4
H
N
NH2
O
OH
O
O
N
H
N
H
O
O
NH
9
n = 10
NH
O
S
H
H
HN
NH
O
Scheme 1. Reagents and conditions: (a) COMU, DIPEA, DMF; (b) TFA, DCM; (c) Et3N, DMF.
H
NH2
n = 10
N
OH
a,b
O
O
O
O
4
N
H
N
H
N
N
H
H
11
10
(TFA salt)
8
c
O
HN
NH
H
H
H
N
H
N
S
O
O
O
n = 10
12
Scheme 2. Reagents and conditions: (a) PyBOP, Et3N, DMF; (b) TFA, DCM; (c) Et3N, DMF.
N
N
H
H

E. Gérard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 586–590
589
A
75
compound 12
compound 9
50
25
0
-25
0
.0
.
.0
0
0.1
1.0
0.1
1
1
1D0 ose (µM)
B
Control
Compound 12
Compound 9
Figure 2. (A) Adhesion of calcein-labeled CCRF-CEM cells to biotinylated molecules 9 and 12. (B) Pictures obtained after incubation of biotinylated molecules 9 and 12 with
CCRF-CEM cells adhering on plates coated with fibronectin (10
addition of fluorescent streptavidin).
lg/mL) and followed by the addition of Alexa 568-conjugated streptavidin (Control = not pre-treated, only
adhesion process itself as determined above). This observation
supports the higher capacity of compound 6 to interfere with the
In conclusion, we have described the synthesis of two LDV pep-
tidomimetics (compounds and 7) equipped with different
6
a
4b1 integrin/fibronectin interaction (see Fig. S2).
Due to its better activity, peptidomimetic 6 was then chosen for
spacer-arms. The most active compound 6, in cell adhesion assays,
featured C12 alkyl chain as linker, which was then conjugated with
biotin. The resulted biotinylated molecule 9 acts as a good inhibitor
the introduction of a biotin molecule on the amine end-function of
the spacer-arm. Biotinylation of 6 and the reference 11 (Schemes 1
and 2) was performed in presence of triethylamine, by reaction
of the
a4b1 integrin-expressing CCRF-CEM cells adhesion on fibro-
nectin (IC50 = 14
l
M). Moreover, we have documented the capacity
with
D
-biotin 8 pre-activated in the form of a N-hydroxysuccinim-
of this peptidomimetic to bind at the surface of the CCRF-CEM cells
and to promote their adhesion on neutravidin-coated support.
Thus, the interaction of biotin-conjugated compound 9 with strep-
tavidin/avidin has the potential to be exploited in biotechnological
applications aiming to label or capture leukemia cells expressing
idyl (NHS) ester (see Supplementary data).25,26 The biotinylated
molecules 9 and 12 were obtained with moderate yields (58%
and 54% respectively).
Compound 9 was a good inhibitor of the adhesion of the CCRF-
CEM cells to fibronectin with an IC50 value of 14
lM, comparatively
the a4b1 integrin.
to 98 M for the reference 12 (Table 1). The addition of biotin im-
l
proved the activity of the initial compounds 6 and 11. The biotin
can favor the solubility of the molecules in the testing conditions
or induce physico-chemical effects.
A propidium iodide-based assay was further used to determine
a possible toxicity of compounds 9 and 12. For that purpose, CCRF-
CEM cells were incubated for 24 h with increasing amounts of 9
and 12 and then stained with fluorescent propidium iodide to
Acknowledgments
This work has been supported by MacoPharma (Mouvaux,
France) and the F.R.S.-FNRS (Belgium). J. Marchand-Brynaert and
O. Feron are FRS-FNRS Research Directors.
Supplementary data
detect dead cells. At 200 lM (final concentration), the results did
not show any significant cytotoxicity of these molecules (Fig. S3
and Supplementary data).
Interestingly, theadhesionofcalcein-labeledCCRF-CEMcellswas
increased when neutravidin-coated plates were pre-treated with
biotin-conjugated peptidomimetic 9( Fig. 2A and Supplementary
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.10.078.
References and notes
data). Saturation of the signal was however obtained with 1 lM of
1. Hemler, M. E.; Elices, M. J.; Parker, C.; Takada, Y. Immunol. Rev. 1990, 114, 45.
2. Stewart, M.; Thiel, M.; Hogg, N. Curr. Opin. Cell. Biol. 1995, 7, 690.
3. Chavakis, E.; Choi, E. Y.; Chavakis, T. Thromb. Haemost. 2009, 102, 191.
4. Woodside, D. G.; Vanderslice, P. Biodrugs 2008, 22, 85.
5. Elices, M. J.; Osborn, L.; Takada, Y.; Crouse, C.; Luhowskyj, S.; Hemler, M. E.;
Lobb, R. R. Cell 1990, 60, 577.
compound 9. Of note, the negative calcein signal from plates treated
with the reference compound 12 (i.e., lower than in control (un-
treated) conditions) suggests repulsion between this compound
and the cells.
6. Komoriya, A.; Green, L. J.; Mervic, M.; Yamada, S. S.; Yamada, K. M.; Humphries,
M. J. J. Biol. Chem. 1991, 266, 15075.
7. Lin, K. C.; Ateeq, H. S.; Hsiung, S. H.; Chong, L. T.; Zimmerman, C. N.; Castro, A.;
Lee, W. C.; Hammond, C. E.; Kalkunte, S.; Chen, L. L.; Pepinsky, R. B.; Leone, D.
R.; Sprague, A. G.; Abraham, W. M.; Gill, A.; Lobb, R. R.; Adams, S. P. J. Med.
Chem. 1999, 42, 920.
In another assay, adherent CCRF-CEM cells (on fibronectin coated
plates), were allowed to interact with the biotin-conjugated mole-
cules 9 and 12 at 100 lM concentration, for 24 h (Supplementary
data). After extensive washing, fluorescent Alexa-conjugated strep-
tavidin was then added to detect the presence of peptidomimetics
on the cell surface (Fig. 2B). Only cells pre-treated with peptidomi-
metic 9 showed an extracellular fluorescent signal. An intracellular
signal was observed in cells exposed or not to the molecules, reflect-
ing the known unspecific binding of streptavidin to some cytosolic
proteins.
8. Tilley, J. W. Expert Opin. Ther. Patents 2008, 18, 841.
9. Vanderslice, P.; Woodside, D. G. Expert Opin. Investig. Drugs 2006, 15, 1235.
10. Xang, G. X.; Hagmann, W. K. Med. Res. Rev. 2003, 23, 369.
11. Steinman, L. Nat. Rev. Drug Disc. 2005, 4, 510.
12. Peng, Li.; Liu, R.; Marik, J.; Wang, X.; Takada, Y.; Lam, K. S. Nat. Chem. Biol. 2006,
2, 381.
13. Diamandis, E. P.; Cristopoulos, T. K. Clin. Chem. 1991, 37, 625.

590
E. Gérard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 586–590
14. Thomas, J. D.; Hofer, T.; Rader, C.; Burke, T. R. Bioorg. Med. Chem. Lett. 2008, 18,
5785.
15. Gérard, E.; Meulle, A.; Feron, O.; Marchand-Brynaert, J. Med. Chem. Commun.
2012. doi:10.1039/c1md00229e.
16. Chen, L. L.; Whitty, A.; Lobb, R. R.; Adams, S. P.; Pepinsky, R. B. J. Biol. Chem.
1999, 274, 13167.
20. Dardonville, C.; Fernandez-Fernandez, C.; Gibbons, S. L.; Ryan, G. J.; Jagerovic,
N.; Gabilondo, A. M.; Meana, J. J.; Callado, L. F. Bioorg. Med. Chem. 2006, 14,
6570.
21. Moreau, J.; Marchand-Brynaert, J. Eur. J. Org. Chem. 2011, 1641.
22. El-Faham, A.; Funosas, R. S.; Prohens, R.; Albericio, F. Chem. Eur. J. 2009, 15,
9404.
17. Singh, J.; van Vlijmen, H.; Liao, Y.; Lee, W. C.; Cornebise, M.; Harris, M.; Shu, I.
H.; Gill, A.; Cuervo, J. H.; Abraham, W. M.; Adams, S. P. J. Med. Chem. 2002, 45,
2988.
18. Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Angew. Chem., Int. Ed.
2010, 49, 6288.
23. Liu, R.; Peng, L.; Han, H.; Lam, K. S. Peptide Sci. 2006, 84, 595.
24. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31, 205.
25. Jiang, X.; Deng, Z.; Narain, R. Bioconjugate Chem. 2009, 20, 994.
26. He, B.; Velaparthi, S.; Pieffet, G.; Pennington, C.; Mahesh, A.; Holzle, D. L.;
Brunsteiner, M.; van Breemen, R.; Blond, S. Y.; Petukhov, P. A. J. Med. Chem.
2009, 52, 7003.
19. Rerat, V.; Dive, G.; Cordi, A. A.; Tucker, G. C.; Bareille, R.; Amédée, J.; Bordenave,
L.; Marchand-Brynaert, J. J. Med. Chem. 2009, 52, 7029.