Solid-phase synthesis of CD40L mimetics

Article abstract of DOI:10.1039/b601528j

The solid phase synthesis of CD40L mimetics and their binding properties and effector functions were analyzed. C₃ symmetry is potentially useful to evaluate the importance of valency in the interaction of CD40L mimetics with CD40 and to investi

Full text of DOI:10.1039/b601528j

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
Solid-phase synthesis of CD40L mimetics†
Alberto Bianco,* Sylvie Fournel, Se´bastien Wieckowski, Johan Hoebeke and Gilles Guichard*
Received 31st January 2006, Accepted 28th February 2006
First published as an Advance Article on the web 10th March 2006
DOI: 10.1039/b601528j
The C3-symmetric molecule 1 has been previously shown to
mimic CD40 ligand (CD40L) homotrimers and to display
effector functions. This molecule consists of a cyclic hexapep-
tide core containing the repetition of the D-Ala-L-Lys motif.
The side chains of the lysine residues have been modified by
appending the CD40L-derived sequence 143Lys-Gly-Tyr-Tyr146
via a 6-aminohexanoic acid residue as a spacer. The present
report describes a general solid-phase synthesis approach to
1 and related trimeric architectures. In addition, their CD40
binding properties as well as their effector functions have been
evaluated.
The interaction between CD40, a member of the tumour necrosis
factor receptor (TNF-R) superfamily, and its ligand CD40L is
Fig. 1 Molecular structures of CD40L mimetics.
key to the differentiation and activation of antigen presenting
cells (APCs) as well as T and B lymphocytes.1 Blockade or
activation of this pathway represents a potentially useful approach
to immunomodulate T-cell-mediated diseases or to enhance
anti-infective/anti-tumour host defence, respectively.2,3 Signalling
through CD40 and other TNF-R members involves ligand-
induced receptor trimerisation and the formation of a 3 : 3
hexameric ligand–receptor complex.4 In this context, we have
described rationally designed CD40L mimetics (<3 kDa) that
can both interact with CD40 and display effector functions.5
The design of these small molecules was based on the crystal
structure of CD40L homotrimers and a homology model of the
CD40L/CD40 complex.6,7 The three-fold axis of the hexameric
complex suggested to conceive trimeric architectures with C3
symmetry.
Large cyclic peptides can be considered as excellent cores
to assemble multivalent ligands.8 The preparation of this type
of structure is highly flexible and is amenable to solid-phase
synthesis.8 For the design of CD40L mimetics we focused on
macrocylic peptides with rigid ring conformation and three
arms at the twelve, four and eight o’clock positions that could
promote radial distribution of a short CD40 binding segment.
Crystallographic studies have shown that cyclic hexapeptides
consisting of alternated L- and D-amino acid residues meet these
criteria and are able to adopt the expected symmetric C3 spatial
arrangement.9–11 For this reason, we prepared a series of molecules
based on the cyclo-(L-Lys-D-Ala)3 core structure (Fig. 1).
The synthesis of molecules 1–3 was performed entirely on a
solid support.12 We followed the strategy for the head-to-tail
cyclisation on polymer beads using Fmoc/t-Bu chemistry.13,14 We
initially functionalised 2-(3,5-dimethoxy-4-formylphenoxy)ethyl
polystyrene resin with the D-Ala residue protected at the carboxylic
group as an allyl ester under reductive amination conditions
(Scheme 1).13 The secondary amine 5 was coupled with Fmoc-
L-Lys(Mtt)-OH, activated with triphosgene in the presence of
collidine.15 Because dipeptide esters made of heterochiral residues
are particularly prone to fast 2,5-diketopiperazine formation,16
we did not attempt to remove the Fmoc group of 6. Instead, the
synthesis was continued towards the C-terminus. The allyl group
(All) was removed by treatment with Pd(Ph3)4 and H-L-Lys(Mtt)-
OAll was coupled to give tripeptide 8. At that stage, the elongation
was continued from the N-terminus to give hexamer 10.17 The
N- and C-protections of the linear peptide were subsequently
removed and cyclisation to 13 was achieved by activation with
DIC (diisopropylethylcarbodiimide) and HOAt (1-hydroxy-7-
azabenzotriazole). The Mtt (4-methyltrityl) groups on the three
lysine side chains were removed in mild acid conditions and the
synthesis of 1 was terminated using Fmoc/t-Bu chemistry and
standard coupling procedures.† 6-Aminohexanoic acid (Ahx) was
inserted as a spacer to assure a favourable interaction between
the CD40L-derived 143Lys-Gly-Tyr-Tyr146 loop sequence and the
residues of the CD40 receptor. After the cleavage from the resin,
the crude compound 1 was obtained in good yield and fairly
good purity (Fig. 2A). After purification by semi-preparative RP-
HPLC (Fig. 2B), we isolated ligand 1 in 16% yield, subsequently
characterised by MALDI-tof.
Institut de Biologie Mole´culaire et Cellulaire, UPR 9021 CNRS, Im-
munologie et Chimie The´rapeutiques, 15 Rue Rene´ Descartes, 67084 Stras-
bourg, France. E-mail: A.Bianco@ibmc.u-strasbg.fr, G.Guichard@ibmc.u-
strasbg.fr; Fax: +33 388 610680; Tel: +33 388 417088 (AB); Tel: +33 388
417025 (GG)
During the initial solid-phase peptide synthesis (SPPS) opti-
misation phase, we were able to identify and separate the ligand
2 (Fig. 1), which was not expected and presented one missing
sequence in one of the arms due to an incomplete removal of
Mtt groups (Scheme 1). This dimeric analogue of 1 lacking
† Electronic supplementary information (ESI) available: detailed experi-
mental procedures. See DOI: 10.1039/b601528j
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 1461–1463 | 1461
©

View Article Online
Scheme 1 Solid-phase synthesis of CD40L mimetics.
The capacity of the CD40L mimetic 1 to interact with CD40
was previously evaluated with surface plasmon resonance (SPR).5
SPR experiments showed a concentration-dependent competition
with binding of recombinant CD40L to CD40. The mean in-
hibitory concentration of 1 corresponded to 78 nM.5 Here we
have measured the inhibitory activity of dimeric ligand 2. The
measurements were performed by immobilising the recombinant
CD40 onto the sensor chip and subsequently injecting a solution
of 0.15 lM of CD40L alone, mixed to 0.1 or 1 lM of 2.† As a
control, we have tested in the same experimental trimeric ligand
1 at 1 lM. Fig. 3 shows the sensorgrams of the competition
Fig. 2 RP-HPLC chromatograms of crude (A) and purified (B) 1.
C3 symmetry is potentially useful to evaluate the importance
of valency in the interaction of CD40L mimetics with CD40
and to investigate possible cooperativity effects.18 Finally, the
circular core 3 was obtained directly by treating part of the
cyclohexapeptide resin 14 with TFA (Scheme 1).
Fig. 3 Inhibition of CD40L binding to CD40 by ligand 1 and 2. Red:
0.15 lM CD40L alone; blue: addition of 0.1 lM of 2; cyan: addition of
1 lM of 2; green: addition of 1 lM of 1.
1462 | Org. Biomol. Chem., 2006, 4, 1461–1463
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
assay. Although ligand 2 is missing one CD40 binding motif
compared to 1, it still displays an inhibitory effect at the highest
concentration. In contrast to 1, however, the inhibition by 2 at
1 lM is not complete (about 40%). This result highlights how
the concomitant presence of the three CD40 binding motifs is
critical for an efficient interaction with CD40. We have formerly
shown that ligand 3, where the three H-Lys-Gly-Tyr-Tyr-Ahx-
OH sequences were completely absent, was unable to block the
formation of the CD40/CD40L complex.5
Finally, we investigated whether or not ligand 2 displays effector
function. Our cellular assay was based on the property of Burkitt
lymphoma cells to enter apoptosis after CD40 ligation.19 As
previously described,5 compound 1 as well as recombinant soluble
CD40L (data not shown) induced a high level of apoptosis of BL41
Burkitt lymphoma cells in a dose-dependent manner (Fig. 4). On
the contrary, the core structure 3 and the short Lys-Gly-Tyr-Tyr-
Ahx linear peptide 4,† used as negative controls, had no effect
(Fig. 4). Interestingly, compound 2 induced significant apoptosis,
suggesting that it could bind to CD40 and trigger an apoptotic
signalling. However, the apoptosis generated by compound 2
was reduced significantly compared with trimeric compound 1,
showing the importance of C3 symmetry in CD40L signalling.
This result is in agreement with the SPR data (Fig. 3), showing a
reduced inhibition capacity of ligand 2 in comparison to 1.
with CD40 as well as a structure–activity relationship should be
greatly facilitated. In addition, this method can be extended to the
conception of other types of mimetics involved in protein/protein
interactions within the family of TNF-R, to whom CD40 belongs.
This work was carried out with financial support of the CNRS
and the ‘Ministe`re de la Recherche’ (ACI ‘Jeunes Chercheurs’), ‘La
Ligue contre le Cancer, Re´gion Alsace’ and the ‘Agence Nationale
de Recherches contre le SIDA’. The authors thank Christophe
Bour for excellent technical assistance, Nathalie Bonnefoy-Berard
and Monique Flacher for providing Burkitt lymphoma cells.
Notes and references
1 C. van Kooten and J. Banchereau, J. Leukocyte Biol., 2000, 67, 17.
2 L. M. Howard, A. J. Miga, C. L. Vanderlugt, M. C. Dal Canto, J. D.
Laman, R. J. Noelle and S. D. Miller, J. Clin. Invest., 1999, 103, 281.
3 L. Diehl, A. T. den Boer, S. P. Schoenberger, E. I. van der Voort, T. N.
Schumacher, C. J. Melief, R. Offringa and R. E. Toes, Nature Med.,
1999, 5, 774.
4 J.-L. Bodmer, P. Schneider and J. Tschopp, Trends Biochem. Sci., 2002,
27, 19.
5 S. Fournel, S. Wieckowski, W. Sun, N. Trouche, H. Dumortier, A.
Bianco, O. Chaloin, M. Habib, J.-C. Peter, P. Schneider, B. Vray, R. E.
Toes, R. Offringa, C. J. Melief, J. Hoebeke and G. Guichard, Nature
Chem. Biol., 2005, 1, 377.
6 M. Karpusas, Y. M. Hsu, J. H. Wang, J. Thompson, S. Lederman, L.
Chess and D. Thomas, Structure (London), 1995, 3, 1031.
7 J. Singh, E. Garber, H. Van Vlijmen, M. Karpusas, Y. M. Hsu, Z.
Zheng, J. H. Naismith and D. Thomas, Protein Sci., 1998, 7, 1124.
8 Z. Zhang, J. Liu, C. L. M. Verlinde, W. G. J. Hol and E. Fan, J. Org.
Chem., 2004, 69, 7737.
9 V. Pavone, E. Benedetti, B. Di Balsio, A. Lombardi, C. Pedone, L.
Tomasich and G. P. Lorenzi, Biopolymers, 1989, 28, 215.
10 M. Saviano, A. Lombardi, C. Pedone, B. Di Blasio, X. C. Sun and G. P.
Lorenzi, J. Inclusion Phenom. Macrocycl. Chem., 1994, 18, 27.
11 V. Dartois, J. Sanchez-Quesada, E. Cabezas, E. Chi, C. Dubbelde, C.
Dunn, J. Granja, C. Gritzen, D. Weinberger, M. R. Ghadiri and T. R.
Parr, Jr., Antimicrob. Agents Chemother., 2005, 49, 3302.
12 M. Goodman, A. Felix, L. Moroder and C. Toniolo, Houben–Weyl
Methods of Organic Chemistry, Thieme, Stuttgart, 2002, vol. E22a.
13 K. J. Jensen, J. Alsina, M. F. Songster, J. Vagner, F. Albericio and G.
Barany, J. Am. Chem. Soc., 1998, 120, 5441.
Fig. 4 Induction of apoptosis by compound 1 and 2. BL41 cells (5 ×
105 cells mL−1) were incubated with compound 1, 2, 3 and 4 at the indicated
concentrations for 16 h. Apoptosis was then measured by detection of
phosphatidylserine externalisation by flow cytometry after co-labelling
with annexin V-FITC and propidium iodide (PI). Results are expressed as
percentage of specific apoptosis (see ESI†).
14 A. Johansson, E. Akerblom, K. Ersmark, G. Lindeberg and A.
Hallberg, J. Comb. Chem., 2000, 2, 496.
15 E. Falb, T. Yechezkel, Y. Salitra and C. Gilon, J. Peptide Res., 1999, 53,
507.
16 M. Del Fresno, J. Alsina, M. Royo, G. Barany and F. Albericio,
Tetrahedron Lett., 1998, 39, 2639.
17 P. Lloyd-Williams, F. Albericio and E. Giralt, Tetrahedron, 1993, 49,
11 065.
In conclusion, we have successfully developed a strategy for the
solid-phase total synthesis of CD40L mimetics. As this approach
is highly versatile, the design of new combinatorial ligands
containing different peptide sequences involved in the interaction
18 M. Mammen, S. K. Choi and G. M. Whiteside, Angew. Chem., Int. Ed.,
1998, 37, 2755.
19 A. W. Tong, B. Seamour, J. Chen, D. Su, G. Ordonez, L. Frase, G. Netto
and M. J. Stone, Leuk. Lymphoma, 2000, 36, 543.
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 1461–1463 | 1463
©