Improving implant materials by coating with nonpeptidic, highly specific integrin ligands

Article abstract of DOI:10.1002/anie.200460770

Giving cells some stick: Osteoblast adhesion to titanium, a common material for implants, is stimulated by coating with an optimized and highly specific nonpeptidic ligand for the αvβ3 integrin (see picture). This new technology is advantageous to conventional coating by peptides or proteins in many practical aspects (selectivity and activity, stability against enzymatic degradation, sterilization, costs).

Full text of DOI:10.1002/anie.200460770

Angewandte
Chemie
Biomaterials
Improving Implant Materials by Coating with
Nonpeptidic, Highly Specific Integrin Ligands**
Claudia Dahmen, Jörg Auernheimer, Axel Meyer,
Anja Enderle, Simon L. Goodman, and Horst Kessler*
Surface modification for enhanced cell adhesion to improve
the properties of the critical interphase between an implant
material and the biological tissue is an ongoing issue. Despite
considerable improvements there is still a challenge to
optimize the following criteria: the efficiency in stimulating
cell adhesion, the selectivity for a specific cell type (e.g.
osteoblast vs. platelet adhesion), the stability under physio-
logical conditions, the stable covalent attachment to the
material, the ease of handling under sterile conditions, and
reasonable costs for the coating. Herein we present a new
solution to these problems by using anchored nonpeptidic,
highly av-selective integrin ligands to coat titanium, a
common implant material.
Osteoblast adhesion can be stimulated by extracelullar
matrix (ECM) proteins (e.g. fibronectin, collagen, laminine,
and bone sialo protein),[1] their fragments, or by RGD
peptides[2] which bind to avb3 integrin on osteoblast cells
but bind to the platelet integrin aIIbb3 as well.[3] Selectivity
for avb3 integrin could be achieved by using optimized cyclic
pentapeptides.[4–6] Coating poly(methyl methacrylate)
(PMMA) with suitable modified cyclic pentapeptides stim-
ulates osteoblast adhesion in vitro[7] and bone formation in
PMMA granulates in vivo (rabbit).[8]
A number of nonpeptidic av-selective RGD mimics have
been developed by us and others[9–12] as potential drugs to
treat cancer, osteoporosis, acute renal failure, restenosis,
arthritis, and retinopathy.[13–18] Recently the X-ray structure of
the avb3 head group containing the cyclic peptide cilengi-
tide[5] was reported.[19] Modeling studies on the nonpeptidic
avb3 ligands elucidated their binding mode.[20] We used this
data to identify suitable positions for anchor groups (linkers)
[*] Dr. C. Dahmen, Dipl.-Ing. J. Auernheimer, Dipl.-Chem. A. Meyer,
Prof. Dr. H. Kessler
Department Chemie
Lehrstuhl Organische Chemie II
Technische Universität München
Lichtenbergstrasse 4, 85747 Garching (Germany)
Fax: (+49)89-289-13210
E-mail: kessler@ch.tum.de
Dr. A. Enderle
Biomet Merck BioMaterials GmbH, Forschung
Frankfurter Strasse F129/250, 64271 Darmstadt (Germany)
Dr. S. L. Goodman
Merck KGaA, Oncology Research
Frankfurter Strasse 250, 64271 Darmstadt (Germany)
[**] This work was supported by the DFG, Sonderforschungsbereich
563, and by the German Federal Ministry of Education and Research
under No. 03N4012.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2004, 43, 6649 –6652
DOI: 10.1002/anie.200460770
ꢀ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 6649

Communications
that could be used to attach ligands to the
surface without interfering with integrin
binding. The guanidine and carboxy
groups of the ligand are essential for binding
to the integrin subunits a and b, respec-
tively.[21] Therefore we chose the two aro-
matic rings of our highly avb3-selective
diacylhydrazine scaffold[10] to position the
anchor groups (Scheme 1). By using the
AutoDock3 program[22,23] two mimetics with
different anchors at R1 and R2 were mod-
eled into the X-ray structure of the avb3–
cilengitide complex[19] after removal of the
peptide ligand. The binding modes were
identical to those of the anchor-free mimetic
(R1 = R2 = H; Figure 1),[20] and the linkers
showed no disturbing interaction with the
integrin, hence we synthesized both variants
with different linker groups.
Scheme 1. Substituted nonpeptidic diacylhydrazines with possible linker
Scheme 2. Synthesis of R1 substituted diacylhydrazines on a solid
phase. a) NH4OAc (2 equiv), malonic acid mono-tert-butyl ester
(1 equiv), EtOH; b) Fmoc-Cl (1.05 equiv), NaHCO3, dioxane; c) TCP-
resin, CH2Cl2, DIEA; d) 20% piperidine in NMP; e) COCl2 (3 equiv;
1.9m solution in toluene), sat.NaHCO3, CH2Cl2;[24] f) 5-(9-H-fluoren-9-
ylmethoxy)-1,3,4-oxadiazol-2-(3H)-one (4 equiv), DMF; g) 20% piperi-
dine in NMP; h) 3-(N-Fmoc)-aminobenzoic acid (2 equiv), HATU
(1.94 equiv), collidine (22 equiv), NMP; i) 20% piperidine in NMP;
k) N,N’-bis(Boc)guanylpyrazole (10 equiv), CHCl3, 508C; l) 20% HFIP
in CH2Cl2; m) linker molecule (1 equiv), HATU (0.97 equiv), HOAt
(1.1 equiv), collidine (11 equiv), DMF; n) 50% TFA, 2% triisopropyl
silane,[30] 2% water in CH2Cl2. DIPEA=N,N-diisopropylethylamine.
positions R1 and R2 for anchoring to surfaces.
Synthesis was performed on solid support (trityl chloride
polystyrene resin = TCP-resin) by an Fmoc strategy (Fmoc =
9-fluorenylmethoxycarbonyl) similar to that described else-
where.[9,10,24] Starting from substituted b-amino acid immobi-
lized on the resin, carbonylated Fmoc-protected hydrazine as
the aza-glycine precursor[24] and 3-(N-Fmoc)aminobenzoic
acid were coupled. Guanidine was successfully incorporated
using an excess of N,N’-bis(Boc)guanylpyrazole (Boc = tert-
butoxycarbonyl; Scheme 2). After cleavage (hexa-
fluoroisopropanol (HFIP)/CH2Cl2) the resulting Boc/OtBu-
protected compound was coupled in solution on position R1
with two thiol linkers of different length—cysteamine and 6-
aminohexanoyl-6’-aminohexanoyl-6’’-aminohexanoyl
cys-
teine—and deprotected by trifluoroacetic acid (TFA). Puri-
fication was done by reverse-phase (RP) HPLC. The high
avb3 affinity of all the mimetics, with linkers (2, 3) or not (1),
was demonstrated in an established IC50 assay.[10,25] Whereas
the dicarboxy compound 1 is only active for the avb3 integrin
in the nanomolar range, the linked molecules are biselective
Figure 1. Results of the docking studies: Two diacylhydrazine mole-
cules with anchors on the aromatic rings (stylized as yellow spheres)
modeled into the crystal structure of the avb3 integrin head group.[19]
6650
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Angew. Chem. Int. Ed. 2004, 43, 6649 –6652

Angewandte
Chemie
for avb3 and avb6 integrins (Table 1) as described for other
nonpeptides of the diacylhydrazine type.[9]
Steric effects cannot be the reason for the high avb3
selectivity of compound 1 as compounds 1–3 are equally
Substances 2–4 were tested for their cell adhesion proper-
ties on surfaces, the thiol linkers enable irreversible immobi-
lization on titanium (an implant material). MC3T3E1 mouse
osteoblasts, expressing the avb3 integrin,[8] were seeded onto
the modified titanium discs
(Ti6Al4V, 11 cm), after 1 h the
Table 1: Ligand affinities of unanchored (free) nonpeptidic RGD mimetics to different integrins.
number of adherent cells was mea-
sured by detecting the hexamini-
dase activity.[27] Our study indicates
that mouse osteoblasts bind to
surfaces coated with compounds 2
or 3 (Figure 2). The plating effi-
ciency was increased up to 42.9%
(100 mm compound 3 in coating
solution) compared to 9.4% on
the unmodified titanium. Com-
pound 3 stimulates cell adhesion
as efficiently as the cyclo-
(-RGDfK[3-mercaptopropionyl]-)
peptide.[28] Compound 2 is slightly
less potent, probably caused by the
Compound
IC50 [nm][a]; inhibition at ligand concentration [nm]
R1
R2
avb3
avb5
avb6
aIIbb3
1
2
3
4
COOH
H
H
H
16
5.7
0.72
0.84
104 (65%)
200
310
103 (54%)
0.24
2.35
104 (22%)
3.20”103
3.15”103
4.15”103
CONH-(CH2)2-SH
COAhx3Cys[b]
Cl
CH2NHAhx3-
245
0.089
CO-(CH2)2-SH[a]
5[10]
H
H
H
0.8
0.1
0.4
n.m.[c]
n.m.[c]
n.m.[c]
n.m.[c]
n.m.[c]
n.m.[c]
8500
5500
>104
6[10] Cl
cyclo(-RGDfK[3-mercaptopropionyl]-)[28]
[a] The data shown represent the mean of at least 2 independent IC50 determinations. As previously
documented the typical variation in such receptor–ligand inhibition measurements is routinely in the
order of the measured value itself.[29] [b] Ahx: 6-Aminohexanoic acid. [c] n.m.: not measured.
sterically demanding. Consistent with our theoretical studies
on avb5 homology models,[26] the residues Lys180 and
Asp252 of the SDL (selectivity determining loop) near the
MIDAS region could effect an unfavored reorien-
tation of ligand 1 in the binding pocket through
electrostatic interactions with the carboxylate.
There are uncharged residues at these positions in
the b3 subunit and this could explain the strong
impact of the carboxylic group in compound 1 for
inhibiting the binding in avb5.
significant shorter linker, which makes the integrin ligand less
accessible to the integrin. Compound 4, although having
comparable activity in the binding assay of isolated avb3
R2-linked compound 4 was synthesized by a
combined solution and solid-phase strategy
(Scheme 3). p-Chlorophenyl-substituted b-alanine
was chosen as the C-terminus of the molecule
because it is found in very potent avb3 integrin
ligands.[10] A complete solid-phase strategy could
not realized, because after coupling of 3-amino-5-
(N-Fmoc)aminomethylbenzoic acid stabilized with
4-methylbenzenesulfonic acid the coupling of e-(N-
Fmoc)-aminohexanoic acid after Fmoc deprotec-
tion (only possible with 2% 1,8-diazabicy-
clo[5.4.0]undec-7-ene (DBU)/2% piperidine in N-
methylpyrrolidone (NMP)) did not work (probably
caused by steric hindrance) even though different
coupling reagents have been used. Therefore 3-
aminomethyl-5-guanidinobenzoic acid was coupled
with 3-(S-Trt)-mercaptopropionyl-Ahx-Ahx-Ahx-
O H (Tr=t trityl) in solution. The product was
activated with O-(7-azabenzoictriazol-1-yl)-1,1,3,3-
tetramethyluroniumhexafluorphosphate (HATU)
and coupled on resin-bound 3-(4-chlorophenyl)-3-
[(hydrazinocarbonyl)-
amino] propionic acid. Cleavage from the resin with
Scheme 3. Synthesis of R2 substituted diacylhydrazine 4. a) SO2(NH2)2 (1.2 equiv), SOCl2
(3.6 equiv), sulfalone, 42 h reflux; b) Pd/C, H2, MeOH; c) SC(NHBoc)2 (1 equiv), NEt3
(4 equiv), HgCl2 (1.3 equiv), MeOH; d) Pd/C, H2 20 bar, 2m NH3/EtOH, 508C; e) LiOH
(3 equiv), MeOH/H2O; f) 40% aqua.TFA (v/v); g) linker molecule (1 equiv), HATU
(1 equiv), HOAt (1 equiv), collidine (10 equiv), DMF; h) HATU (1 equiv); i) 20% HFIP in
CH2Cl2; k) 40% TFA, 2% triisopropyl silane,[30] 2% water in CH2Cl2.
HFIP/CH2Cl2, complete deprotection (TFA/
CH2Cl2), and subsequent purification by RP-
HPLC gave in the integrin ligand 4 that is biselec-
tive for avb3/avb6 with an affinity in the sub-
nanomolar range (Table 1).
Angew. Chem. Int. Ed. 2004, 43, 6649 –6652
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Communications
[6] R. Haubner, D. Finsinger, H. Kessler, Angew. Chem. 1997, 109,
1440 – 1456; Angew. Chem. Int. Ed. Engl. 1997, 36, 1374 – 1389.
[7] M. Kantlehner, D. Finsinger, J. Meyer, P. Schaffner, A. Jonczyk,
B. Diefenbach, B. Nies, H. Kessler, Angew. Chem. 1999, 111,
587 – 590; Angew. Chem. Int. Ed. 1999, 38, 560 – 562.
[8] a) M. Kantlehner, P. Schaffner, D. Finsinger, J. Meyer, A.
Jonczyk, B. Diefenbach, B. Nies, G. Hölzemann, S. L. Goodman,
H. Kessler, ChemBioChem 2000, 1, 107 – 114; b) U. Hersel, C.
Dahmen, H. Kessler, Biomaterials 2003, 24, 4385-4415.
[9] C. Gibson, G. A. G. Sulyok, D. Hahn, S. L. Goodman, G.
Hölzemann, H. Kessler, Angew. Chem. 2001, 113, 169 – 173;
Angew. Chem. Int. Ed. 2001, 40, 165 – 169.
[10] G. A. G. Sulyok, C. Gibson, S. L. Goodman, G. Hölzemann, M.
Wiesner, H. Kessler, J. Med. Chem. 2001, 44, 1938 – 1950.
[11] G. Hölzemann, IDrugs 2001, 4, 72 – 81.
[12] J. S. Kerr, A. M. Slee, S. A. Mousa, Expert Opin. Invest. Drugs
2000, 9, 1271 – 1279.
[13] P. A. DꢀAmore, R. W. Thompson, Annu. Rev. Physiol. 1987, 49,
453 – 464.
Figure 2. Adhesion of MC3T3E1 mouse osteoblasts on uncoated and
[14] J. Folkman, Y. Shing, J. Biol. Chem. 1992, 267, 10931 – 10934.
[15] J. Folkman, Nat. Med. 1995, 1, 27 – 31.
coated titanium surfaces. The mean values of each point given is the
result of triplicate determinations, the error bars represent standard
deviations. [Pm]=mimic concentration in the coating solution.
[16] W. H. Miller, D. P. Alberts, P. K. Bhatnagar, W. E. Bondinell,
P. K. Callahan, R. R. Calvo, R. D. Cousins, K. F. Erhard, D. A.
Heerding, R. M. Keenan, C. Kwon, P. J. Manley, K. A. New-
lander, S. T. Ross, J. M. Samanen, I. N. Uzinskas, J. W. Venslav-
sky, C. C.-K. Yuan, R. C. Haltiwanger, M. Gowen, S.-M. Hwang,
I. E. James, M. W. Lark, D. J. Rieman, G. B. Stroup, L. M.
Azzarano, K. L. Salyers, B. R. Smith, K. W. Ward, K. O.
Johanson, W. F. Huffman, J. Med. Chem. 2000, 43, 22 – 26.
[17] M. W. Lark, G. B. Stroup, S. M. Hwang, I. E. James, D. J.
Rieman, F. H. Drake, J. N. Bradbeer, A. Mathur, K. F. Erhard,
K. A. Newlander, S. T. Ross, K. L. Salyers, B. R. Smith, W. H.
Miller, W. F. Huffman, M. Gowen, J. Pharmacol. Exp. Ther.
1999, 291, 612 – 617.
[18] P. A. Burke, S. J. DeNardo, L. A. Miers, K. R. Lamborn, S.
Matzku, G. L. DeNardo, Cancer Res. 2002, 62, 4263 – 4272.
[19] J.-P. Xiong, T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S. L.
Goodman, M. A. Arnaout, Science 2002, 296, 151 – 155.
[20] L. Marinelli, A. Lavecchia, K.-E. Gottschalk, E. Novellino, H.
Kessler, J. Med. Chem. 2003, 46, 4393 – 4404.
[21] K.-E. Gottschalk, H. Kessler, Angew. Chem. 2002, 114, 3919 –
3927; Angew. Chem. Int. Ed. 2002, 41, 3767 – 3774.
[22] G. M. Morris, D. S. Goodsell, A. J. Olson, AutoDock3 3.0 beta
ed., 1993.
[23] G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E.
Hart, R. K. Belew, A. J. Olson, J. Comput. Chem. 1998, 19, 1639 –
1662.
[24] C. Gibson, S. L. Goodman, D. Hahn, G. Hölzemann, H. Kessler,
J. Org. Chem. 1999, 64, 7388 – 7394.
integrin (Table 1), yielded no stimulation of osteoblast
adhesion when bound to the titanium surface in repeated
testing. An explanation could be that in spite of its huge linker
the immobilized ligand has an unfavored orientation for
integrin binding; the possibility that compound 4 does not
immobilize on titanium is unlikely since we have investigated
many peptidic derivatives of cyclo(-RGDfK[3-mercaptopro-
pionyl]-) in the past (unpublished data) and all of them coated
well on the titanium surfaces (checked by ELISA and cell
adhesion assays, data not shown). In the case of the RGD
mimic, immobilization could not be directly measured by
ELISA because the antibody used recognizes only the cyclic
RGD peptide and not the mimetics.
In conclusion, compounds 2 and 3 are the first nonpeptidic
av-selective integrin ligands for surface coating which exhibit
a potency for stimulated osteoblast adhesion similar to that of
cyclo(-RGDfK[3-mercaptopropionyl]-) when immobilized on
titanium. Compounds 2 and 3 are more stable to enzymatic
degeneration, pH variations, and heat and their synthesis is
much cheaper than that of the cyclic peptide.
Spectroscopic and analytical data for compounds 1–4 are
included in the Supporting Information.
[25] G. Thumshirn, U. Hersel, S. L. Goodman, H. Kessler, Chem. Eur.
J. 2003, 9, 2717 – 2725.
[26] L. Marinelli, K.-E. Gottschalk, A. Meyer, E. Novellino, H.
Kessler, J. Med. Chem. 2004, 47, 4166 – 4177.
[27] U. Landegren, J. Immunol. Methods 1984, 67, 379 – 388.
[28] B. Jeschke, J. Meyer, A. Jonczyk, H. Kessler, P. Adamietz, N. M.
Meenen, M. Kantlehner, C. Goepfert, B. Nies, Biomaterials
2002, 23, 3455 – 3463.
[29] S. L. Goodman, G. Hölzemann, G. A. G. Sulyok, H. Kessler, J.
Med. Chem. 2002, 45, 1045 – 1051.
Received: May 25, 2004
Keywords: cell adhesion · cell recognition · materials science ·
.
peptide mimetics · titanium
[1] J. M. Seeger, N. Klingman, J. Surg. Res. 1985, 38, 641 – 647.
[2] A. Wierzba, U. Reichl, R. F. B. Turner, R. A. J. Warren, D. G.
Kilburn, Biotechnol. Bioeng. 1995, 46, 185 – 193.
[30] D. A. Pearson, M. Blanchette, M. L. Baker, C. A. Guindon,
Tetrahedron Lett. 1989, 30, 2739 – 2742.
[3] E. Ruoslahti, Annu. Rev. Cell Dev. Biol. 1996, 12, 697 – 715.
[4] M. Aumailley, M. Gurrath, G. Müller, J. Calvete, R. Timpl, H.
Kessler, FEBS Lett. 1991, 291, 50 – 54.
[5] M. A. Dechantsreiter, E. Planker, B. Mathä, E. Lohof, G.
Hölzemann, A. Jonczyk, S. L. Goodman, H. Kessler, J. Med.
Chem. 1999, 42, 3033 – 3040.
6652
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Angew. Chem. Int. Ed. 2004, 43, 6649 –6652