Ligand Binding Analysis for Human r51
Integrin: Strategies for Designing New
r51 Integrin Antagonists
Luciana Marinelli,
‡
Axel Meyer,
†
Dominik Heckmann,
†
Antonio Lavecchia,
‡
Ettore Novellino,
‡
and Horst Kessler*
,†
Department Chemie, Technische Universita ¨ t Mu ¨ nchen,
Lichtenbergstrasse 4, D-85747 Garching, Germany, and
Dipartimento di Chimica Farmaceutica e Tossicologica,
Universita ` di Napoli “Federico II”, Via D. Montesano,
49-80131 Napoli, Italy
Received December 29, 2004
Abstract: We report a three-dimensional model of the R51
integrin headgroup bound to the most potent and selective
ligand (SJ749) known to date. The model was built using the
comparative protein modeling method, and it is consistent with
experimental data. From this study, we identified two poten-
tially important regions in the R51 receptor that are peculiar
to this integrin and might be worth considering for drug
targeting.
Integrins are ubiquitous cell adhesion receptors that
bind ligands on the surface of other cells and in the
extracellular matrix and are involved in bidirectional
signaling across the plasma membrane, regulating cell
adhesion, differentiation, migration, grow, and sur-
vival.
1
Evidence exists that such receptors are linked
to pathological conditions including tumor progression,
thrombosis, immune dysfunction, inflammation, and
osteoporosis, so integrins have been attractive thera-
peutic targets for several diseases.
2,3
Integrin R51 and
especially Rv integrins have attracted attention as
targets for antiangiogenic therapy.
4
Brooks et al. have
reported that various low molecular weight ligands,
which are recognized by Rv3 and Rv5 integrins, block
angiogenesis in response to growth factors in tumors
and suppress the cancer growth and metastasis.
5
On
the basis of these findings, the Rv3 receptor has been
the main integrin target in the search for new antican-
cer drugs in the past 2 decades, and significant progress
has been made in the identification of selective and
potent Rv3 integrin antagonists.
6
As a result of our
research, the cyclic peptide cyclo(-RGDf[NMe]V-),
known as cilengitide, is now in phase II clinical trials
for patients with glioblastoma.
7,14
Nonetheless, recent evidence that mice lacking of 3
or 5 integrins exhibit in some cases enhanced angio-
genesis
8
led to a reevaluation of Rv3 and Rv5 integrin
function in angiogenesis.
9
The different hypothesis made
in an effort to explain the discrepancy between the
genetic results and those obtained using low-molecular-
weight ligands are not fully convincing, and the debate
is still open. Reconsidering each integrin as a regulator
of angiogenesis, an important issue came out. The R51
receptor is the only unambiguously proangiogenic in-
tegrin; genetic ablation experiments and pharmacologi-
cal results are consistent and strongly support its
importance in neovascularization processes.
10
Hence,
the R51 integrin is expected to move into the forefront
of research for new effective anticancer drugs.
The research of R51 antagonists is not as advanced
as that of Rv3, and only few ligands are known to bind
the R51 integrin.
11
This constitutes a considerable
obstacle to the rational ligand-based drug design.
Moreover, the 3D structure of membrane spanning
receptors such as R51 is hard to obtain through X-ray
crystallography or NMR methods. Consequently, few
detailed structural information about ligand-receptor
interactions have been obtained until now. However, it
is well-know that integrins, which are heterodimers of
different combinations of noncovalently bound R and
chains, share extensive structural homology. It was also
demonstrated that the ligand binding to Rv, R51, and
RIIb3 integrins is mediated through the Arg-Gly-Asp
(RGD) recognition motif.
12,13
A first glimpse at the binding between integrins and
the RGD tripeptide sequence was provided by the
crystal structure
13
of the extracellular domain of Rv3
integrin in complex with cyclo(-RGDf[NMe]V-).
14
Ex-
periments using X-ray scattering and single-particle
electron microscopy pointed out that the overall shape,
the domain organization, and the way in which the R
and subunits assemble are very similar between Rv3
and R51.
13,15
Fibronectin, which is the physiological
ligand of R51, was found to bind its receptor through
the RGD-containing module 10 (Fn10) in a manner
similar to cyclo(-RGDf[NMe]V-) in the Rv3 crystal
complex.
13
These findings, the high sequence similarity
between R51 and Rv3 receptors (Rv:R5 53% identity;
3:1 55% identity in the integrin’s headgroup), and the
need to get more information about the R51 selectivity
requirement prompted us to build a 3D model of the
R51 receptor. A multiple sequence alignment was
performed utilizing evolutionary information of all R and
subunits in different organisms. The crystal structure
of Rv3 in the bound conformation was used as a
template, obtaining 10 3D models of R51 integrin by
means of comparative protein modeling methods. The
models, which differ mostly in the side chain orientation,
were used one at a time for ligand docking studies using
the Autodock program (see Supporting Information).
Compound 1 (SJ749) was chosen as ligand because of
its potency, receptor selectivity, conformational restric-
tions, and the recently published SARs (Table 1).
16
Docking results obtained for the 10 receptor models
were carefully inspected to evaluate the agreement with
the experimental data (mutagenesis, cross-linking, SARs)
and the convergence and the binding free energy
achieved for each simulation. SJ749 was found to fit in
one R51 model preferentially over the others. Such a
complex was energetically minimized using 3000 steps
of steepest descent algorithm with the CVFF force field,
permitting only the ligand and the side chain atoms of
the protein within a radius of 5 Å around the ligand to
relax. The stereochemical quality of the resulting model
was checked with the program PROCHECK. The ma-
jority of the residues occupied the most favored regions
(81.7%) of the Ramachandran plot. Other residues
* To whom correspondence should be addressed. Phone: +49-89-
289 13300. Fax: +49-89-289 13210. E-mail: kessler@ch.tum.de.
‡
Universita ` “Federico II” di Napoli.
†
Technische Universita ¨t Mu ¨ nchen.
4204 J. Med. Chem. 2005, 48, 4204-4207
10.1021/jm040224i CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/01/2005