Synthesis and evaluation of bivalent, peptidomimetic antagonists of the αvβ3 integrins

Article abstract of DOI:10.1016/j.bmcl.2010.09.035

Targeting the integrin α_vβ₃ by directly interfering with its function is considered to be an effective and non-cytotoxic strategy for the treatment of tumor. In this study, a series of bivalent analogs of peptidomimetic integrin antagonists IA 1 and IAC 2 were designed, synthesized, and evaluated for their ability to inhibit the integrin α_vβ₃. All the bivalent ligands exhibited increased potency compared to that of their monomeric counterparts for the integrin α_vβ₃ with low nanomolar range binding affinity. The best bivalent ligand 6 tested in the series has an IC₅₀ = 0.09 nM evaluated by ELISA assay. We conclude that multivalency is providing a useful template for the development novel integrin α_vβ ₃ antagonists as potential therapeutics.

Full text of DOI:10.1016/j.bmcl.2010.09.035

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6577–6580
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of bivalent, peptidomimetic antagonists
of the avb3 integrins
Feng Li a, Gouri S. Jas b, Guoting Qin a, King Li a, , Zheng Li a,
⇑
⇑
a Department of Radiology, The Methodist Hospital Research Institute, Houston, TX 77030, United States
b Department of Chemistry and Biochemistry, Baylor University, 101 Bagby Avenue, Waco, TX 76706, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Targeting the integrin
avb3 by directly interfering with its function is considered to be an effective and
Received 25 August 2010
Revised 3 September 2010
Accepted 7 September 2010
Available online 15 September 2010
non-cytotoxic strategy for the treatment of tumor. In this study, a series of bivalent analogs of peptidom-
imetic integrin antagonists IA 1 and IAC 2 were designed, synthesized, and evaluated for their ability to
inhibit the integrin
monomeric counterparts for the integrin
lent ligand 6 tested in the series has an IC50 = 0.09 nM evaluated by ELISA assay. We conclude that mul-
a
vb3. All the bivalent ligands exhibited increased potency compared to that of their
a
vb3 with low nanomolar range binding affinity. The best biva-
Keywords:
Multivalency
Computer modeling
Integrin antagonists
tivalency is providing a useful template for the development novel integrin
therapeutics.
avb3 antagonists as potential
Ó 2010 Elsevier Ltd. All rights reserved.
The integrins are a family of cell-surface glycoproteins with
critical roles in regulating both physiological and pathological pro-
cesses. They are involved in a variety of cell signaling pathways by
mediating cell adhesion, migration, and proliferation through ex-
plicit non-covalent interactions with endogenous extra cellular
agent for gene delivery to angiogenic blood vessels (Fig. 1).9 We
also reported on IAC 2, an IA carbamate derivative which enhanced
the
a
vb3 binding affinity about 10 times in comparison with IA.10
Using IA as the parent compound, we successfully developed a
bivalent near-infrared fluorescent imaging probe with carbon
chain linker which selectively targeted integrin avb3 and showed
matrix (ECM) proteins.1 19 different integrin
ferent b subunits have been identified, forming at least 25
a
subunits and 8 dif-
b het-
a
promising in vitro and in vivo results for tumor detection.11 Since
the optimal linker plays an important role in multivalent ligand–
receptor interactions we decided to explore this aspect of the
formulation. In this study, we chose IA 1 and IAC 2 as parent
compounds for the bivalent ligand construction employing rational
structure design using computer simulation. Since polyethylene
glycol (PEG) has low toxicity and immunogenicity, and has good
solubility in both aqueous and organic solvents, it has been used
as a carrier for various drugs. These favorable physicochemical
properties make PEG a good linker material for constructing multi-
valent ligands. Herein we describe the synthesis of bivalent ligands
3–8 using PEG linker with different lengths to study structure
function relationship using in vitro assays (Fig. 1).
erodimers and making the integrins a structurally and functionally
diverse family of cell adhesion molecules.2 One of the most impor-
tant members of the integrin family is the integrin avb3, which is a
heterodimeric transmembrane receptor protein that has been pro-
ven to be involved in the formation of angiogenesis, a phenomenon
that occurs in major diseases such as cancer, osteoporosis, rheuma-
toid arthritis, and macular degeneration.3,4 The distinct biological
role of
avb3 makes it an attractive target for the development of
therapies for a variety of diseases.5 To date, six integrin inhibitors
are being evaluated in clinical trials as anti-angiogenic agents for
tumor imaging and therapy, which demonstrate remarkable affin-
ity and selectivity to a
vb3.6
Design of new multivalent therapeutic and imaging agents has
received a lot of attention because of their potential for increased
binding affinity to biological receptors.7 Bivalency, the most basic
form of multivalency has already been adopted as a strategy in
pharmaceutical research to improve interactions of designed li-
The bivalent ligands 3–8 were synthesized using the procedure
described in Figure 2, starting with commercially available trieth-
ylene glycol 9, tetraethylene glycol 10, and octaethylene glycol 11.
The PEGs were first activated as biscarbonylimidazoles derivatives
13–15, and their chemical structure and purity was verified by
NMR. The activated alcohols were then coupled to the free amine
1 and 2, respectively, in DMSO to provide bivalent ligands 3–8.
All compounds were purified to homogeneity by semi-preparative
RP-HPLC (Phenomenex C18 column), and their structures were
confirmed by NMR and ESI ion trap mass spectrometry (Supple-
mentary data).
gands with natural receptors.8 A peptidomimetic integrin
avb3
antagonist IA 1, has been successfully exploited as the targeting
⇑
Corresponding authors. Tel.: +1 28 17575518 (Z.L.).
E-mail addresses: Kli@tmhs.org (K. Li), Zli@tmhs.org (Z. Li).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.035

6578
F. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6577–6580
H
N
H
H
N
N
N
N
N
O
O
O
HN
HN
HN
HN
O
HN
O
HN
O
CO2H
CO2H
O
S
CO2H
O
S
H
N
H
N
O
S
O
NH
HN
O
O
HN
NH2
O
O
n
O
O
O
O
H
N
3: n=1; 4: n=2; 5: n=6
H
N
IA 1
H
N
N
O
N
N
O
O
HN
HN
HN
HN
O
CO2H
HN
O
CO2H
O
S
O
HN
O
CO2H
O
S
O
O
H
N
H
N
O
S
O
H
N
H
H
N
NH
O
HN
O
NH2
O
O
O
N
HN
O
O
n
O
O
O
O
IAC 2
6: n=1; 7: n=2; 8: n=6
Figure 1. Schematic structure of IA 1, IAC 2, and bivalent ligands 3–8.
O
O
O
O
O
DIEA
N
O
O
N
HO
O
N
N
N
N
+
n
O
OH
N
N
N
n
DCM, 80-90%
9
10
11
: n=1; :n=2; : n=6
13
14
15
: n=1; :n=2; : n=6
CDI 12
H
H
N
N
N
O
O
HN
HN
HN
O
HN
O
CO2H
CO2H
O
S
H
N
H
O
S
O
NH
HN
O
O
N
O
O
n
O
O
O
3: n=1; 4: n=2; 5: n=6
, DIEA, DMSO
1
70 ºC, 18h, 70-90%
H
N
H
N
N
O
N
O
HN
HN
O
CO2H
HN
HN
O
CO2H
O
S
O
H
N
H
N
O
S
O
H
H
N
NH
O
O
O
O
N
HN
O
O
n
O
O
O
O
6: n=1; 7: n=2; 8: n=6
Figure 2. General procedure for the synthesis of bivalent ligands 3–8.
All final compounds were tested for their ability to competi-
tively inhibit the attachment of the natural ligand vitronectin to
purified human
binding of the integrin antagonist and biotinylated human vitro-
varying linker lengths and compositions were constructed and
molecular dynamics simulation was performed with CHARMM
35 force field (Supplementary data). From calculations of binding
energies and ligand spatial distributions, the prediction of favored
protein–ligand binding modes, interaction strengths, and binding
specificity can be obtained with AutoDock simulations. Ten (10)
LGA (Lamarckian genetic algorithm) docking runs were performed
for each protein–ligand pair, with each run producing one possible
a
vb3 by ELISA assay.12 It measured competitive
nectin for the immobilized receptor
Table 1.
avb3. The results are listed in
The computer modeling used molecular dynamics (MD) with
explicit solvent. In the modeling study, bivalent IA and IAC with

F. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6577–6580
6579
Table 1
In silico conformational energy and free energy of PEG-linked integrin antagonists and in vitro evaluation
a
Compds
n
In silico conformational energy (kcal/mol)
In silico free energy (kcal/mol)
In vitro ELISA IC50 (nM)
IA 1
IAC 2
—
—
1
2
6
1
2
6
À17.0 ( 2.0)
À41.5 ( 2.0)
À100.9 ( 2.0)
À98.0 ( 2.0)
À99.3 ( 2.0)
À140.9 ( 2.0)
À138.7 ( 2.0)
À153.4 ( 2.0)
À3.1 ( 0.8)
À10.2 ( 1.6)
À6.9 ( 1.2)
À6.1 ( 1.2)
À5.7 ( 1.2)
À8.2 ( 1.2)
À1.6 ( 0.6)
À2.1 ( 0.6)
22.3 ( 4.5)
2.07 ( 0.9)
0.16 ( 0.12)
0.16 ( 0.05)
0.16 ( 0.10)
0.09 ( 0.08)
0.11 ( 0.02)
0.32 ( 0.09)
3
4
5
6
7
8
a
Values are averages of at least three determinations.
binding mode or solution. The solutions were first sorted in terms
of the binding mode, i.e., the position and orientation of the ligand
relative to the protein target. The solutions were clustered based
on rms (root mean square) deviations in ligand atomic positions,
with structures with rmsd of <0.5 Å grouped into a cluster. The to-
tal number of generated low-energy clusters measures the speci-
ficity of binding.13–16 A small number of clusters indicates that
the ligand has only a few possible binding modes and interacts
with a specific site (or sites) on the target protein. On the other
hand, a large number of clusters imply existence of a wide range
of binding modes and lack of specific ligand–target interactions.
The second step in sorting solutions involves identification of the
solution of lowest binding energy within each cluster and ranking
the different clusters according to this energy value. The solution
with the lowest energy in the top-ranked cluster and all solutions
with energies higher by up to 5.0 kcal/mol were considered as pos-
sible binding modes for ligand to target. The docking of each antag-
particular, bivalent ligands 3–5 (IC50 = 0.16 nM) exhibited remark-
able potency for the vb3, and showed 139-fold higher binding
affinity as compared to that of the monomer 1 (IC50 = 22.3 nM).
Bivalent ligand 6 (IC50 = 0.09 nM) had almost 23-fold higher affin-
ity as compared to the corresponding monomer 2. In fact, 6 is one
a
of the most potent
avb3 antagonists reported to date
(IC50 = 0.09 nM). Although the binding affinity of parent monomer
2 is 10 times more potent than monomer 1, the binding affinity of
corresponding dimers did not show much difference. This observa-
tion suggests that the structure of the parent lead compound is not
the only basis for the improved integrin avb3 binding of the dimers.
So, it is theoretically possible to design multivalent ligands with
high binding affinity based on weak parent compounds. The
hypothesis for the significantly increased binding affinity of dimers
as compared to the corresponding monomer is that the local IA
concentration is significantly ‘enriched’ in the vicinity of the neigh-
boring integrin
avb3 sites. Once the first IA motif is bound to an
onist to the integrin
avb3 produced 3 clusters out of 10 runs. There
integrin vb3, bivalency might lead to a faster rate of receptor bind-
a
were five solutions in the first cluster with an average docking en-
ergy of IA, IAC, and its dimers. A summary of the AutoDock results
is presented in Table 1 and the docked structures of the first and
second clusters are shown in Figure 3.
ing and/or a slower rate of dissociation from the receptor. In com-
puter modeling, the binding site defined by the cluster is in the
cleft between the RGD binding domains in integrin avb3. It was ob-
served that all the ligands stay at that site in the presence of the
In general, all bivalent ligands 3–8 showed significantly im-
whole protein. This cluster presented the best model for a possible
proved affinity for the
a
vb3 as compared to monomer 1 and 2. In
antagonist interaction to the integrin avb3. The modeling results
Figure 3. Binding of antagonists with the integrin. The corresponding structural antagonist conformations that are interacting with integrin are offset and enlarged to show
molecular details. (A) IA monomer 1, (B) IA dimer 4 (n = 2), (C) IA dimer 5 (n = 6), (D) IAC monomer 2, (E) IAC dimer 7 (n = 2), and (F) IAC dimer 8 (n = 6).

6580
F. Li et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6577–6580
suggested that IA dimers should have higher binding affinity than
IA monomer because they have lower conformational energy and
free energy compared with IA monomer. It also suggested IA di-
mers with PEGs linker (n = 1, 2, 6) should have similar binding
affinity because they have similar energy level in silico. Further-
more, among the bivalent conformations, the simulations suggest
that IAC dimer 6 (n = 1) has a higher specificity and better fit into
the known active site than other conformations. The ELISA results
correlates well with these predictions. However, ELISA results did
not correlate perfectly with the molecular modeling results. For
example, although IAC had the lowest free energy on computer
modeling the ELISA result was much less impressive. The micro-
scopic reason for these effects appears to be a lack of fit between
different IA conformation and the inhibitor/substrate binding site.
The docking results are approximate. The scoring is based on an
empirical energy function, solvation effects treated with a highly
simplified model, and only ligand flexibility taken into account,
with the protein structure kept fixed.13–15 Thus, the AutoDock re-
sults should only be considered as qualitative.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.035.
References and notes
1. Hynes, R. O. Cell 1992, 69, 11.
2. Takagi, J.; Springer, T. A. Immunol. Rev. 2002, 186, 141.
3. Folkman, J. Nat. Med. 1995, 1, 27.
4. Schnitzer, J. E. N. Engl. J. Med. 1998, 339, 472.
5. Arap, W.; Pasqualini, R.; Ruoslahti, E. Science 1998, 279, 377.
6. Schottelius, M.; Laufer, B.; Kessler, H.; Wester, H. Acc. Chem. Res. 2009, 42, 969.
7. Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37,
2754.
8. Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am.
Chem. Soc. 2002, 124, 14922.
9. Hood, J. D.; Bednarski, M.; Frausto, R.; Guccione, S.; Reisfeld, R. A.; Xiang, R.;
Cheresh, D. A. Science 2002, 296, 2404.
10. Burnett, C. A.; Xie, J.; Quijano, J.; Shen, Z.; Hunter, F.; Bur, M.; Li, K. C. P.; Danthi,
S. N. Bioorg. Med. Chem. 2005, 13, 3763.
11. Li, F.; Liu, J.; Jas, G. S.; Zhang, J.; Qin, G.; Xing, J.; Cotes, C.; Zhao, H.; Wang,
X.; Diaz, L. A.; Shi, Z. Z.; Lee, D. Y.; Li, K. C. P.; Li, Z. Bioconjug. Chem. 2010,
21, 270.
In summary, we have successfully designed and synthesized a
series of bivalent antagonists of the avb3 integrin through tethering
IA and IAC with polyethylene glycol, respectively. Biological evalu-
ation of six bivalent ligands shows that all dimers inhibit integrin
12. In vitro binding affinity assay: purified integrin
International, Temecula, CA) was applied to 96-well polystyrene microtiter
plates at 1 g/well. After overnight incubation at 4 °C the plates were washed,
avb3 protein (Chemicon
l
and then blocked with milk solution (KPL, Inc., Gaithersburg, MD) at room
temperature for 2 h. The blocking buffer was removed, and the plates were
inoculated in quadruplicate with bivalent IAs with
concentration of 125 nM. Serial dilutions were prepared in the 96-well plates
using multichannel pipettes. Biotinylated vitronectin solution (0.1 g/well)
was added to each well as a standard competitor. The plates were incubated at
room temperature for 3 h, washed, and the bound vitronectin was detected
using NeutrAvidin-HRP conjugate at 0.01 lg/well (Pierce, Rockford, IL) and
LumiGlo chemiluminescent substrate system (KPL, Inc., Gaithersburg, MD). The
luminescence was read using a FLUOstar OPTIMA Microplate Reader (Durham,
NC). The concentration of inhibitor producing 50% inhibition (IC50
vitronectin binding to vb3 was calculated based on a curve fitting model
using KaleidaGraph 3.5 (Synergy Software, Reading, PA).
avb3 with increased potency as compared to that of their mono-
a typical starting
meric counterparts IA and IAC. The bivalent ligands 3–5 of IA with
different linker length showed similar binding affinity for integrin
l
avb3 with IC50 = 0.16 nM, 139 times higher in comparison with IA
(IC50 = 22.3 nM). In addition, the bivalent ligand 6, with triethylene
glycol linker, has the highest binding affinity in the series based on
ELISA assay (IC50 = 0.09 nM), the therapeutic study and develop-
ment of these potent avb3 integrin antagonists into imaging probes
for optical and PET imaging is now in progress in our group.
) of
a
13. Goodsell, D. M.; Olson, A. J. Proteins 1990, 8, 195.
14. Morris, G. M.; Goodsell, D. S.; Huey, R.; Olson, A. J. J. Comput. Aided Mol. Des.
1996, 10, 293.
Acknowledgment
15. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
16. Xiong, J. P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.; Goodman, S. L.;
Arnaout, M. A. Science 2002, 296, 151.
This project was supported by The Methodist Hospital Research
Institute, the M.D. Anderson Foundation, and the Vivian L. Smith
Foundation.