Arylamide derivatives as allosteric inhibitors of the integrin α2β1/type I collagen interaction

Article abstract of DOI:10.1016/j.bmcl.2006.04.037

We herein report a group of allosteric inhibitors of integrin α₂β₁ based on an arylamide scaffold. Compound 4 showed an IC₅₀ of 4.80 μM in disrupting integrin I-domain/collagen binding in an ELISA. These arylamide compound

Full text of DOI:10.1016/j.bmcl.2006.04.037

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3380–3382
Arylamide derivatives as allosteric inhibitors of the integrin
a2b1/type I collagen interaction
Hang Yin,a Lars Ole Gerlach,a Meredith W. Miller,a David T. Moore,a Dahui Liu,a
Gaston Vilaire,b Joel S. Bennettb and William F. DeGradoa,c,
*
aDepartment of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
bHematology-Oncology Division, Department of Medicine, Philadelphia, PA 19104, USA
cDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA
Received 16 March 2006; accepted 5 April 2006
Available online 5 May 2006
Abstract—We herein report a group of allosteric inhibitors of integrin a2b1 based on an arylamide scaffold. Compound 4 showed an
IC50 of 4.80 lM in disrupting integrin I-domain/collagen binding in an ELISA. These arylamide compounds are able to block
collagen binding to integrin a2b1 on the platelet surface. Further we find that compound 4 recognizes a hydrophobic cleft on the
side of the a2 I-domain, suggesting an alternative targeting site for drug development.
Ó 2006 Elsevier Ltd. All rights reserved.
Integrins are homologous heterodimeric proteins con-
sisting of two trans-membrane subunits, a and b, that
non-covalently interact.1 Each ab combination has its
own binding specificity and signaling properties.2 The
a2b1 integrin is a primary platelet adhesion receptor that
can complex with collagen through the MIDAS motif
within the a2 I-domain.3 Despite the significant thera-
peutic potential, only a few peptide-based agents have
been reported to regulate the integrin a2b1 functions
by targeting the a2 I-domain.4 Herein, we report a group
of synthetic inhibitors based on an arylamide scaffold
identified from screening a focused small molecule li-
brary. These compounds bind to an allosteric site on
the a2 I-domain and inhibit the I-domain/collagen (type
I) binding both in vitro and in whole cells.
intra-molecular hydrogen binding lowers the entropy
penalty upon binding to the receptors.5a
We used an enzyme-linked immunosorbent assay (ELI-
SA) for fast initial screening to identify the lead com-
pounds.6 In this assay, soluble type I collagen was
immobilized on a 96-well plate, and a recombinant
GST-tagged a2 I-domain construct was added, along
with increasing concentrations of various arylamide
derivatives. The amount of I-domain binding in the
presence of the arylamide derivatives was measured by
the activity of a peroxidase that was conjugated to an
anti-GST antibody using a colorimetric reaction that
generates absorbance at 405 nm.
The IC50 values of the arylamide derivatives are listed in
Table 1. Compound 4, with two flanking L-2-naphthyl-
alanine (Nal) residues and tert-butyl groups on the
center phenyl rings, potently disrupted the collagen/I-
domain binding with an IC50 value of 4.8 lM. As a
comparison, compound 1, with D-2-Nal residues
appended, showed about 4-fold weaker potency, sug-
gesting the L-isomer provides better spatial complemen-
tarity. However, the tert-butyl groups appear to be not
essential in the target recognition. In general, arylamides
with aromatic, hydrophobic residues at both termini (1,
4, 5, 7, 9, and 12) are more potent inhibitors, suggesting
the target area on the a2 I-domain has the same proper-
ty. However, the limited solubility of these compounds
precluded more detailed studies. Arylamide derivatives
Arylamide derivatives have been previously reported as
a good template for membrane-binding proteins.5 Other
advantages of using these arylamide derivatives in drug
development include: (1) they are prepared via straight-
forward modular synthesis, readily permitting modifica-
tion of backbone and termini with different functional
groups and (2) the rigid conformation enforced by the
Keywords: Integrin; Protein–protein interaction; Platelet adhesion;
Allosteric inhibitor.
*
Corresponding author. Tel.: +1 215 898 4590; fax: +1 215 573
7229; e-mail: wdegrado@mail.med.upenn.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.037

H. Yin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3380–3382
Table 1. Results of the ELISA and adhesion assays for the arylamide derivatives
3381
NH2
H2N
R4
R5
O
R5
O
S
S
H
N
H
N
H
N
H
N
R1
R2
R3
R3
Compound
R1
R2
R3
R4
R5
IC50 (lM)
Adhesion
ELISA
1
2
D-2-Nal1
D-3-Pya2
3-Pya
L-2-Nal
D-2-Nal
D-2-Nal
L-2-Nal
L-2-Nal
D-Phe
Gaba3
D-Arg
D-Trp
D-Ala
Gly
D-2-Nal
D-3-Pya
H
t-Bu
t-Bu
H
H
H
H
H
H
H
H
H
H
>20
>50
>50
4.80
19.7
>50
6.23
>50
>50
>50
>50
>20
>50
>50
>50
>50
14.4
H
>100
>100
3.40
3
H
4
L-2-Nal
D-2-Nal
H
t-Bu
H
H
5
H
16.9
64.6
6
H
H
7
L-2-Nal
H
H
H
10.1
85.3
8
H
H
9
D-Phe
Gaba
D-Arg
D-Trp
D-Ala
Gly
H
O(CH2)2NH2
H
43.2
10
11
12
13
14
15
16
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
H
H
H
H
H
H
H
H
>100
>100
17.34
>100
>100
>100
>100
H
H
H
H
H
H
O(CH2)2NH2
O(CH2)2Gua4
H
H
(1) Nal, naphthylalanine; (2) Pya, pyridinylalanine; (3) Gaba, gamma-amino butyric acid; (4) Gua, guanidium.
with small amino acid residues attached (10, 13, and 14)
showed few inhibitory effects. Compounds with only one
amino acid residue attached (3, 6, and 8) showed signif-
icantly lowered potency, indicating the necessity of the
intact arylamide scaffold. It is also suggested that the to-
tal number of charges that these arylamide derivatives
carry does not affect their binding to the a2 I-domain
to a large extent.
atives in phospholipid vesicles. Compound 4 failed to
induce a fluorescent dye, Tb(DPA)3, to leak from
phospholipid vesicles at lipid/compound ratio up to
10:1 (Supporting information Fig. 3), suggesting that
these arylamide compounds did not significantly affect
the cell membrane. Further toxicity tests for these com-
pounds were conducted using a lactate dehydrogenase
(LDH) release assay. Platelets release LDH when their
plasma membrane is perturbed. In the presence of
30 lM of arylamide 4, the level of LDH released by plate-
lets is not significantly higher than the negative control.
The inhibitory effects of these arylamide derivatives in
disrupting collagen/integrin a2b1 association in whole
cells were tested using a previously described platelet
adhesion assay.7 The inhibitory effects of the arylamide
derivatives were determined by measuring the number of
gel-filtered human platelets bound to immobilized type I
collagen on a 96-well plate. Consistent with the ELISA
results, compound 4 showed the strongest inhibitory
potency with an IC50 of 3.4 lM in disrupting collagen
binding to intact platelets. This finding indicated that
these arylamide derivatives are able to block type I col-
lagen binding to integrin on the platelet surface (Sup-
porting information Fig. 2).
In order to study the binding mode of these arylamide
derivatives to integrin a2 I-domain, we have conducted
(1H,15N)-2D-HSQC NMR experiments. NMR samples
containing 15N-labeled I-domain protein (0.25 mM), in
the presence of 0, 0.5, and 1.0 equivalence of arylamide
derivative 4, were prepared. Figure 1 shows the results
of the HSQC experiments, with the amino acid residues
that showed significant and moderate chemical shift
changes upon addition of arylamide 4 colored in magen-
ta and orange, respectively. Five amino acids (Lys294,
Lys301, Y311, Ser316, and Asp317, labeled in Fig. 1)
showed significant chemical shift changes upon addition
of the arylamide derivatives and 12 other amino acids
(Val208, Ala209, Thr246, Gln269, Asp273, Asn289,
Lys298, Ser305, Ile306, Arg307, Phe313, and Ala319)
showed moderate chemical shift changes. Noticeably,
12 out of these 17 affected residues are located around
a hydrophobic cleft on one side of the a2 I-domain, sug-
gesting that arylamide 4 inhibits the collagen/a2 I-do-
main association by targeting an allosteric site that
is different from the collagen-binding MIDAS site.
Furthermore, the collagen-induced platelet aggregation
can be blocked by compound 4. Addition of soluble type
I collagen to a suspension of washed platelets results in
platelet aggregation caused by the activation of integrin
a2b1. We found this aggregation is blocked in the pres-
ence of 5.0 lM 4, confirming that the collagen/integrin
a2b1 interaction is the target for these inhibitors.
Because amphiphilic compounds can cause cell lysis,8 we
examined the leakage potential of these arylamide deriv-

3382
H. Yin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3380–3382
We also thank Dr. Gregory A. Caputo’s help in the
Tb(DPA)3 release assay.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.04.037.
References and notes
1. Giancotti, F. G.; Ruoslahti, E. Science 1999, 285, 1028.
2. Arnaout, M. A.; Mahalingam, B.; Xiong, J. P. Annu. Rev.
Cell Dev. Biol. 2005, 21, 381.
3. (a) Emsley, J.; Knight, C. G.; Farndale, R. W.; Barnes, M.
J.; Liddington, R. C. Cell 2000, 191, 47; (b) Auger, J. M.;
Kuijpers, M. J.; Senis, Y. A.; Watson, S. P.; Heemskerk, J.
W. M. FASEB J. 2005, 19, 825.
4. For examples, see: (a) Baronas-Lowell, D.; Lauer-Fields,
J. L.; Fields, G. B. J. Biol. Chem. 2004, 279, 952; (b)
Kim, J. K.; Xu, Y.; Xu, X.; Keene, D. R.; Gurusidd-
appa, S.; Liang, X.; Wary, K. K.; Ho¨o¨k, M. J. Biol.
Chem. 2005, 280, 32512; (c) Sweeney, S. M.; DiLullo,
G.; Slater, S. J.; Martinez, J.; Iozzo, R. V.; Lauer-
Fields, J. L.; Fields, G. B.; San Antonio, J. D. J. Biol.
Chem. 2003, 278, 30516; (d) Ivaska, J.; Ka¨pyla¨, J.;
Pentika¨inen, O.; Hoffren, A. M.; Hermonen, J.; Hutt-
unen, P.; Johnson, M.; Heino, J. J. Biol. Chem. 1999,
274, 3513.
5. (a) Yin, H.; Frederick, K. K.; Liu, D.; Wand, A. J.;
DeGrado, W. F. Org. Lett. 2006, 8, 223; (b) Tew, G. N.;
Liu, D.; Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll, P.
J.; Klein, M. L.; DeGrado, W. F. Proc. Natl. Aca. Sci.
U.S.A. 2002, 99, 5110; (c) Choi, S.; Clements, D. J.;
Pophristic, V.; Ivanov, I.; Vemparala, S.; Bennett, J. S.;
Klein, M. L.; Winkler, J. D.; DeGrado, W. F. Angew.
Chem., Int. Ed. 2005, 44, 6685; (d) Liu, D.; Choi, S.; Chen,
B.; Doerksen, R. J.; Clements, D. J.; Winkler, J. D.; Klein,
M. L.; DeGrado, W. F. Angew. Chem., Int. Ed. 2004, 43,
1158.
Figure 1. (1H,15N)-2D-HSQC NMR experiment results on I-domain/
arylamide binding. The residues that showed significant and moderate
chemical shift changes upon the addition of 4 (1.0 equiv) are shown in
magenta and orange, respectively. The amino acid residues that
showed significant chemical shift changes upon addition of 4 were
labeled. The integrin a2 I-domain is shown in green cartoon represen-
tation with mesh. The type I collagen triple-helix bundle ligand is
shown in blue tubes.
Furthermore, this cleft overlaps with the a7–b6 loop,
which plays an essential role in the integrin conforma-
tional change in the signaling pathway (Supporting
information Fig. 5).9 Interestingly, it has been recently
reported that a marketed drug, lovastatin, complexes
with the analogous aL I-domain at a similar site.10 This
finding may suggest an alternative strategy to target the
integrin I-domains and provide therapeutically useful
agents. Along these lines, the compounds described here
may serve as research tools or early leads for subsequent
optimization.
6. Xu, Y.; Gurusiddappa, S.; Rich, R. L.; Owens, R. T.;
Keene, D. R.; Mayne, R.; Ho¨o¨k, A.; Ho¨o¨k, M. J. Biol.
Chem. 2000, 275, 38981.
7. Basani, R. B.; D’Andrea, G.; Mitra, N.; Vilaire, G.;
Richberg, M.; Kowalska, M. A.; Bennett, J. S.; Poncz, M.
J. Biol. Chem. 2001, 276, 13975.
8. Bechinger, B. J. Membr. Biol. 1997, 156, 197.
9. Takagi, J.; Springer, T. A. Immunol. Rev. 2002, 186, 141.
10. Weitz-Schmidt, G.; Welzenbach, K.; Dawson, J.; Kallen,
J. J. Biol. Chem. 2004, 279, 46764.
Acknowledgments
This work was supported in part by NIH Grant
HL62250. We thank Dr. Kathleen G. Valentine’s
generous help and discussion in the HSQC NMR
experiments; Prof. Mark Kahn for helpful discussion.