The pharmacophore of a peptoid VEGF receptor 2 antagonist includes both side chain and main chain residues

Article abstract of DOI:10.1016/j.bmcl.2008.07.023

Here we identify the pharmacophore in a peptoid that antagonizes Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) in vitro and in vivo. Only three of the side chains in the peptoid are required for activity. Surprisingly, however, main chain atoms also form critical interactions with the receptor.

Full text of DOI:10.1016/j.bmcl.2008.07.023

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5892–5894
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The pharmacophore of a peptoid VEGF receptor 2 antagonist includes
both side chain and main chain residues
D. Gomika Udugamasooriya a, Geoff Dunham a, Caroline Ritchie a, Rolf A. Brekken b, Thomas Kodadek a,
*
a Department of Internal Medicine and Molecular Biology, Division of Translational Research, University of Texas Southwestern Medical Center,
5323 Harry Hines Boulevard, Dallas, TX 75390-9185, USA
b Department of Surgery and Pharmacology, Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-9185, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we identify the pharmacophore in a peptoid that antagonizes Vascular Endothelial Growth Factor
Receptor-2 (VEGFR2) in vitro and in vivo. Only three of the side chains in the peptoid are required for
activity. Surprisingly, however, main chain atoms also form critical interactions with the receptor.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 10 June 2008
Revised 7 July 2008
Accepted 8 July 2008
Available online 10 July 2008
Keywords:
Peptoid
Minimum pharmacophore
Glycine scan
Sarcosine scan
Rapid identification of the ‘minimum pharmacophore’ of a lead
compound is a vital step in the drug development process since it
sets the stage for subsequent optimization. With peptide-based
agents, this exercise is simplified by the regular structure of the
molecule. A common practice is to evaluate a series of derivatives
in which each residue in turn is replaced with a glycine or alanine
(alanine scanning).1,2 Recently, we reported the effective applica-
tion of glycine scanning to a peptoid (N-substituted oligoglycine)
inhibitor of the 19S regulatory particle of the proteasome. This
allowed us to create a minimal derivative of the original hit with
about half the mass, and thus increased cell permeability and
potency.3 We have also reported the isolation of highly specific
peptoid ligands for the extracellular domain of the Vascular
Endothelial Growth Factor Receptor-2 (VEGFR2),4 an integral
membrane receptor that triggers angiogenesis when bound by its
cognate hormone VEGF.5 A dimerized derivative (GU40C4) of one
of these nine residue peptoids (GU40C; see Fig. 1) is a low nM
ligand for the receptor’s extracellular domain, and is a potent
antagonist of angiogenesis in vivo.4 Inhibition of VEGFR2-mediated
angiogenesis is a validated strategy to slow the growth of tumors
as well as to treat ‘wet’ macular degeneration.6–14 Thus, this
peptoid is of potential therapeutic interest, and its optimization
is an important goal. Therefore, we sought to identify the minimal
pharmacophore in GU40C as the initial step in this effort.
O
O
O
H2N
HN
H2N
O
H2N
O
O
O
O
O
4
4
8
N
4
6
N
4
2
N
N
7
N
5
N
3
N
N
NH2
1
9
O
O
4
4
H2N
H2N
GU40C
Figure 1. Structure of GU40C. Residues are numbered starting from C-terminus.
First, nine derivatives of GU40C were synthesized in which each
of the nine residues in the parent peptoid was replaced with a gly-
cine. All these derivatives were synthesized with a C-terminal cys-
teine to facilitate fluorescein attachment via maleimide chemistry.
The affinity of each of these derivatives for the extracellular
domain (ECD) of VEGFR2 was then determined using an ELISA-like
binding assay described in our previous report.4 The results are
shown in Figure 2 (black bars). Only two side chains (the 6th and
8th from the C-terminus) appeared to be important for binding
of GU40C to the VEGFR2 ECD.
To buttress these data, we repeated the analysis, but replaced
each monomer in the peptoid with sarcosine rather than with gly-
cine. Since secondary amides have a strong preference for a tran-
soid configuration about the peptide bond, while tertiary amides
do not, it is possible that glycine substitution could introduce con-
formational constraints not present in the parent peptoid, and thus
the comparison of the derivative to the parent molecule might re-
* Corresponding author. Tel.: +1 214 648 1239; fax: +1 214 648 4156.
E-mail address: thomas.kodadek@utsouthwestern.edu (T. Kodadek).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.023

D. G. Udugamasooriya et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5892–5894
5893
250
200
150
100
50
GU40C(1)
A
O
O
N
OH
H2N
O
O
O
S
4
H
N
OH
H2N
N
N
N
NH2
R =
O
O
O
R
O
O
B
GU40C
GU40C(1)
Control
39500
29500
19500
9500
0
Peptoid
Figure 2. Glycine (black bars) sarcosine (gray bars) scan binding results of GU40C.
Please refer Fig. 1 for residue numbers.
-500
-3
-2
-1
0
1
2
3
log [peptoid] μM
Figure 3. Shortest derivative of GU40C, which contains only the important side
chains and its binding isotherm. (A) Structure of GU40C(1). (B) Binding isotherms of
GU40C, GU40C(1), and a control peptoid that does not bind selectively to VEGFR2
ECD.
flect issues other than simply deleting the side chain. For example,
if the preferred binding conformation of a peptoid involved a cisoid
conformation about a particular peptide bond in the molecule,
then replacement of the side chain with a hydrogen would
discriminate against this conformation and presumably inhibit
binding, even though the side chain was not involved directly. A
sarcosine scan has the effect of replacing each of the side chains
in turn with a methyl group rather than with a hydrogen, preserv-
ing the tertiary amide bond, but removing the bulk of the side
chain. Therefore, we decided to conduct a sarcosine scan in the
region of the molecule identified as being critical for binding by
the glycine scan.
Therefore, we decided to reintroduce the full backbone, but
leave out the side chains, except those important residues at posi-
tions 6–8 [GU40C(2)—Fig. 4A, first structure]. Interestingly, this
compound recognized the receptor ECD with an affinity similar
to that of the GU40C parent peptoid (Fig. 4B, square and circle data
points), confirming the conclusion derived from the scanning
experiments that the side chains at positions 1–5 are not involved
in receptor binding. This observation, combined with the sarcosine
As shown in Figure 2 (gray bars), substitution of the methyl
group for isobutyl moiety at position 8 or the
a-methylbenzyl
group at position 6 weakened binding of the peptoid for the
VEGFR2 ECD which is significantly consistent with the glycine scan
results. However, in contrast with the glycine scanning result, sub-
stitution of the lysine-like side chain at position 7 with methyl also
reduced binding affinity. This result was confirmed by competition
binding assays that compared directly the relative affinities of the
peptoids with glycine and sarcosine substitution at position 7 (see
Supplementary Figure 5). We do not fully understand the basis of
the different results obtained using the two scanning methods at
position 7. One possibility might be that a polar substituent capa-
ble of donating a hydrogen bond to solvent might be favorable
there. In any case, the combined data from the glycine and
sarcosine scans indicate that the N-terminal region of GU40C, spe-
cifically positions 6–8 (see Fig. 1), is important for binding of the
peptoid to VEGFR2.
Based on these data, it seemed reasonable to speculate that a
trimeric peptoid including positions 6–8 would be a good ligand
for the VEGFR2 ECD, an interesting possibility since this molecule
would have a mass of less than 450 Da. To test this idea, we synthe-
sized a fluorescein-conjugated tetramer peptoid GU40C(1) that
contains the three original residues at 6–8 positions along with
an N-terminal glycine (Fig. 3A).
S
R
H2N
O
A
O
O
O
O
O
H
N
H
N
H
N
4
4
H2N
N
N
NH2
N
N
N
N
H
N
H
N
H
O
O
O
O
GU40C(2)
H2N
O
O
O
O
O
S
H
H
N
H2N
N
N
N
H
NH2
R
O
O
GU40C(3)
B
GU40C
GU40C(2)
GU40C(3)
Control
37000
27000
17000
7000
The affinity of this minimized GU40C derivative for the receptor
ECD was then tested, again using the ELISA-like binding assay.
Somewhat surprisingly, this molecule showed no detectable bind-
ing to the receptor ECD at any of the concentrations tested (Fig.
3B). Combined with the glycine scanning data shown in Figure 2,
this result suggested the possibility that some of the main chain
atoms in the parent peptoid might be involved in receptor
binding.
-3000
-2.5 -1.5 -0.5
0.5
1.5
2.5
log [peptoid] μM
Figure 4. Structures and binding isotherms of GU40C and its derivatives. (A)
Structures of GU40C(2) and GU40C(3). R: please see Figure 3. (B) Binding isotherms
of GU40C and its derivatives. Only GU40C and GU40C(2) are able to display binding
to VEGFR2 ECD.

5894
D. G. Udugamasooriya et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5892–5894
O
GU40C
O
A
O
O
O
H2
N
H2N
O
H2N
H2
N
H2N
O
H2N
O
O
O
O
4
4
O
O
O
O
O
4
4
4
4
HN
N
N
N
N
HN
N
N
N
N
N
N
N
N
NH2
N
N
N
N
NH2
O
O
O
4
4
O
O
O
H2N
H2N
4
4
H2N
H2N
8-mer
7-mer
6-mer
Figure 6. Structure of the GU40C with highlighted residues that are proposed to
constitute the minimal pharmacophore.
5-mer
4-mer
B
5500
4500
3500
2500
1500
500
tions 1 and 5 that contact the receptor ECD. This model is further
supported by the fact that the nine residue peptoid GU40C(2),
which contains only the side chains at positions 6–8 but is other-
wise composed of glycines, binds the receptor ECD about as well
as the GU40C parent.
Figure 6 highlights the residues that are proposed to constitute
the minimal pharmacophore of GU40C based on these results. This
study also highlights how we can take advantage of the regular
structure of peptoids to rapidly identify the pharmacophore of a
bioactive molecule. Current efforts are focused on optimizing the
fit of the important side chains with the receptor ECD to improve
the potency of the compound.
-500
-4
-3
-2
-1
0
1
2
3
4
μ
log [unlabeled peptoid]
M
Acknowledgments
Figure 5. GU40C truncation study results. (A) Downward arrows show GU40C
truncation positions (B) Competitive binding assay results; increasing concentra-
tions of unlabeled GU40C and truncated derivatives were competed with a constant
amount of fluoresceinated GU40C. Symbols represent; GU40C (h), 8-mer (N), 7-mer
(.), 6-mer (e), 5-mer (s), 4-mer (j), control (Â).
This work was supported by a contract from the National Heart,
Lung, and Blood Institute (NO1-HV-28185) for the UT Southwest-
ern Center for Proteomics Research, a grant from the Welch Foun-
dation (I-1299), and the Effie Marie Cain Scholarship for
Angiogenesis Research.
scanning data, confirms that some of the backbone amide bonds
within the first five residues participate in the binding event. We
also synthesized and tested GU40C(3) (Fig. 4A), a compound con-
taining the essential side chains and two amide groups C-terminal
to these residues, but which are out of register with the amides in
the parent peptoid. This compound had no detectable affinity for
the receptor ECD (Fig. 4B).
To identify the main chain amides important for binding, we
synthesized five different truncated versions of GU40C, each
containing all four of the N-terminal residues (Fig. 5A). Increasing
concentrations of these truncated versions were competed with a
constant amount of fluorescein-conjugated GU40C for binding to
the VEGR2 ECD. The results are shown in Figure 5B. Unlabeled
GU40C competed efficiently with its labeled counterpart as
expected (Fig. 5B). Elimination of the first C-terminal residue
weakened binding about 10-fold (Fig. 5B). Further elimination of
the next three C-terminal residues further diminished binding only
slightly. However, deletion of the next residue essentially
abolished binding of the peptoid to the VEGFR2 ECD.
In summary, the data described above have defined the minimal
pharmacophore of the peptoid VEGFR2 antagonist GU40C4. Only
three side chains are important for peptoid-receptor binding (the
6th through 8th counting from the C-terminus; Fig. 6) as shown
by glycine and sarcosine scanning (Fig. 2). However, a smaller
peptoid containing only these residues is inactive (Fig. 3). Further-
more, an analysis of truncated derivatives of GU40C showed that
elimination of the first C-terminal residue reduced the affinity of
the peptoid by about 10-fold and removal of the fifth residue
essentially abolished binding. Combined with the insensitivity of
the binding affinity to the removal of the side chains at these
residues, these data argue that it is the main chain residues at posi-
Supplementary data
Experimental details, structures of glycine, sarcosine and trun-
cated derivatives of GU40C and their MALDI-TOF confirmation
data. Competitive binding assay data. Supplementary data associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2008.07.023.
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