Profile and molecular modeling of 3-(indole-3-yl)-4-(3,4,5- trimethoxyphenyl)-1H-pyrrole-2,5-dione (1) as a highly selective VEGF-R2/3 inhibitor

Article abstract of DOI:10.1021/jm0609871

We report on selectivity profiling of 1 in a panel of 20 protein kinases and molecular modeling indicating 1 to be highly active and selective for VEGF-R2/3. Sequence alignment analysis and detailed insights into the ATP binding pockets of targeted protei

Full text of DOI:10.1021/jm0609871

J. Med. Chem. 2006, 49, 7549-7553
7549
Profile and Molecular Modeling of 3-(Indole-3-yl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrrole-2,5-
dione (1) as a Highly Selective VEGF-R2/3 Inhibitor
Christian Peifer,*,† Agata Krasowski,† Nina Ha¨mmerle,† Oliver Kohlbacher,‡ Gerd Dannhardt,§ Frank Totzke,#
Christoph Scha¨chtele,# and Stefan Laufer†
Department of Pharmacy, Eberhard Karls UniVersity, Auf der Morgenstelle 8, D-72076 Tu¨bingen, Germany, Center for Bioinformatics,
Eberhard Karls UniVersity, Sand 14, D-72076 Tu¨bingen, Germany, Department of Pharmacy, Johannes Gutenberg UniVersity, Staudingerweg
5, D-55099 Mainz, Germany, and ProQinase GmbH, Breisacherstrasse 117, D-79106 Freiburg, Germany
ReceiVed August 16, 2006
We report on selectivity profiling of 1 in a panel of 20 protein kinases and molecular modeling indicating
1 to be highly active and selective for VEGF-R2/3. Sequence alignment analysis and detailed insights into
the ATP binding pockets of targeted protein kinases from the panel result in a unique structural architecture
of VEGF-R2 mainly caused by the hydrophobic pocket I, determining the molecular basis for activity and
selectivity of 1.
Introduction
cancer-relevant kinases.14 Tyrosine kinases,21 including vascular
endothelial growth factor receptor (VEGF-Ra) 2/3, are known
to play an important role in oncology and are useful in the
development of clinicaly effective inhibitors. Therefore, inhibi-
tors of the tyrosine group of PKs are the focus of vibrant
research activities.22,23
Protein kinases (PKs) comprise 22% of the druggable genome
and are currently among the most important and promising target
classes for drug discovery.1 PKs regulate signal transduction
by phosphorylating tyrosine, threonine, and serine residues in
key proteins involved in signal pathways with significant
relevance to many diseases. Thus, small-molecule inhibitors of
these pathways can be used to treat diseases such as cancer,2
diabetes, and inflammation.3,4 Since all 518 PKs of the kinome
use ATP as a cofactor to carry out the phosphorylation of
specific signal proteins, they share a highly conserved ATP
binding pocket that serves as the molecular binding site of most
inhibitors.5 To make things even more complex, there are
different expression levels of hundreds of PKs and cell signaling
proteins in different organs. Furthermore, the phosphorylation
status depends on complex physiological and pathophysiological
situations in patients.6 Thus, for drug candidates that interfere
with PKs, it is essential to have an understanding of the
structural basis for selectivity and specifity within the kinome
(and beyond7) and a method for the robust evaluation of potency
and selectivity.8,9 Currently, there is no consensus whether the
concepts of single-targeting, group-targeting, multitargeting, or
even promiscous inhibitors across kinase space may result in
potential therapeutical benefits.10,11 To extensively address the
topic of kinase selectivity of a preclinical compound, it would
be optimal to screen and characterize its activity against all 518
PKs of the kinome. However, an appropriate approach in early
drug discovery would be to evaluate potency against a significant
panel of selected members of PK families12 and to evaluate its
activity in cellular assays.10 Clinical data will ultimately tell us
if custom-made or promiscous inhibitors of PKs can provide
therapeutic benefits.13 Most research activities regarding the
development of small-molecule inhibitors of PKs address cancer-
related tyrosine kinases (TK),14 and to date, six ATP-competitive
small-molecule inhibitors with very different selectivity profiles
have been approved as anticancer drugs (Table 1).
In our recent study, 1 (Figure 1), with remarkable antiangio-
Figure 1. Structures of 1, 2, and staurosporine.
genic activity, was discovered from a small set of 2,3-
diarylmaleimide derivatives tested in the in vivo chick embryo
assay.24,25 To specify the molecular target, 1 was initially
screened in a panel of 12 selected protein kinases and was
consequently found to have an IC50 of 2.5 nM for the angiogenic
key kinase VEGF-R226 and therefore determined to be a highly
potent VEGF-R2 tyrosine kinase inhibitor.25 Because this
approach revealed the molecular target for 1 but with no detailed
information about kinase selectivity, we were interested in
investigating the inhibitory profile of 1 with respect to other
kinases.
Selectivity Profiling of 1
Here, we report on selectivity profiling of 1 in a panel of 20
therapeutically relevant PKs of different families of the kinome
(Figure 2) and molecular modeling studies indicating 1 to be
highly active and selective for VEGF-R2 and VEGF-R3. The
PKs have been selected for the determination of an IC50 profile
of 1 because they are involved in malignant diseases and because
they represent different PK families. As can be seen in Table
2, 1 is actually selective over the homologous TKs including
EGF-R, ERbB2,27 TIE2,28 PDGFRâ,29 SRC,30 and FAK.31,32
In addition, many PK inhibitors are currently in preclinical
development and in clinical trials, mainly as inhibitors for
* To whom correspondence should be addressed. Phone: 0049 7071 29
75278. Fax: 0049 7071 29 5037. E-mail: Christian.Peifer@uni-tuebin-
gen.de.
a Abbreviations: VEGF-R, vascular endothelial growth factor receptor;
EGFR, epidermal growth factor receptor; BCR-ABL, chromosomes encod-
ing the kinase; c-KIT, protooncogene encoding KIT (a TK receptor for the
mast cell growth factor); mPDGFR-â, platelet derived growth factor
receptor; FLt3, FMS-like receptor tyrosine kinase 3; p38R MAPK, mitogen
activated PK; SRC, family of protooncogenes encoding nonreceptor tyrosine
kinases; EPHA2, ephrin receptor A2.
† Department of Pharmacy, Eberhard Karls University.
‡ Center for Bioinformatics, Eberhard Karls University.
§ Johannes Gutenberg University.
# ProQinase GmbH.
10.1021/jm0609871 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/14/2006

7550 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Brief Articles
Table 1. Protein Kinase Inhibitors Currently Approved as Drugs, Their Predominantly Target Kinases, and Their Clinical Use
Table 2. IC50 Values (M) of 1 in a Panel of 20 Kinase Assays
compounds in the kinome space, the selectivity profile needs
to be expanded.
assay
IC50 (M)
assay
IC50 (M)
assay
IC50 (M)
EPHB4
IGF1-R
SRC
1.6 × 10-5 TIE2
9.9 × 10-6 ABL1
2.0 × 10-5 INS-R
2.8 × 10-6 GSK3-â
9.3 × 10-5 NEK2
2.3 × 10-5 FAK
1.1 × 10-6
>1 × 10-4
9.7 × 10-7
>1 × 10-4
9.9 × 10-6
2.5 × 10-5
Binding mode of 1 in VEGF-R2
VEGF-R2 2.5 × 10-9 PLK1
>1 × 10-4
AKT1
Molecular modeling studies revealed an interesting and
reasonable binding mode of 1 in the ATP pocket of VEGF-R2,
which fully explains the in vitro selectivity for VEGF-R2 over
the other PKs. In detail, hydrogen bonds to the hinge backbone
carbonyl of Glu915 and to the backbone amine of Cys917 are
formed. The indole ring is located in the hydrophobic region I
with π-π stacking interactions to Phe1045, whereas the
trimethoxyphenyl moiety is situated in the hydrophobic region
II. No direct hydrogen bond acceptor within a radius of 4 Å of
the indole-NH could be found in the protein structure.
However, SAR (in vitro VEGF-R2 assay) revealed that the
free indole NH in 1 as optimal and methylation (2, Figure 1)
decreased activity 100-fold, indicating an important ligand-
protein interaction for the NH moiety.25 Since this is the only
detail in the modeled binding mode that is in conflict with the
SAR data, we postulate that a water molecule can perhaps
mediate NH-protein hydrogen bonding that is not detected by
the docking experiments (Figure 3). First, one can argue that
the sugar pocket consisting of Arg1030 and Asn1031 is located
within the 6 Å distance required for water-mediated interactions
and is therefore available to accept H-bonds. Second, the π-π
stacking interactions of 1 to Phe1045 position the indole moiety
in such a way that the indole-NH points in a geometrically
convenient direction toward the sugar pocket. Third, this
exposed area of the ATP binding pocket is supposed to be
accessible to water molecules. Furthermore, the effect of water
molecules on ligand binding interactions in the ATP pocket of
the cyclin-dependent kinase 2 (CDK2) was found to be pivotal.42
The water hypothesis in this study was examined using the
program GRID by calculating preferred water H-bond interac-
tion spots toward the ATP binding site of VEGF-R2. Indeed,
between Arg1030, Asn1031 and the position of indole NH, a
VEGF-R3 5.0 × 10-9 PDGFR-â
6.8 × 10-7 Aurora-A
8.2 × 10-6 Aurora-B
9.9 × 10-6
EGF-R
ERbB2
3.2 × 10-5 CDK2/CycA
5.0 × 10-5 CDK4/CycD1
Compound 1 inhibited VEGF-R2/3 in the low nanomolar range,
but compared to VEGF-R2, the kinases PDGFRâ inhibited in
an almost 300-fold higher concentration (IC50 ) 6.8 × 10-7
M), FAK (IC50 ) 9.7 × 10-7 M) and the other PKs (EPHB4,33
IGF1-R,34 ABL1,35 INS-R,36 PLK1,37 CDK2/4,38 GSK3-â,39
NEK2,40 Aurora A/B41) in only the micromolar range. In
particular, the selectivity of 1 for VEGF-R2/3 over INS-R is
important because a critical requirement for a PK inhibitor to
be further developed is to avoid disturbing glucose homeosta-
sis.36 However, for an optimal characterization of this class of
Figure 2. Panel of 20 therapeutically relevant PKs of the kinome for
selectivity profiling of 1. Key targets VEGF-R2/3 are highlighted. The
phylogenetic tree of kinases (kinome) was adapted from the literature.5

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 7551
Figure 3. Crystallographically determined ATP binding site of VEGF-
R2 (PDB code 1y6a) and modeled binding mode of 1 (yellow). Key
residues and hydrogen bonds are labeled. For clarity, the water molecule
is shown in a hypothetical position. The working hypothesis of water-
mediated ligand-protein interactions was subsequently investigated by
methods of molecular modeling.
Figure 5. Crystallographically determined ATP binding site of FAK
(PDB code 1MP8) and modeled binding mode of 1 (white). Key
residues and hydrogen bonds are labeled. Compared to the ATP pocket
of VEGF-R2, hydrophobic region I in FAK is less spacious (indicated
by red arrow), causing a twisted and suboptimal binding of the indole
moiety.
Figure 6. Sequence alignment of ATP binding pocket areas of targeted
PKs (with PDB code and name) for key residues in VEGF-R2, (F)
Phe1045 in a DFGL motif, and (R) Arg1030/(N) Asn1031 in an ARN
motif. Positions in TKs are as follows: SRC (F)333 (N)319; FAK (F)-
565 (N)551; EGFR (F)856 (N)842; ABL1 (F)382 (N)368. Positions in
AGC kinase (containing PKA, PKG, PKC families) are Aurora-B (F)-
235 (N)221. Positions in CMGC kinases (containing CDK, MAPK,
GSK3, CLK families) are CDK2 (F)146 (N)132, GSK3 (F)201 (N)-
187.
Figure 4. Crystallographically determined ATP binding site of VEGF-
R2 (PDB code 1Y6A). Binding mode of 1 (green) was modeled and
minimized involving a flexible complex of one molecule of water, 1,
and Glu915/Cys917/Arg1031/Asn1031. Key residues and hydrogen
bonds are labeled.
formed and the trimethoxyphenyl moiety is situated in hydro-
phobic region II. But significantly different from the ATP
binding site of VEGF-R2, in FAK the indole moiety of 1 is
twisted out of the minor hydrophobic region I, presumably
forming a cation-π stacking with protonated Lys454 (which
itself is fixed by a salt bridge with Glu471). Taken together,
because of the small dimensions of hydrophobic region I and
the resulting suboptimal orientation of the indole moiety in FAK,
the key water-mediated hydrogen bond to the sugar pocket
(Arg550/Asn551) is prevented. This binding mode is in ac-
cordance with an almost 400-fold loss of activity for 1 in the
FAK assay compared to VEGF-R2.
Even if accurate and realistic in silico docking remains a
challenge, the binding modes of 1 in VEGF-R2 and FAK are
consistent with SAR in the in vitro selectivity profile and can
serve as rational models explaining the high activity for VEGF-
R2 and the selectivity over FAK. As mentioned above, in this
binding mode, residue Phe1045 of hydrophobic pocket I in
VEGF-R2 is key, fixing the indole moiety into a water-mediated
interaction to Arg1030/Asn1031, accounting for high activity
and selectivity of 1 in VEGF-R2. To evaluate the position of
the conserved Phe in the other PKs of the panel, we aligned
the sequences of the targeted PKs for which PDB structures
were published (Figure 6).
favorable water location was found. On the basis of the
calculated location, we placed the virtual water molecule and
subsequently minimized a flexible complex of Arg1030/Asn1031/
water and 1, maintaining the H-bonds to Glu915 and Cys917.
This approach revealed an expedient involvement of water in
the binding mode (Figure 4) with water/carbonyl-Arg1030 )
2.14 Å, water/carbonyl-Asn1031 ) 1.92 Å, and water/NH-1
) 2.12 Å.
Binding mode of 1 in FAK
Having determined a possible binding mode for 1 in VEGF-
R2, which is supported by SAR data of the compound for this
enzyme, we were particularly interested in the issue of selectivity
over the other protein kinases. Since 1 is structurally related to
the unselective “pankinase” inhibitor staurosporine9 (Figure 1),
we investigated whether the binding modes of 1 in VEGF-R2
and other TKs account for the unexpected selectivity profile.
To address this, the binding mode of 1 in VEGF-R2 (PDB code
1y6a)43 was compared to docking results of 1 in FAK. We used
the ATP pocket of FAK (PDB code 1MP844 (IC50(1) ) 1 µM)
for the docking experiments since there is no crystallographically
based protein structure published for PDGFRâ (IC50(1) ) 680
nM).
Furthermore, by overlaying the backbone of the ATP binding
pocket of VEGF-R2 with targeted PKs of the panel (for which
PDB structures were published), we examined the orientation
of Phe and Asn (key residues in the binding mode of 1 in VEGF-
R2) in each of the kinases (Figure 7). Although highly conserved
in all these PK sequences, the Phe residue can be found in quite
Thus, a binding mode of 1 in the ATP binding site of tyrosine
kinase FAK was modeled (Figure 5). Comparable to the binding
mode in VEGF-R2, hydrogen bonds to the hinge backbone
carbonyl of Glu500 and to the backbone amine of Cys502 are

7552 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25
Brief Articles
conserved architecture of the ATP binding pocket, it is unlikely
that an ATP-competitive compound with high activity against
a single PK will leave the other 517 PKs unaffected. Potent
VEGF-R inhibitors such as SU5416, SU6668, PTK787, ZK-
222584, CGP-79787(D), CP-547632, and AZD2171 have been
developed with different in vitro selectivities but “reasonable
side effect profiles” and with in vivo efficacy.46 Furthermore,
approved multikinase inhibitors like sunitinib and vatalanib are
also highly active inhibitors of VEGF-R2, and the potent orally
active VEGF-R2 inhibitor ZD6474/vandetanib with inhibitory
activity for EGF-R and RET kinase is currently in clinical
development.47 Therefore, 1 can be useful as a pharmacological
tool for inhibiting VEGF-R2/R3 in signal transduction studies
and a reference compound in in vivo angiogenesis assays.24
Considering the high in vitro potency and selectivity for VEGF-
R2/3, as well as being bioavailable in the chick embryo model,25
1 may have potential for development as a potent and selective
VEGF-R2/3 inhibitor in angiogenic-relevant diseases. However,
an enhanced selectivity profile in the kinome space and
evaluation in further in vivo angiogenic models are underway
to characterize the preclinical potential of 1. Even though 1
shows promising in vitro efficiency with selectivity for VEGF-
R2/3, future studies will answer the question if selective or
promiscuous PK inhibitors can result in clinical benefits.
Experimental Section
Chemistry. Compounds 1 and 2 were synthesized by one-pot
condensation of 3,4,5-trimethoxyphenylacetamide with (indolyl-
3)-ethylglyoxylate for 1 and ethyl-(1-methylindolyl-3)-glyoxylate
for 2 using KOBut and 4 Å molecular sieves in THF.25
Figure 7. Backbone overlay of ATP binding pocket areas of VEGF-
R2 with targeted PKs (PDB structures). Conserved key residues
Phe1045 and Asn1031 in VEGF-R2 are maintained in red and colored
differently in each case. For FAK, the position of Phe565 is not exactly
defined in the PDB structure, and therefore, immediate neighbor residue
Asp564 is highlighted.
Molecular Modeling. The modeling for 1 was performed on a
Fedora Core 3 Linux system. For visualization and building the
structures, Sybyl 7.1 (Tripos Inc., St. Louis, MO) was used. The
Connolly surface was calculated using the MOLCAD module in
Sybyl and colored according to lipophilicity (from very hydrophobic
to hydrophilic, corresponding to brown to green to blue). The active
site in VEGF-R2 and FAK was defined as 6.5 Å surrounding of
the ligand. Compound 1 was docked into the active site of VEGF-
R2 and FAK using the FlexX docking program.48 The FlexX scoring
function was applied during the placement and construction phase
of the ligands, and DrugScore was used for the final ranking.49
The 3D coordinates of the VEGF-R2 catalytic core in complex with
a 2-anilino-5-aryloxazole inhibitor were taken from the Brookhaven
Protein Databank (PDB code: 1Y6A). The 3D coordinates of the
FAK catalytic core in complex with ligand adenosine 5′-diphosphate
were taken from the Brookhaven Protein Databank (PDB code:
1MP8).
Favorable water interaction regions in the ATP binding pocket
of VEGF-R2 (PDB code: 1Y6A) were calculated using the program
GRID 2a (2004).50 Between Arg1030, Asn1031, and the position
of the indole NH, a water spot was found with a minimum
interaction energy of -6.113 kcal/mol.
The overlay and visualization of structures were performed with
BALLView,51 version 1.1.1 (07.01.2006) using the 3D coordinates
of the Brookhaven Protein Databank PDB structures 1Y6B (VEGF-
R2), 1BYG (SRC), 1MP8 (FAK), 1XKK (EGFR), 1M52 (ABL1),
2BFY (AUR-B), 2C6I (CDK2), 1UV5 (GSK3).
Figure 8. Sequence analogy of highly homologous ATP binding pocket
areas of VEGF-R2 and VEGF-R3. Key residues in VEGF-R2 are as
follows: (F) Phe1045 and (R) Arg1030/(N) Asn1031. Key residues in
VEGF-R3 are as follows: (F) Phe1056 and (R) Arg1041/(N) Asn1042.
different orientations whereas residue Asn remains in almost
the same position, as indicated in Figure 7. However, in VEGF-
R2 only, Phe1045 is situated in a suitable position for the indole
moiety to make water-mediated interaction, as discussed in the
binding mode. Thus, in combination with the other ligand-
protein interactions, Phe1045 especially provides the molecular
basis for activity and selectivity of 1 in this important drug
target. Finally, X-ray crystallographic data of a ligand-protein
complex will confirm the modeled binding mode in this study.
Compound 1 was also found to be active for VEGF-R3 in
the low nanomolar range (IC50 ) 5 nM), but no crystal structure
was published to perform modeling experiments at the ATP
binding site of this kinase. Sequence comparison of VEGF-R2/
VEGF-R3 (Figure 8) shows significant homology, and therefore,
the orientation of Phe and Asn can be considered to be
comparable between these kinases, resulting in a comparable
binding mode in VEGF-R3 for 1.
Acknowledgment. Financial support by Fonds der Chemis-
chen Industrie is gratefully acknowledged.
Supporting Information Available: Results of 1 (at 10 µM)
in an initial screening panel of 12 kinase assays and experimental
section for selectivity profiling 1 using 20 kinases by IC50 values.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Pharmacological and Clinical Relevance of VEGF-R
Inhibitors
In light of the biological role of VEGF-R2 as an essential
factor for the function of endothelial cells and VEGF-R3 for
lymphendothelial cells,45 the inhibition of both kinases can result
in a strong antiangiogenic effect. However, because of the
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