Novel inverse binding mode of indirubin derivatives yields improved selectivity for DYRK kinases

Article abstract of DOI:10.1021/ml300207a

DYRK kinases are involved in alternative pre-mRNA splicing as well as in neuropathological states such as Alzheimer's disease and Down syndrome. In this study, we present the design, synthesis, and biological evaluation of indirubins as DYRK inhibitors wi

Full text of DOI:10.1021/ml300207a

Letter
pubs.acs.org/acsmedchemlett
Novel Inverse Binding Mode of Indirubin Derivatives Yields
Improved Selectivity for DYRK Kinases
Vassilios Myrianthopoulos,†,∇ Marina Kritsanida,†,∇ Nicolas Gaboriaud-Kolar,† Prokopios Magiatis,†
Yoan Ferandin,‡ Emilie Durieu,‡ Olivier Lozach,‡ Daniel Cappel,§ Meera Soundararajan,⊥
Panagis Filippakopoulos,⊥ Woody Sherman,∥ Stefan Knapp,⊥ Laurent Meijer,‡,# Emmanuel Mikros,*,†
and Alexios-Leandros Skaltsounis*,†
†Department of Pharmacy, University of Athens, Panepistimiopolis Zografou, GR-15771, Athens, Greece
‡Station Biologique de Roscoff, CNRS, 'Protein Phosphorylation and Human Disease' Group, Place G. Teissier, BP 74, 29682
Roscoff, Bretagne, France
§Schrodinger GmbH, Dynamostrasse 13, D-68165 Mannheim, Germany
̈
∥Schrodinger Inc., 120 West 45th Street, 17th Floor, New York, New York 10036, United States
̈
⊥Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research
Building, Roosevelt Drive, Oxford OX3 7DQ, United Kingdom
#ManRos Therapeutics, Centre de Perharidy, 29680 Roscoff, France
S
* Supporting Information
ABSTRACT: DYRK kinases are involved in alternative pre-mRNA splicing as well as in
neuropathological states such as Alzheimer's disease and Down syndrome. In this study, we
present the design, synthesis, and biological evaluation of indirubins as DYRK inhibitors with
enhanced selectivity. Modifications of the bis-indole included polar or acidic functionalities at
positions 5′ and 6′ and a bromine or a trifluoromethyl group at position 7, affording analogues
that possess high activity and pronounced specificity. Compound 6i carrying a 5′-carboxylate
moiety demonstrated the best inhibitory profile. A novel inverse binding mode, which forms
the basis for the improved selectivity, was suggested by molecular modeling and confirmed by
determining the crystal structure of DYRK2 in complex with 6i. Structure−activity
relationships were further established, including a thermodynamic analysis of binding site
water molecules, offering a structural explanation for the selective DYRK inhibition.
KEYWORDS: DYRK inhibitors, indirubins, kinase selectivity, pre-mRNA splicing, docking calculations, WaterMap
he dual-specificity tyrosine phosphorylation-regulated
development of potent kinase inhibitors.11−17 Regardless of the
Tkinase (DYRK)/minibrain family of protein kinases notable selectivity of the various indirubin analogues, they all
share a common interaction pattern with their target enzyme
active site (for example, see PDB structures 1E9H, 2BHE,
1UV5, and 1UNH). Recently, the structural basis of selective
inhibition of CLK3, a closely related kinase, by leucettamine-B
derivatives was reported.6 Interestingly, the cocrystal structure
revealed a significantly different binding mode from the typical
ATP-competitive interaction pattern. A similar binding
orientation was subsequently reported as the structural basis
for selective DYRK1a inhibition by harmine.7 Those studies
prompted us to investigate whether selective indirubin
analogues targeting the closely related DYRK2 could be
designed.
represents a relatively unexplored portion of the human
kinome in terms of inhibitor development. The DYRK1a
gene is localized in the Down syndrome (DS) critical region of
chromosome 21, and its overexpression has been related to the
development of DS features.1 DYRK2 regulates p53 to induce
apoptosis in response to DNA damage and serves as a scaffold
for an E3 ubiquitin ligase complex.2,3 It is mainly expressed
during development, but it has been found to be overexpressed
in adenocarcinomas of the esophagus and lung.4 DYRK2 has
been recently identified as the priming phosphorylation enzyme
for oncogenes c-Jun and c-Myc, although the precise role of
DYRK2 in cancer needs further investigation.5 As such, DYRKs
can be regarded as emerging targets, and selective inhibitors
could assist in elucidating the various biochemical mechanisms
in which they are implicated.
The initial series of inhibitors had minor modifications on
the indirubin core that were designed with the objective of
generating a noncanonical binding mode and subsequently an
A limited number of compounds demonstrate selective
inhibition toward DYRK kinases, although binding with cdc2-
like kinases (CLKs) is frequently observed.6−10 The bis-indole
indirubin has been utilized as an interesting lead for the
Received: July 19, 2012
Accepted: November 1, 2012
Published: November 1, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Table 1. IC50 Values (μM) of Indirubin Analogues Binding to Five Kinases
IC50 (μM)
compd
R1
R2
R3
R4
R5
R6
CDK5
GSK3
CK1
DYRK1a
DYRK2
6BIO
7BIO
5a
5b
5c
5d
5e
5f
NOH
NOH
O
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.083
>10
>10
>10
3.40
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.20
>10
>10
0.52
0.53
>10
>10
>10
>10
>10
1.6
0.005
>10
>10
>10
1.10
25
1.2
1.7
2.1
Br
H
>10
>10
0.58
1.40
>10
>10
>10
>10
>10
1.70
>10
>10
9.30
>10
>10
0.80
>10
>10
0.30
0.42
>10
>10
>10
>10
>10
4.2
1.9
1.3
Br
CH3
H
>10
>10
2.00
>10
>10
>10
>10
>10
2.60
>10
>10
1.30
>10
>10
1.10
>10
>10
>10
0.31
>10
>10
>10
>10
0.21
0.41
>10
0.13
0.63
>10
0.11
0.60
7.0
>10
>10
1.80
>10
>10
>10
>10
>10
1.10
>10
>10
1.30
>10
>10
ND
>10
>10
>1
O
COOCH3
COOH
COOCH3
COOCH3
CN
H
O
H
H
O
Br
H
O
CF3
CF3
CF3
Br
H
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.20
>10
>10
0.40
0.07
>10
>10
>10
>10
>10
1.2
O
H
5g
5h
5i
O
tetrazole
COOCH3
H
H
O
CH3
H
O
COOH
H
5j
O
H
COOCH3
Br
H
5k
5l
O
H
COOCH3
CF3
Br
H
O
H
COOH
H
5m
5n
5o
6a
6b
6c
6d
6e
6f
O
H
COOH
CF3
Br
H
O
CH2OH
CHNOH
H
H
H
O
H
Br
H
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
NOH
H
CF3
Br
H
H
H
CH3
H
COOCH3
COOH
COOCH3
COOCH3
CN
H
H
H
H
H
0.35
>10
>10
>10
>10
0.13
ND
>10
0.22
1.90
>10
ND
1.70
>10
ND
ND
H
Br
H
H
CF3
CF3
CF3
Br
H
6g
6h
6i
H
H
tetrazole
COOH
COOH
COOCH3
COOH
H
H
H
H
H
6j
H
CF3
Br
H
6k
6l
H
CH3
CH3
H
>10
0.2
>10
>10
6.10
>10
0.1
>10
>10
0.80
>10
5.0
H
Br
6m
6n
6o
6p
6q
6r
COOH
COOCH3
COOCH3
COOH
COOH
H
H
3.30
>10
1.7
H
Br
H
H
CF3
Br
H
H
H
>10
3.1
>10
>10
0.40
0.60
7.40
>10
>10
7.40
H
CF3
Br
H
CH2OH
CHNOH
H
0.40
0.60
1.60
2.10
6s
H
Br
H
a
All measurements were determined in triplicate, and mean values are reported. The standard error of determination in all cases does not exceed
10%. The Km for ATP with DYRK2 is 7.7 μM.
improved selectivity profile toward DYRKs. A bulky mod-
ification such as bromine or trifluoromethyl was introduced at
position 7 based on previous studies of the analogue 7BIO (7-
bromoindirubin-3′ oxime; Table 1) that showed a minor
negative influence on affinity due to a putative steric clash
between the inhibitor and the kinase hinge.15 In addition, the
removal of a hydrogen bond donor from the indirubin core by
methylating the lactam nitrogen at position 1 was explored as a
more drastic way to perturb the interactions. Such methylated
analogues are totally inactive against CDKs and glycogen
synthase kinase 3β (GSK3β) and serve as negative control
compounds.13 However, the low water solubility of the bis-
indole system necessitated the introduction of polar or
ionizable functionalities such as carbomethoxy, methylene
hydroxy, aldehyde, carboxy, tetrazole, cyano, and oxime groups
at positions 5′ and 6′. Moreover, positions 5′ and 6′ represent
the least explored sites of this scaffold. Thus, results would
contribute toward a systematic exploration of the indirubin
SAR landscape. The routes employed for derivative synthesis
are summarized in Schemes S1 and S2 in the Supporting
Information. All compounds were evaluated against a panel of
five kinases (Table 1).
7BIO showed significant gains in selectivity toward DYRKs
as compared with 6BIO. Derivatives 5c, 5i, 6d, and 6m lacking
a 7-bromo substitution showed a diminished selectivity toward
DYRKs. Results also suggested an anionic functionality to
maintain efficient inhibition, as protection of the acidic moiety
abolished DYRK activity of 7-substituted methylesters 5d, 5e,
5h, 6e, and 6f. The difference of IC50 values between analogues
6d and 6m indicated 5′ rather than 6′ as the preferred position
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ACS Medicinal Chemistry Letters
Letter
for an acidic substitution to maintain DYRK inhibition. The
presence of a 3′-oxime moiety generally improved potency with
minor effects on selectivity. Finally, the influence of methyl-
capping N1 was unfavorable for DYRK activity (7BIO as
compared to 6b). Inspection of the SAR landscape suggested
that the combination of the 7-bromo, 3′-oxime, and 5′-carboxy
substitutions would be optimal for DYRK selective inhibition.
The corresponding 7-bromo-5′-carboxyindirubin-3′ oxime (6i)
proved to be a potent inhibitor of DYRK1a and DYRK2 (IC50
values 210 and 130 nM, respectively). DYRK activity was
accompanied by a marked selectivity, as 6i was inactive toward
cyclin-dependent kinase 5 (CDK5), GSK3β, and casein kinase
1 (CK1). Interestingly, the introduction of the bulky bromine
in position 7 enhanced selective DYRK inhibition only when
combined with the carboxylate substitution, which was evident
comparing 6d and 6i. Quite unexpectedly, the carboxylate
bioisostere tetrazole compounds (5g and 6h) were inactive.
Moreover, analogues where the carboxylate moiety was
replaced by a polar but neutral oxime group (5o and 6s)
showed good DYRK inhibition but poor selectivity, despite of
the concurrent 7-bromine substitution. The optimal effect of
bromine in 6i was denoted by the lack of selectivity of 6j
carrying the closely related substituent CF3 at position 7.
Surprisingly, the N1-methylated analogue of 6i (compound 6l)
retained selectivity and affinity for DYRK kinases with small
variations in IC50 values determined for the two family
members (IC50 of 130 nM for DYRK1a and 220 nM for
DYRK2).
To rationalize the affinity and selectivity profiles, we analyzed
poses from previous docking studies to a DYRK1a-derived
homology model of DYRK2, which showed that compound 6i
may adopt a binding mode that is significantly different from
the experimentally established indirubin-kinase binding mode.12
Two distinct modes were observed in the docking studies,
distinguished by a 180° flip of the indirubin core with respect to
either its primary or secondary axis (Figures S1A and S1B in
the Supporting Information, respectively). In both cases, the
usually observed hydrogen bond triplet between the indirubin
pharmacophore and the kinase hinge was either disrupted
(mode I) or inverted (mode II). In both cases, the 5′
carboxylate formed a salt bridge with either the Lys165 (mode
I) or the catalytic Lys178 (mode II).
To confirm the inverse binding mode, we determined the
crystal structure of 6i bound to DYRK2 at a resolution of 2.28
Å (PDB ID 3KVW). The structure (Figures 1A and S2 in the
Supporting Information) revealed that the binding mode was
indeed inverted with respect to the geometry of all previously
reported indirubin-kinase complexes and consistent with the
predicted binding mode II. The predicted salt bridge anchoring
the anionic 5′-carboxylate to the side chain of Lys178 was
observed in the crystal structure. An additional hydrogen bond
was present between the 5′ carboxylate and the NH backbone
of Asp295. Furthermore, three water molecules participated in
the hydrogen bond network by bridging the inhibitor
carboxylate and Lys178 ammonium groups to the side chains
of Asp295 and Glu193. At the hinge, two hydrogen bonds were
formed between the lactam carbonyl of 6i and the NH
backbone of Leu231 (n + 3 from the gatekeeper) and between
the lactam nitrogen of the inhibitor and the backbone carbonyl
of the same residue. The solvent-accessible surface area buried
upon binding was 417 Å2 (229.4 Å2 nonpolar) for the ligand
and 254.8 Å2 (222.8 Å2 nonpolar) for the protein, underscoring
the hydrophobic nature of this binding site. The bromine at
Figure 1. (A) Crystal structure of the 6i-DYRK2 complex revealed a
nontypical binding mode, in which the orientation of the indirubin
core was in good accordance with the predicted binding mode. The
water molecule displaced by the carboxylate group of 6i is depicted as
a yellow sphere. (B) Unfavorable DYRK2 hydration sites (>1.4 kcal/
mol) from WaterMap calculations on the DYRK2 apo crystal
structure. Hydration sites are colored by free energy, with red being
the most unfavorable. The hydration site adjacent to Lys178 (ΔG =
1.6 kcal/mol) is shown as a large semitransparent blue sphere, and the
crystallographic water is shown as a small opaque blue sphere.
Compound 6i is superimposed to show the displacement of the
hydration sites. The view has been rotated to improve visibility of the
key water.
position 7 was almost half-exposed to the solvent. Encourag-
ingly, docking calculations using Glide with the crystal structure
of DYRK2 predicted mode II as the best pose of 6i.
Interestingly, docking in the crystal structure of DYRK2
showed that 7BIO would also adopt the inverted mode, while
6BIO was predicted to bind in the usual indirubin-kinase
orientation, further supporting the putative role of the flipped
binding mode in determining selectivity.12
A superposition of the inhibitor cocrystal structure of
DYRK2 with that of the apoenzyme (PDB ID 3K2L,
manuscript in preparation) revealed that the 5′-carboxylate
moiety of 6i overlaps with a crystallographic water molecule
appearing in the active site of the apoenzyme and interacting
with Lys178 (Figure 1A). To understand the thermodynamic
characteristics of the system and that particular water molecule,
calculations were run with the WaterMap program, which
combines molecular dynamics, solvent clustering, and statistical
thermodynamics to assess the enthalpy, entropy, and free
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ACS Medicinal Chemistry Letters
Letter
energy of water “hydration sites”.18 WaterMap has been
successfully applied to study selectivity in kinases and PDZ
domains, as well as several studies of understanding binding
affinity and SAR series.19−21 Figure 1B shows the predicted
WaterMap hydration sites in the apoenzyme. The site near
Lys178 is in near-perfect accordance with the crystallographic
water. This hydration site has a thermodynamic profile making
the total free energy slightly worse than bulk water (+1.5 kcal/
mol). While the site is highly unfavorable entropically (+3.4
kcal/mol) due to the localization around Lys178, it is
enthalpically favorable (−1.9 kcal/mol) due to the interactions
with Lys178. Displacement of this hydration site by a ligand
functional group that also replaces the water interactions is
predicted to improve potency, in agreement with the
experimental 10-fold affinity difference between 7BIO and its
carboxylated analogue 6i. This shows the importance of
including water molecules in the analysis and assessment of
binding energies.
The selectivity of 6i and 6l toward DYRKs prompted us to
investigate their inhibition profile over a broader panel of
protein kinases. Compounds 6i and 6l along with 6BIO and
7BIO were assayed in vitro against a panel of 42 kinases (Table
S1 in the Supporting Information). Compounds 6i, 6l, and
7BIO were inactive against all assayed kinases. In contrast,
6BIO showed a broad inhibitory profile. Apart from its well-
established target, GSK-3β, 6BIO was weakly active toward the
receptor tyrosine kinases fibroblast growth factor receptor 3
(FGFR3) and platelet-derived growth factor receptor
(PDGFR) and showed a notable inhibition of proto-oncogene
tyrosine-protein kinase receptor (RET). Although the crystal
structure of 6l-DYRK2 was not determined, the similar
specificity profile of 6i and 6l suggests that the presence of
the N1-methyl does not induce an alternative binding mode.
To conclude, the combined presence of a bromine
substitution at position 7 and an acidic functionality at position
5′ of the indirubin scaffold turns the nonselective bis-indole
indirubin into a potent and selective DYRK inhibitor. Structural
insights offered by docking, crystallographic studies, and solvent
thermodynamic calculations suggest that selective DYRK
inhibition can be attributed to a nonstandard kinase binding
mode where the indirubin core adopts an inverted pose. Data
indicate that the driving force for the inverted binding
orientation is the occurrence of a steric clash between the
bulky halogen of position 7 and the kinase hinge. The acidic
substitution at position 5′ further enhances activity by
displacing an unstable water and establishing a salt bridge
between the 5′-carboxylate and the Lys178. The need for the
simultaneous substitutions at position 5′ and 7 was evident by
the fact that neither of the two substitutions alone resulted in
the desired activity-selectivity profile. As a result, the desired
selectivity profile was achieved with an ATP-competitive but
not ATP-mimetic inhibitor using a variety of rational design
strategies.22
Accession Codes
The 6i-DYRK2 structure has been deposited to the PDB with
accession code 3KVW.
AUTHOR INFORMATION
Corresponding Author
*Tel: +302107274813. E-mail: mikros@pharm.uoa.gr (E.M.).
Tel: +302107274598. E-mail: skaltsounis@pharm.uoa.gr
(A.-L.S.).
■
Author Contributions
∇These authors contributed equally.
Funding
This research was supported by grants from the “Fonds Unique
Interminister
“Association France-Alzheimer (Finister
́ ̂
Sante Bretagne” (L.M.), “Fondation Jero
́
iel” (FUI) PHARMASEA project (L.M.), the
e)” (L.M.), “CRITT-
me Lejeune” (L.M.),
̀
́
and EU-FP7REGPOT-2011 project INsPiRE (284460) (V.M.).
S.K. and M.S. are supported by the SGC, a registered charity
(number 1097737) that receives funds from the Canadian
Institutes for Health Research, the Canada Foundation for
Innovation, Genome Canada, GlaxoSmithKline, Pfizer, Eli Lilly,
the Novartis Research Foundation, Takeda, the Ontario
Ministry of Research and Innovation, and the Wellcome
Trust. P.F. is supported by a Wellcome Trust Career
Development Fellowship (095751/Z/11/Z).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
FGFR3, fibroblast growth factor receptor 3; PDGFR, platelet-
derived growth factor receptor; RET, proto-oncogene tyrosine-
protein kinase receptor; CDK5, cyclin-dependent kinase 5;
GSK3β, glycogen synthase kinase 3β; CK1, casein kinase 1
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