NVP-BHG712: Effects of Regioisomers on the Affinity and Selectivity toward the EPHrin Family

Article abstract of DOI:10.1002/cmdc.201800398

Erythropoietin-producing hepatocellular (EPH) receptors are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates intracellular kinase activity. EPHs are key players in bid

Full text of DOI:10.1002/cmdc.201800398

Accepted Article
Title: NVP-BHG712: Effects of regioisomers on the affinity and
selectivity towards the EPHrin family
Authors: Harald Schwalbe, Alix Tröster, Stephanie Heinzlmeir,
Benedict-Tilman Berger, Santosh L. Gande, Krishna Saxena,
Sridhar Sreeramulu, Verena Linhard, Amir H. Nasiri, Michael
Bolte, Susanne Müller, Bernhard Kuster, Guillaume Médard,
and Denis Kudlinzki
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To be cited as: ChemMedChem 10.1002/cmdc.201800398
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NVP-BHG712: Effects of regioisomers on the affinity and
selectivity towards the EPHrin family
Alix Tröster,[a] Stephanie Heinzlmeir,[b,c] Benedict-Tilman Berger,[d] Santosh L. Gande,[a,c] Krishna
Saxena,[a] Sridhar Sreeramulu,[a] Verena Linhard,[a] Amir H. Nasiri,[a] Michael Bolte,[e] Susanne Müller,[d]
Bernhard Kuster,[b,c] Guillaume Médard,*[b] Denis Kudlinzki,*[a,c] Harald Schwalbe*[a,c]
[a]
A. Tröster, Dr. S. L. Gande, Dr. K. Saxena, Dr. S. Sreeramulu, V. Linhard, Dr. A. H. Nasiri, Dr. D. Kudlinzki, Prof. Dr. H. Schwalbe
Center for Biomolecular Magnetic Resonance (BMRZ)
Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe-University, Max-von-Laue-Straße 7, 60438 Frankfurt am Main (Germany)
E-mail: kudlinzki@nmr.uni-frankfurt.de, schwalbe@nmr.uni-frankfurt.de
[b]
Dr. S. Heinzlmeir, Prof. Dr. B. Kuster, Dr. G. Médard
Chair of Proteomics and Bioanalytics, Technical University of Munich, Emil-Erlenmeyer-Forum 5, 85354 Freising (Germany)
E-mail: g.medard@tum.de
[c]
[d]
[e]
Dr. S. Heinzlmeir, Dr. S. L. Gande, Prof. Dr. B. Kuster, Dr. D. Kudlinzki, Prof. Dr. H. Schwalbe
German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
B.-T. Berger, Dr. S. Müller
Structural Genomics Consortium, Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main (Germany)
Dr. M. Bolte
Institute for Inorganic Chemistry, Goethe University Frankfurt, Frankfurt am Main, Germany
Abstract: EPH receptors are transmembrane receptor tyrosine
kinases. Their extracellular domains bind specifically to ephrin
A/B ligands, and this binding modulates the intracellular kinase
activity. EPHs are key players in bidirectional intercellular
signaling, controlling cell morphology, adhesion and migration.
They are increasingly recognized as cancer drug targets. We
analyzed the binding of the Novartis inhibitor NVP-BHG712 (NVP)
to EPHA2 and EPHB4. Unexpectedly, all tested commercially
available NVP samples turned out to be a regioisomer (NVPiso)
of the inhibitor, initially described in a Novartis patent application.
They only differ by the localization of a single methylation on
either one of two adjacent nitrogen atoms. The two compounds of
identical mass revealed different binding modes. Further, both in
vitro and in vivo experiments showed that the isomers differ in
their kinase affinity and selectivity.
promising pharmacophores for further inhibitor development
using the so-called Kinobeads technology.[15] 24 inhibitors were
shown to bind EPHA2 with submicromolar affinity. We solved
structures of the EPHA2 kinase domain (D596-G900) with nine
different bound inhibitors.
In the context of this off-target screening with Kinobeads, we
could identify NVP-BHG712 (NVP), a nanomolar EPHB4 inhibitor
claimed by a Novartis patent application in 2007,[16,17] to inhibit
EPHA2 with similar affinity than EPHB4. NVP consists of two
parts, a methylated N-phenylbenzamide moiety and the adenine-
related pyrazolo[3,4-d]pyrimidine moiety (Scheme 1).
Erythropoietin-producing
hepatocellular
(EPH)
receptors
represent the largest family of receptor tyrosine kinases (RTKs).
There are two EPH subfamilies based on sequence conservation
that exhibit different ephrin A/B ligand affinities (EPHA1-10,
EPHB1-6).[1] 14 out of 16 EPH receptors can be found in humans
(EPHA1-8 & 10; EPHB1-4 & 6).[2] Upon interaction with the
corresponding ephrin ligands, these RTKs act as key elements in
bidirectional intercellular signal transduction pathways, controlling
cell morphology, adhesion and migration.[3] Moreover, EPHs are
involved in cancer development: As a result of different signaling
modalities, several partly opposing effects (promotion and
suppression of tumorigenicity) can be observed.[4,5] Within the
EPH family, particularly EPHA2 and EPHB4 are overexpressed in
cancer cells. Downregulation of their expression can inhibit
tumorigenicity, suggesting an important role in tumor cancer
biology.[4] For example EPHA2 is overexpressed in head and
neck,[6] breast,[7] or non-small cell lung cancer[8] and associated
with poor patient prognosis.[9,10] To further evaluate the role of
EPHA and EPHB receptors as therapeutic targets, specific
inhibitors for these kinases are in demand.[11–14]
Scheme 1: Constitution of NVP-BHG712 (NVP) and its isomer (NVPiso).
Based on previous structural data, we anticipated that the
N-phenylbenzamide moiety would lead to a binding of NVP to the
DFG-out conformation of EPHA2, comprising a conserved Asp-
Phe-Gly (DFG, in EPHA2: D757, F758, G759) motif at the
proximal end of the activation loop of kinases.[18,19] This prediction
was verified by 1H,15N correlation spectra of selectively 15N-
phenylalanine labeled EPHA2 kinase domain expressed in Sf9
cells.[20] The spectra showed a dynamic behavior of the DFG-motif
in the apo state of the kinase.[20,21] Addition of NVP gave rise to a
single additional amide signal at δ(15N)
= 124.29 ppm,
δ(1H) = 7.95 ppm, which could be assigned to F758. This
resonance is characteristic for EPHA2 locked in a single, DFG-
out conformation (see Figure S6 in the SI).[20] We next co-
crystallized the EPHA2 kinase with the commercially available
NVP compound.
Recently, we investigated the off-target binding and activity of 235
clinically evaluated kinase inhibitors on EPHA2 to identify
1
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Scheme 2: Synthesis of NVP-BHG712 (4 steps) and its structural isomer NVPiso using the new improved synthesis strategy. (a) Reaction to the N-phenylbenzamide
fragment; b) Synthesis of the patented Novartis compound NVP-BHG712; c) Synthesis route of the structural isomer NVPiso).
confirmed by small molecule X-ray crystal structure analysis (see
SI for details).
Crystallographic data at a resolution of 1.04 Å confirmed the
conformational arrest of the activation loop and the DFG-out
binding mode of the inhibitor·EPHA2 complex. Unexpectedly,
however, the data revealed that the commercially available
inhibitor possessed a wrong constitution. It was only possible to
fit a regioisomer of NVP (NVPiso, Scheme 1) of identical mass
with shifted position of a single methyl group into the electron
density. Further NMR analysis of inhibitor samples from six
different suppliers identified all of those as the same NVPiso
regioisomer (see Figure S4 in the SI). Therefore, we decided to
synthesize and analyze the original compound described by
Novartis and its structural isomer in-house.
The regioisomer NVPiso, which is the compound that one can
purchase from six vendors, can be synthesized applying a minor
modification in the synthesis (Scheme 2). Under neutral reaction
conditions, the 5-aminopyrazole derivative
4
is formed
predominantly, whereas the addition of a base yields the
3-aminopyrazole derivative 6 as the main product.[22] Both
structural isomers can be distinguished by ROESY NMR
spectroscopy: A close distance was observed between CH3 and
NH2 in 4, the derivative 6 showed however a cross peak between
CH3 and CH2 (see Figure S12 and Figure S17 in the SI). The two
subsequent steps were identical to the synthesis of NVP (small
molecule X-ray crystal structure of NVPiso available in the SI).
Due to the different substituent pattern, both isomers show
different UV/Vis absorption spectra (Figure S34 in the SI). This
offers the opportunity to differentiate both compounds, without
access to more sophisticated analytical techniques.
Thus, a small modification in the reaction conditions led to the two
different regioisomers. By starting the synthesis of pyrazole 4 from
hydrazine hydrochloride as described by Novartis, the use of an
excess of base could easily lead to the isomeric form 6 instead of
the desired product.
The Novartis patent application describes a seven step synthesis
for NVP. An aniline synthon, (compound S3, Scheme S1 in the
SI, depicted in red, obtained in two steps) is coupled to a nitrogen-
rich chloro-heterocycle (pyrazolo[3,4-d]pyrimidine S6, Scheme
S1 in the SI, depicted in blue, obtained in four steps), to yield the
final product.[17] In our synthesis, we relied on Buchwald-Hartwig
chemistry for the final step in order to shorten the synthetic route
(Scheme 2). Instead of an aniline, we prepared a bromoaryl
derivative (compound 2) and moved the amine substituent to the
pyrazolo[3,4-d]pyrimidine building block (compound 5), which
could be obtained in two steps. Overall, this new synthesis route
reduced the number of reaction steps from seven to four and
avoided pyrophoric (Raney-Ni) and highly corrosive (POCl3)
reagents. Our synthesized NVP batch was characterized by
standard analytical techniques (MS, 1H-, 13C- and 19F-NMR).
Additionally, its molecular structure was unambiguously
Crystal structures of EPHA2 and EPHB4 with NVP and NVPiso
revealed very deep binding in the ATP pocket and its adjacent
region (see Figure 1) for both compounds. The regioisomers show
conserved non-bonded binding to EPHA2 and EPHB4 and
2
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feature typical characteristics of kinase inhibitors: hydrogen
bonds to the gate keeper T692 (EPHB4: T693), to D757 (D758)
of the activation loop (DFG-motif) and to M695 (M696) of the
hinge region, either to nitrogen atoms of the NVP pyrazole or the
NVPiso pyridine moiety. Both regioisomers arrest the DFG-loop
in an inactive out conformation. Interestingly, the salt bridge
between E663 (E664) and K646 (K647), which is usually
characteristic for an active kinase state, is maintained, which
stabilizes the αC-helix and preserves a pseudo-active kinase
conformation. Another 13 (15) hydrophobic contacts and further
water-mediated interactions stabilize the binding of both
regioisomers.
an alternative hydrogen bond between M695 (M696) and the
nitrogen atom of the pyridine ring. Although the flip does not
induce major changes of the interaction pattern, a detailed
interaction surface analysis (PISA analysis in the SI) revealed a
reduction of overlapping areas between the isomer and M695 by
more than 50%.
To further characterize the preferred binding mode of the
pyrazolo[3,4-d]pyrimidine moiety we were able to co-crystallize
the NVP fragment 5. Indeed, the binding mode of 5 is highly
similar to the one of NVP, the interactions with T692 (EPHB4:
T693) and M695 (M696) are conserved (Figure 1, g+h). Due to
the absence of the N-phenylbenzamide moiety, the kinases adopt
the DFG-in conformation.
By NMR, we could characterize relevant interactions of the
fragments of NVP and NVPiso using waterLOGSY NMR
experiments (Figure S7 in the SI). Briefly, fragment 5 and the N-
phenylbenzamide fragment (typically present in DFG-out ligands)
displace the ATP analogue AMP-PNP. In addition, both fragments
bind simultaneously to the kinase, most likely because they can
occupy two different binding sites. Whereas fragment 5 strongly
displaces AMP-PNP from EPHA2, the “DGF-out-N-
phenylbenzamide fragment” displaces AMP-PNP only partially.
The more detailed account, including binding to EPHB4, is given
in the SI (Figure S8 and Figure S9).
The affinities of NVP, NVPiso and of the DFG-in inhibitor dasatinib
as control were then investigated by microscale thermophoresis
(MST),[23] Kinobeads[24–26] and NanoBRET assays[27]. MST uses
the fluorophore-labeled kinase domain to determine in vitro
changes of the diffusion constant in an inhibitor concentration-
dependent manner. The Kinobeads assay investigates the
competition of inhibitors to binding of broad-band, ATP-
competitive unspecific immobilized tool compounds within native
cellular lysates. For the cellular NanoBRET assays, EPH
receptors are fused to luciferase and
a bioluminescence
resonance energy transfer (BRET) signal is induced upon binding
of an energy transfer probe. ATP-competitive kinase inhibitors
prevent binding of the BRET probe in the live cell and lead to an
inhibitor concentration-dependent signal decrease.
Using MST, we determined dissociation constants of NVP and
dasatinib binding to EPHA2 in the low nanomolar range but a
sixfold lower binding affinity to NVPiso (Table 1). This trend of
lower EPHA2 affinity for NVPiso was confirmed by the Kinobeads
and NanoBRET assays, indicating low affinity binding also in a
cellular context. However, we also observe differences between
the two cellular assays that could be explained by different
experimental setups of the assays. The Kinobeads assay is
performed with cell lysates from several cancer cell lines
containing the endogenous full-length kinase receptor. The life
cell NanoBRET assay targets the ectopically expressed full-
length receptor fused to an intracellular luciferase reporter in
intact HEK293T cells. Higher dissociation constants as in the
Kinobeads assays are often observed in lysate-based binding
assays[28] where the full-length target proteins are present in
different activation states, PTM (posttranslational modifications)
signatures and protein complexes. Nevertheless, both assays
agree in that NVPiso has a different profile of targeting the two
EPH receptors compared to NVP.
Figure 1: Crystal structures of NVP, NVPiso and the fragment 5 in complexes
with EPHA2 and EPHB4. a & b) Crystal structures of NVP·EPHA2 (a), PDB ID:
6FNF) and NVP·EPHB4 (b), PDB ID: 6FNI); the nitrogen of the pyrazole ring is
forming the hydrogen bond towards M695 or M696. c & d) Crystal structures
NVPiso·EPHA2 (c), PDB ID: 6FNG) and NVPiso·EPHB4 (d), PDB ID: 6FNJ);
the hydrogen bond towards the M695 and M696 is formed by the nitrogen atom
in the pyridine substituent. e & f) Comparison of the binding mode of both
isomers in EPHA2 (e) and EPHB4 (f); the pyrazolo[3,4-d]pyrimidine moiety
shows a rotation around 180°. g & h) Crystal structures of the fragment
5·EPHA2 (g), PDB ID: 6FNH) and 5·EPHB4 (h), PDB ID: 6FNK); the fragments
bind in comparable poses as the pyrazolo[3,4-d]pyrimidine moiety of NVP to the
kinases, which here adopt a DFG-in conformation.
Although the interaction network for both regioisomers is
conserved, the binding mode is surprisingly not. While the N-
phenylbenzamide moiety of both compounds extends deep into
the hydrophobic pocket between activation loop and αC-helix, for
NVPiso the pyrazolo[3,4-d]pyrimidine moiety rotates by 180° (see
Figure 1). The methyl group at the NVPiso pyrazole ring prevents
the formation of a hydrogen bond to the hinge region backbone.
Instead the planar ring system flips and enables the formation of
3
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We then expanded the NanoBRET assay to probe for differential
binding affinities of NVP over NVPiso to other members of the
EPH receptor family. In fact, NVP targeted the majority of all
tested EPH receptors with excellent affinities between 0.3 nM
(EPHA3) and 303 nM (EPHA1); only EPHA7 and the
pseudokinase EPHB6 were not or only slightly bound by this
inhibitor. In comparison, NVPiso affinities ranged from 50 nM
(EPHA3) to 630 nM (EPHB2) and no or low inhibitory effects were
determined for half of the tested EPH receptors (EPHA1, EPHA4,
EPHA6, EPHA7, EPHB4, EPHB6). NanoBRET data further
showed highest difference in binding affinity of NVP over NVPiso
for EPHA4 (~1300) and EPHB4 (~550) (see Table S1 in the SI).
Using the Kinobeads assay, we could not reach a similar
coverage of the different members of the receptor class, but could
gain additional information about the proteome-wide selectivity
profiles of both isomers (see Table S1 and Figure S5 in the SI).
While NVP primarily targets EPH family members, the main target
of NVPiso is the cancer relevant receptor tyrosine kinase DDR1.
Experimental Section
All experimental details are described in the Supporting
Information.
Acknowledgements
We would like to thank Kerstin Witt for her technical assistance.
Diffraction data have been collected on beamline P13 operated
by EMBL Hamburg at the PETRA III storage ring (DESY,
Hamburg, Germany), beamline ID30B at the European
Synchrotron Radiation Facility (ESRF, Grenoble, France) and
BL14.1 at the BESSY II electron storage ring operated by the
Helmholtz-Zentrum Berlin, Germany. We would particularly like to
acknowledge the help and support of Thomas Schneider, Gordon
Leonard and Manfred Weiss during the experiments. The work
has been supported by the German consortium for translational
cancer research (DKTK), by DFG (SFB807), EU research
infrastructure iNEXT. Work at BMRZ is supported by the state of
Hesse. The SGC is a registered charity (number 1097737).
Table 1: Comparison of affinities for NVP and the isomer NVPiso toward EPHA2
and EPHB4 using different techniques (MST, NanoBRET and Kinobeads assay).
All values are denoted in nM.
Selectivity
factor Sf
NVP-BHG712 (NVP)
NVPiso
Keywords: Medicinal Chemistry • Structural Biology • Eph
receptors • Xray • NMR spectroscopy
References:
EPHA2
EPHB4
13
3.3
3.0
240
695
73
163
2546
49.4
553
5.7
142
1660 1113
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10.1002/cmdc.201800398
ChemMedChem
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A small change can make a big difference: A modification in the substitution pattern of the published kinase inhibitor NVP-BHG712
from Novartis leads to different binding modes as well as binding affinities towards EPH receptor tyrosine kinases. Additionally also the
selectivity profiles of the two compounds for binding to kinases are substantially changed.
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This article is protected by copyright. All rights reserved.