Fragment-Based Discovery of a Dual pan-RET/VEGFR2 Kinase Inhibitor Optimized for Single-Agent Polypharmacology

Article abstract of DOI:10.1002/anie.201501104

Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen led to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 a

Full text of DOI:10.1002/anie.201501104

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201501104
German Edition: DOI: 10.1002/ange.201501104
Polypharmacology
Hot Paper
Fragment-Based Discovery of a Dual pan-RET/VEGFR2 Kinase
Inhibitor Optimized for Single-Agent Polypharmacology**
Brendan Frett, Francesca Carlomagno, Maria Luisa Moccia, Annalisa Brescia, Giorgia Federico,
Valentina De Falco, Brittany Admire, Zhongzhu Chen, Wenqing Qi, Massimo Santoro,* and
Hong-yu Li*
Abstract: Oncogenic conversion of the RET (rearranged
during transfection) tyrosine kinase is associated with several
cancers. A fragment-based chemical screen led to the identifi-
cation of a novel RET inhibitor, Pz-1. Modeling and kinetic
analysis identified Pz-1 as a type II tyrosine kinase inhibitor
that is able to bind the “DFG-out” conformation of the kinase.
Importantly, from a single-agent polypharmacology stand-
point, Pz-1 was shown to be active on VEGFR2, which can
block the blood supply required for RET-stimulated growth. In
cell-based assays, 1.0 nm of Pz-1 strongly inhibited phosphor-
ylation of all tested REToncoproteins. At 1.0 mgkgÀ1 dayÀ1 per
os, Pz-1 abrogated the formation of tumors induced by RET-
mutant fibroblasts and blocked the phosphorylation of both
RETand VEGFR2 in tumor tissue. Pz-1 featured no detectable
toxicity at concentrations of up to 100.0 mgkgÀ1, which
indicates a large therapeutic window. This study validates the
effectiveness and usefulness of a medicinal chemistry/poly-
pharmacology approach to obtain an inhibitor capable of
targeting multiple oncogenic pathways.
profiles for critical disease-promoting kinases, including
crucial mutant targets. A single agent with an appropriate
inhibitory profile can possess the benefits of combination
therapy and effectively target tumor growth while preventing
resistance formation.[5] Herein, we have applied MCP using
a novel fragment-based approach to identify a pan-RET/
VEGFR2 dual inhibitor (RET= rearranged during trans-
fection, VEGFR2 = vascular endothelial growth factor recep-
tor 2). The inhibitor is capable of effectively treating both the
parenchyma (RET) and stroma (VEGFR2) of RET-driven
tumors, while maintaining activity on all tested RET onco-
gene mutants.
RET is a transmembrane tyrosine kinase (TK) receptor
that has emerged as a molecular target for the treatment of
several cancer types, primarily medullary thyroid carcinoma
(MTC).[6–8] In a tumor environment, VEGFR2-mediated
angiogenesis provides oxygen and the nutrients necessary
for RET oncogenic signaling. Vandetanib (Caprelsa) and
cabozantinib (Cometriq) were originally discovered as inhib-
itors of different tyrosine kinases, including VEGFR2, and
have been approved to treat MTC because of their capability
to prolong progression-free survival compared to placebo
treatments.[9,10] Both agents possess activity on wild-type and
oncogenic RET mutants, but not on RET gatekeeper (V804)
mutants. Therefore, our goal was to implement the concept of
single-agent polypharmacology (SAP) to design a RET/
VEGFR2 dual inhibitor that is selective and equipotent on
both kinases, including clinically relevant mutants, to provide
a new targeted agent for RET-driven cancers.
T
argeted treatments are aimed at providing cancer patients
with agents, such as tyrosine kinase inhibitors (TKIs), that are
designed to target tumor cells or the tumor microenviron-
ment. Unfortunately, drug resistance to TKIs invariably
develops owing to the inability to sustainably knock out
tumor-survival pathways or to maintain activity on the target
as additional mutations form.[1–3] Resistance can be reduced
by identifying, through medicinal chemistry/polypharmacol-
ogy[4] (MCP), personalized TKIs with optimized inhibitory
[*] B. Frett,[+] Z. Chen, H.-y. Li
B. Frett,[+] H.-y. Li
Medicinal Chemistry Division, Pharmacology and Toxicology
College of Pharmacy, The University of Arizona
1703 E. Mabel, Tucson, AZ 85721 (USA)
E-mail: hongyuli@pharmacy.arizona.edu
F. Carlomagno,[+] M. L. Moccia, A. Brescia, G. Federico, V. De Falco,
M. Santoro
Synactix Pharmaceuticals, Inc.
6510 N. Camino Arturo, Tucson, AZ 85718 (USA)
E-mail: contact@synactixpharma.com
Z. Chen
Chongqing University of Arts and Sciences
Chongqing, 402160 (China)
[+] These authors contributed equally to this work.
Dipartimento di Medicina Molecolare e Biotecnologie Mediche
Università di Napoli “Federico II”/Istituto di Endocrinologia ed
Oncologia Sperimentale del CNR
80131 Naples (Italy)
E-mail: masantor@unina.it
[**] RAT1 cells expressing RET mutants were kindly donated by M.
Billaud, and VEGFR2/KDR-expressing vectors by S. De Falco. This
study was supported by the Associazione Italiana per la Ricerca sul
Cancro (AIRC), by a MERIT grant, and MIUR (Italy). Furthermore,
this work was supported by a training grant from The National
Institutes of Health (T32 GM008804), start-up funding from the
University of Arizona, a Caldwell Health Sciences Research Fellow-
ship, and an ACS IRG grant. This is part I of a synergistic medicinal
chemistry series.
B. Admire
Pharmaceutical Sciences, College of Pharmacy
The University of Arizona
1703 E. Mabel, Tucson, AZ 85721 (USA)
W. Qi
The University of Tennessee, Health Science Center
Memphis, TN 38163 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201501104.
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Table 1: Compound activities on RET, VEGFR2, and RETV804M
.
Compound design was largely guided by computational
modeling and the use of a modified fragment-based approach.
For the modeling studies, the RET tyrosine kinase domain
that had previously been crystallized in complex with small
type I inhibitors, which bind to the “DFG-in” (native active
fold) conformation of the kinase, was used.[11] To search for
more selective RET TKIs, a “DFG-out” (inactive fold)
homology model capable of interacting with type II inhibitors
was generated by utilizing an available RET sequence (PDB:
2IVU) and a VEGFR2 DFG-out template (PDB: 2OH4; see
the Supporting Information, Figure S1). The model displayed
the appropriate shift in the DFG loop, which opened the
allosteric pocket and was used for molecular docking and
structure–activity relationship (SAR) studies.
Kinase fragment libraries are traditionally composed of
small hinge binders that consist of one or two fused aryl and/
or heteroaryl ring systems with hydrogen-bond donors or
acceptors. Such fragments are highly polar and can aggregate
at the millimolar concentrations that are often necessary to
achieve activity in a biochemical fragment screen. As a result,
achieving a good noise-to-signal ratio is a major challenge
with this method. A kinase-directed fragment (KDF) library
was designed to enhance the sensitivity and more effectively
Compound
RET
VEGFR2
RETV804M
1
(28Æ3)%,[b]
(59Æ3)%[c]
–
(63Æ5)%[c]
1a
2
3
3b
(18.2Æ0.81) mm[a]
(2.1Æ0.24) mm[a]
0.006 mm[d]
(89Æ4)%[c]
(93Æ2)%[c]
0.018 mm[d]
(97Æ2)%[c]
<0.001 mm[a]
–
–
0.006 mm[d]
(0.047Æ0.007) mm[a]
–
Pz-1 (4)
<0.001 mm[a]
<0.001 mm[a]
[a] IC50 values were determined using a microfluidic separation based
assay. The data represent means from at least three independent
experiments. [ATP]=190 mm. [b] Residual activity at 100.0 mm deter-
mined in triplicate. [ATP]=190 mm. [c] Residual activity at 20.0 mm
determined in triplicate. [ATP]=190 mm. [d] Kd values were determined
using an active-site-directed competition binding assay that was out-
sourced to KINOMEScan.
compound 1 in the RET kinase showed that heterocyclic
substitution at the bromine would be tolerated (compound
1a; Figure 2A,D). This region is solvent-exposed and occu-
pied by the adenine ring system when ATP is bound.
Compound 1a displayed improved RET potency with an
IC50 value of 18.2 Æ 0.81 mm (Table 1). Further modeling
showed that the DFG pocket of the RET kinase could be
accessed with the addition of a bridge connected to an
allosteric group such as a meta-trifluoromethylphenyl moiety
(compound 2; Figure 2A,C); compound 2 indeed showed
improved potency against the RET kinase with an IC50 value
of 2.1 Æ 0.24 mm (Table 1). The combination of SAR informa-
tion from compounds 1, 1a, and 2 resulted in compound 3
with RET, VEGFR2, and RETV804M Kd values of 0.006, 0.018,
and 0.006 mm, respectively (Table 1). Compound 3 was further
optimized on RET and VEGFR2 at both the allosteric region
and the 5-position of the benzimidazole, which led to
compound 4 (Pz-1; Figure 2A,E, see also Figure S2). Specif-
ically, a pyrazole at the solvent region and an isoxazole in the
allosteric pocket were highly favored for strong RET
inhibition. Pz-1 was found to be active on RET, RET mutants,
and VEGFR2 with similar IC50 values (all < 1.0 nm, Table 1).
The equal potency was later confirmed in cell-based assays.
Pz-1 is expected to simultaneously block VEGFR2-mediated
nutrient accumulation and RET/RET-mutant cell prolifera-
tion.
interrogate binding functionalities at
a kinase hinge
(Figure 1).[12] This KDF library contained a diverse set of
Molecular modeling demonstrated that Pz-1 could bind
RETand VEGFR2 with different binding geometries because
of free rotation of the methylene linker that bridges the hinge
region to the allosteric pocket (Figure S3). In complex with
RET, the linker amide is in the “up” position, whereas with
VEGFR2, the linker amide is in the “down” position. The
free rotation in Pz-1 presumably helps the inhibitor to achieve
similar potencies on RET, RET mutants, and VEGFR2.
Accordingly, Pz-1 exhibited IC50 values of < 1.0 nm for both
wild-type RET and VEGFR2 kinases (Table 1). Computa-
tional modeling of Pz-1 within the RET kinase showed that
binding occurred > 3.5 Š away from the gatekeeper residue
(V804; Figure 2E). As a result of the large binding distance
from the gatekeeper region, Pz-1 exhibited a strong affinity
for the gatekeeper mutant RETV804M (Table 1). RETV804M is
Figure 1. Concept for increasing the potency and mitigating the
aggregation liability in a novel kinase fragment library by using kinase-
directed fragments (KDFs). KDFs are composed of a hinge region
tethered to a lipophilic region for increased potency.
heterocyclic hinge-region binders along with moieties that can
engage lipophilic pockets or the ribose sugar pocket. Accord-
ingly, KDFs have larger molecular weights and are generally
more active than the fragments contained in traditional
libraries, permitting screening in the micromolar range. From
such a library, a benzimidazole analogue was identified as
a promising starting platform for a RET TKI (compound 1;
Figure 2A, Table 1). Compound 1 effectively inhibited RET
at both 100.0 and 20.0 mm (Table 1). Molecular modeling of
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refractory to both vandetanib and
cabozantinib,[13] suggesting that Pz-
1 has a novel RET binding mode.
Vandetanib is located approxi-
mately 2.8 Š away from the gate-
keeper residue (V804), almost 1.0 Š
closer than Pz-1; the vandetanib
affinity for RETV804 m and RETV804L
is likely abrogated because of steric
hindrance with the larger mutant
residues at the gatekeeper region.
To identify the type of inhibition
exhibited, the ATP Km value for
RET was determined and found to
be 12.00 Æ 0.26 mm (Figure S4). As
the utilized RET kinase assay is not
suitable for precisely determining
the activity of highly potent inhib-
itors such as Pz-1 (IC50 < 1.0 nm),
compound 3b was selected for
inhibition studies because its activ-
ity was within the limits of assay
reliability. The IC50 value of 3b was
determined at ATP concentrations
of 6.2, 12.5, 50.0, and 100.0 mm.
Although there was a slight increase
in IC50 values with an increase in
ATP concentration, compound 3b
remained a strong inhibitor at highly
saturating ATP concentrations
(Table 2, see also Figure S5). This
finding suggests that the benzimida-
zole scaffold of Pz-1 is somewhat
competitive with ATP for binding as
inhibition was relieved but not com-
pletely abrogated.
In cell-based assays, consistent
with the biochemical SAR studies,
Pz-1 displayed a higher RET inhib-
itory activity towards oncogenic
RETC634R than its predecessor com-
pounds (1, 2, and 3; Figure 3A).
Pz-1 was also highly effective
against other common oncogenic
RET point mutants, including
Figure 2. Design and molecular modeling of Pz-1 (compound 4). A) Progression from KDF 1 to
clinical candidate Pz-1 (4). From compound 1, the solvent region as well as the allosteric pocket were
optimized to furnish compounds 1a and 2, respectively. Compounds 1a and 2 were combined to
generate lead candidate 3. Further medicinal-chemistry efforts generated the optimized RET/VEGFR2
dual inhibitor Pz-1 (4). B) Computational modeling of compound 1 in the RET DFG-in crystal
structure (PDB: 2IVU). The modeling identified two possible points for optimization: 1) the solvent
pocket and 2) the allosteric pocket. C) Computational modeling of compound 2 in the RET DFG-out
homology model. Compound 2 is predicted to bind to A807 of the hinge and form two hydrogen
bonds on the RET bridge. One hydrogen bond is predicted to form with D892 of the DFG motif and
the other one with E775 of the C helix at the back of the allosteric pocket. The interactions with an
aspartic acid of the DFG motif and a glutamic acid residue of the C helix are similar to the
interactions of imatinib with c-ABL (PDB: 3K5V). D) Computational modeling of compound 1a in the
RET DFG-in crystal structure (PDB: 2IVU). Substitution at the solvent pocket is predicted to be
tolerated. E) Computational modeling of Pz-1 (4) in the RET DFG-out homology model. Pz-1 is
predicted to make a contact with D892 at the DFG loop but not with E775 of the C helix. The binding
geometry of Pz-1 places the compound far away from the gatekeeper region avoiding a steric clash
with bulky residues at position 804.
RETM918T
,
the most common
mutant associated with the inherited
multiple endocrine neoplasia type
2B (MEN2B) syndrome and spora-
dic MTC, as well as RETV804M and
RETV804L mutants (Figure 3B).
Moreover, Pz-1 was also confirmed
to be equally potent on VEGFR2,
inhibiting
VEGFR2
VEGFA-induced
phosphorylation at
a
1.0 nm dose in transfected
HEK293 cells and, at the endoge-
nous level, in HUVEC cells (Fig-
ure 3C, see also Figure S6).
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Table 2: Inhibition values of compound 3b.[a]
Table S1). Pz-1 preferentially inhibited RET- over Ras-driven
tumors (Figure 4). Indeed, whereas this treatment completely
prevented the formation of tumors induced by RETC634Y, it
reduced, but did not abrogate, the formation of tumors driven
by HRasG12V (Figure 4, see also Table S1). Only in RET-
driven tumors was Pz-1 able to inhibit MAPK and mTOR
mitogenic signaling cascades (Figure S10). Even at a dose of
10.0 mgkgÀ1, Pz-1 had no
ATP [mm]
IC50 [nm]
6.2
12.5
50.0
100.0
20.6Æ4.3
27.4Æ1.1
33.7Æ3.18
46.5Æ6.9
[a] IC50 values were determined using a microfluidic separation based
assay. The data represent means from at least three independent
experiments.
effect on MAPK signaling
in Ras tumors. However, in
both RET- and Ras-driven
tumors, Pz-1 (1.0 mgkgÀ1
)
inhibited VEGFR2 phos-
phorylation. The preferential
efficacy of Pz-1 for RET-
driven tumors may be
explained by its dual effect
on both the parenchyma
(RET)
and
stroma
(VEGFR2) supporting the
polypharmacological strat-
egy to target these tumor
types. Pz-1 was highly toler-
ated at daily doses of up to
100.0 mgkgÀ1 for one week
in mice (Figure S11). The
amount of alanine transami-
nase (ALT), which can be
used as a monitorable toxic-
ity marker, increased in
Figure 3. A) SAR evolution from KDF 1 to Pz-1 (4). The indicated compounds were tested in cell-based
assays (RAT1 fibroblasts transfected with RETC634R); RET phosphorylation (pRET) was measured at two sites
(Y1062 and Y905) by immunoblot analysis. Data is reported in micromolar concentrations. B) Pz-1 (4)
mediated inhibition of pRET in RET-mutant-transfected RAT1 fibroblasts (see A for details). C) Pz-1 mediated
inhibition of pVEGFR2 in VEGFR2 transiently transfected HEK293 cells (see A for details). All data is reported a linear fashion with the
in micromolar concentrations. NT=no treatment.
Pz-1 dose, but remained
Pz-1 was subjected to kinome screening to determine the
global kinase selectivity. At a concentration of 50.0 nm,
Pz-1 was screened against a 91 kinase panel representing
each kinome cluster (Figure S7). Pz-1 featured good global
kinase selectivity with activity on only seven additional
kinases (TRKB, TRKC, GKA, FYN, SRC, TAK1, MUSK),
which is consistent with the low toxicity that was observed in
the cell-based assays (Figure S9).
To address in vivo RET-driven effects, Pz-1 was tested on
tumors induced by oncogenic RET that were compared to
a constitutively active control Ras oncogene. To this end,
NIH3T3 fibroblasts transformed by either RETC634Y or
HRasG12V were used. In in vitro cell assays, NIH3T3
Figure 4. Antitumor activity of Pz-1 (4) in nude mice implanted with
RET- or Ras-transformed NIH3T3 fibroblasts. NIH3T3 RETC634Y or
RETC634Y cell proliferation was inhibited by Pz-1 with an
NIH3T3 HRasG12V cells were inoculated subcutaneously into nu/nu
IC50 value of 0.5 nm, a dose significantly lower than that
mice. After four days, animals were randomly assigned to receive
required to inhibit NIH3T3 HRasG12V cell proliferation
Pz-1 (1.0, 3.0, or 10 mgkgÀ1 daily) by oral gavage (ca. 7 mice) or left
(IC50 = 34.4 nm; Figure S8B). This was paralleled by the
untreated (8 mice). Average tumor volumes are reported. Standard
deviations are given in Table S1.
efficient inhibition of RETC634Y tyrosine phosphorylation
(Figure S8A). At concentrations of up to 100.0 nm,
Pz-1 exerted only a minimal growth inhibition on parental
NIH3T3 cells (Figure S9).
within a normal range (Figure S11). Furthermore, Pz-1 dis-
played highly favorable pharmacokinetic properties
(Table S2).
In summary, we have identified Pz-1 as a dual kinase
inhibitor using a KDF library screen in conjunction with
Immunodeficient (nu/nu) mice were then injected with
NIH3T3 RETC634Y or NIH3T3 HRasG12V cells and treated
per os with Pz-1 (1.0, 3.0, or 10.0 mgkgÀ1 dayÀ1) before the
tumors had appeared, or left untreated (Figure 4; see also
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Otto, K. Brennan, R. Murali, M. Garrido, V. A. Miller, J. S. Ross,
computational modeling. In cell-based assays, 1.0 nm of
Pz-1 strongly inhibited tyrosine phosphorylation of
VEGFR2 and clinically relevant RET mutants, including
those refractory to vandetanib and cabozantinib (RETV804M
and RETV804L). Pz-1 completely blocked RET-driven tumor
formation at 1.0 mgkgÀ1 with no detectable toxicity at doses
of up to 100.0 mgkgÀ1. The high activity and low toxicity of
Pz-1 can be explained by the selective dual inhibition of both
RET and VEGFR2. In conclusion, this study validates
medicinal chemistry and single-agent polypharmacology
methods to guide the development of compounds with well-
defined and balanced activities against multiple cancer-
relevant targets for synergistic outcomes. The clinical devel-
opment of Pz-1 may offer a promising therapeutic approach
for patients afflicted with RET-driven malignancies and
represents a paradigm shift for targeted therapies.
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Keywords: inhibitors · kinases · medicinal chemistry ·
polypharmacology
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Received: February 5, 2015
Revised: March 24, 2015
Published online: June 30, 2015
Angew. Chem. Int. Ed. 2015, 54, 8717 –8721
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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