Bioisosteric discovery of npa101.3, a second-generation ret/vegfr2 inhibitor optimized for single-agent polypharmacology

Article abstract of DOI:10.1021/acs.jmedchem.9b01336

RET receptor tyrosine kinase is a driver oncogene in human cancer. We recently identified the clinical drug candidate Pz-1, which targets RET and VEGFR2. A key in vivo metabolite of Pz-1 is its less active demethylated pyrazole analogue. Using bioisosteri

Full text of DOI:10.1021/acs.jmedchem.9b01336

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Article
Bioisosteric discovery of NPA101.3, a second generation RET/
VEGFR2 inhibitor optimized for single-agent polypharmacology
Marialuisa Moccia, Brendan Frett, Lingtain Zhang, Naga Rajiv Lakkaniga, David
C Briggs, Rakhee Chauhan, Annalisa Brescia, Giorgia Federico, Wei Yan,
Massimo Santoro, Neil Q McDonald, Hong-yu Li, and francesca carlomagno
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01336 • Publication Date (Web): 16 Apr 2020
Downloaded from pubs.acs.org on April 18, 2020
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Bioisosteric discovery of NPA101.3, a second generation RET/VEGFR2 inhibitor
optimized for single-agent polypharmacology
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Marialuisa Moccia,1,† Brendan Frett,2,6,† Lingtian Zhang, Naga Rajiv Lakkaniga,
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David C. Briggs, Rakhee Chauhan, Annalisa Brescia, Giorgia Federico, Wei Yan,
Massimo Santoro,1 Neil Q. McDonald,3,4 Hong-yu Li,2,6* and, Francesca
Carlomagno1,5*
†Marialuisa Moccia and Brendan Frett contributed equally to this work.
1Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli
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“
Federico II", 80131, Napoli, Italia; Department of Pharmaceutical Sciences, College
of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205,
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USA; Signalling and Structural Biology Laboratory, The Francis Crick Institute,
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London, NW1 1AT UK; Institute of Structural and Molecular Biology, Department of
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Biological Sciences, Birkbeck College, London, WC1E 7HX, UK; Istituto di
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Endocrinologia ed Oncologia Sperimentale del CNR, 80131, Napoli, Italia; Synactix
Pharmaceuticals, Inc., Tucson, AZ 85718, USA.
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*
Corresponding Authors: Hong-yu Li, Department of Pharmaceutical Sciences,
College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR
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2205, United States; (Ph. 5012961154; HLi2@uams.edu); Francesca Carlomagno,
Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli
“Federico II" via S. Pansini 5, 80131 Napoli-Italy (Ph. +390815455561; Fax:
+390817462685; francesca.carlomagno@unina.it)
Keywords: kinase; tyrosine kinase inhibitor; targeted therapy; thyroid cancer; lung
adenocarcinoma.
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ABSTRACT
RET receptor tyrosine kinase is a driver oncogene in human cancer. We recently
identified the clinical drug candidate Pz-1, which targets RET and VEGFR2. A key in
vivo metabolite of Pz-1 is its less active demethylated pyrazole analogue. Using
bioisosteric substitution methods, here, we report the identification of NPA101.3,
lacking the structural liability for demethylation. NPA101.3 showed selective inhibitory
profile and an inhibitory concentration 50 (IC ) of <0.001 M for both RET and
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VEGFR2. NPA101.3 inhibited phosphorylation of all tested RET oncoproteins as well
as VEGFR2 and proliferation of cells transformed by RET. Oral administration of
NPA101.3 (10 mg/kg/day) completely prevented formation of tumours induced by
RET/C634Y-transformed cells, while it decreased, but did not abrogate, formation of
tumours induced by a control oncogene (HRAS/G12V). The balanced synchronous
inhibition of both RET and VEGFR2, as well the resistance to demethylation, renders
NPA101.3 a potential clinical candidate for RET-driven cancers.
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INTRODUCTION
RET (REarranged during Transfection) is the receptor tyrosine kinase (RTK) for
neurotrophic factors of the GDNF (glial cell line-derived neurotrophic factor) family
(1). In several human cancers, RET rearrangements lead to the formation of chimeric
oncoproteins, containing the RET tyrosine-kinase (TK) domain fused to the N-
terminal region of heterologous proteins. These include: papillary thyroid carcinoma
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(PTC) (2), medullary thyroid carcinoma (MTC) (3), lung adenocarcinoma (4-6),
chronic myeloproliferative disorders (7, 8), Spitz melanoma (9), colon (10, 11), breast
(12) and salivary duct (13) carcinomas. CCDC6 (coiled-coil domain containing 6)-
RET rearrangement has also been found in EGFR (epidermal growth factor receptor)
mutant lung adenocarcinoma patients who had progressed upon EGFR TKI
treatment (14). In addition, various types of germline point mutations activating the
RET kinase are the causative event of hereditary MTC, in the frame of multiple
endocrine neoplasia type 2 (MEN2A, MEN2B) syndromes. Somatic RET mutations,
mostly RET/M918T, are commonly associated (around 50% of cases) to sporadic
MTC (1, 15). Finally, RET is overexpressed in several malignancies including breast
(16, 17) and pancreatic (18, 19) carcinoma.
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RET targeting with tyrosine kinase inhibitors (TKIs) has emerged as a
promising molecular approach for the treatment of cancer (20, 21). Two TKIs,
Vandetanib and Cabozantinib, were found to exhibit VEGFR2 (vascular endothelial
growth factor receptor 2) and RET activity (22, 23). These two drugs have been
approved to treat MTC because of their capability to prolong progression-free survival
(24, 25). In addition, Sorafenib and Lenvatinib, two multikinase inhibitors with activity
against RET, have been approved for the treatment of radioiodine-refractory
differentiated thyroid cancer (26, 27). Novel TKIs, BLU-667, LOXO-292 and RXDX-
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105, have recently demonstrated promising clinical activity in RET-driven cancers
(28-30).
Toxicity, due to on- or off-targets effects, and resistance formation may limit
efficacy of TKIs in clinical practice. We sought out to set up a fragment-based
chemical screen to identify novel RET inhibitors. This approach lead to the clinical
candidate Pz-1, a type-2 TKI directed against RET and VEGFR2 (31).
In vivo stability is a critical factor that directly determines efficacy (32). Through
completion of investigative new drug (IND) studies, a demethylated less active
metabolite of Pz-1 was identified. Here, by applying bioisosteric substitution of the
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Pz-1 site susceptible to demethylation, we report the identification of NPA101.3, as a
novel clinical TKI candidate and its characterization through in vitro and in vivo
assays.
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RESULTS
Discovery of NPA101.3. During IND-studies of Pz-1, a series of in vivo metabolites
were identified; via Phase 1 metabolic systems, Pz-1 is oxidized, hydrolysed, or
demethylated generating a less active derivative (Fig. 1 and data not shown).
Hydrolysis and initial oxidation occur at the selectivity region, which is the region of
Pz-1 that enters the allosteric pocket of both RET and VEGFR2. Any modification to
reduce metabolism at this region can alter selectivity and inhibitory profiles and,
therefore is likely to alter the optimized RET/VEGFR2 inhibitory profile. Another
major, metabolic pathway of Pz-1 is initiated via the Phase 1 demethylation of
methylpyrazole to generate Pz-1a. In turn, Pz-1a is further biotransformed via
oxidation (to Pz-1b) or oxidation and sugar conjugation through Phase 2 systems (to
Pz-1c). Interestingly, the methylpyrazole substituent of Pz-1 is oriented towards the
solvent and is not expected to contribute significantly to selectivity profiles (31, 32)
(Fig. 1).
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Bioisosteric replacement of the methylpyrazole of Pz-1 was investigated to
generate an alternative clinical candidate with similar polypharmacological profile but
resistance to demethylation. In both RET and VEGFR2, the methylpyrazole binds to
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solvent exposed regions, which can accommodate a variety of substituents. SAR
(structure-activity relationship) studies have shown that replacement of
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methylpyrazole with various five-member ring systems decreases inhibitory activity
(31). Therefore, substitution with polar, electron-deficient (methylsulfonyl)benzene
was investigated. The methylsulfonyl group occupies the same chemical space as
the methyl pyrazole and has the same hydrogen binding pattern at the computer
modelling (data not shown); therefore, it is expected not to affect the pharmacological
activity of the drug as compared to methylpyrazole. Importantly, analogues containing
(methylsulfonyl)benzene do not carry the structural liability of demethylation.
Therefore, the (methylsulfonyl)benzene was chosen as an appropriate bioisosteric
replacement for methylpyrazole, to obtain the novel compound designated NPA101.3
(Fig. 1). The chemical synthesis of NPA101.3 is described in the Supporting
information.
Computational modelling of NPA101.3. NPA101.3 was modelled in the RET kinase
DFG (aspartic acid [D892], phenylalanine [F893], glycine [G894])-out computational
model as previously described (Fig. 2) (31). The model suggested that NPA101.3
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binds to RET kinase in its inactive (DFG-out) conformation, as it is characteristic of
type-2 TKIs (33). The benzimidazole moiety acts as the ‘warhead’, making a key
hydrogen bond with A807 at the RET ATP binding site (hinge-region). The
benzimidazole also forms two π-π stacking interactions with Y806. The p-sulfone
substituent displays orientation towards the solvent and engages K728 through a
cation-π interaction. The amide region interacts with D892 from the DFG motif
through a hydrogen bond. This interaction opens-up a novel lipophilic pocket, which
is effectively filled by tert-butyl isoxazole. The modelling predicted NPA101.3 to bind
RET with high affinity and with a projected ΔG of -11.9 k/cal mol. Moreover, the model
indicated that binding of NPA101.3 was not influenced by substitutions of V804, which
is the gatekeeper residue whose replacement with a methionine or a leucine confers
resistance to Vandetanib, Cabozantinib, and several other inhibitors (Fig. 2 and
Supporting Information, Table S1) (29, 34-36). NPA101.3 was also predicted to bind
VEGFR2 with comparable high affinity (Fig. 2). Indeed, similar to Pz-1, the free
rotation of the methylene linker permits tailored orientation of the isoxazole in the
allosteric pocket of both RET and VEGFR2 enabling balanced inhibition of both
kinases (31).
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To further address the type of binding, we docked sorafenib in the RET model
and compared docking poses between the RET-sorafenib and VEGFR2-sorafenib
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(PDB #3WZE) complexes. The poses were near-identical, further suggesting
NPA101.3 is a type-2 inhibitor (Supporting information, Fig. S1).
Kinase inhibitory activity of NPA101.3. We tested the ability of NPA101.3 to inhibit
RET, the RET/V804M mutant, and VEGFR2 in an in vitro kinase assay. To determine
if NPA101.3 tightly bound the kinase, we used high concentration of ATP (190 µM).
Such a high concentration is orders of magnitude greater than the Km for either RET
or VEGFR2 and reflects the high intracellular concentration of ATP (1-10 mM).
NPA101.3 exhibited an inhibitory concentration 50 (IC ) of 0.001 µM for RET, 0.008
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µM for RET/V804M, and 0.003 µM for VEGFR2. Thus, NPA101.3 represents a novel
RET/VEGFR2 dual inhibitor with a pharmacological profile similar to Pz-1.
To further investigate NPA101.3 binding to RET, a thermal shift assay was
performed to monitor protein melting temperature. This assay determines the drug-
induced increase in the melting temperature (ΔTm) of the isolated RET kinase
domain, which reflects the stability of the kinase-ligand complex (37, 38) and
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correlates with the inhibitor IC (39). Addition of NPA101.3 increased the thermal
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stability of active and phosphorylated RET by a dramatic ΔTm of 16 °C ± 1.1 °C, over
a time course of 240 minutes (Fig. 3C; a representative experiment is reported in Fig.
3A). This was consistent with values seen for type-2 inhibitors, such as Sorafenib,
stabilising a DFG-out inactive conformer (40). We observed the same magnitude of
thermal shift by NPA101.3 for wild type and V804M (Fig. 3B), confirming that the
compound is insensitive to gatekeeper mutation. In contrast, a substantially smaller
ΔTm shift of 6.1°C ± 3.3 °C was observed with the type-1 compound PP1 (Fig. 3C),
consistent with our previous published data (38). As expected, the type-1 inhibitor
PP1 was sensitive to gatekeeper mutation (Fig. 3C). We conclude that NPA101.3
binds tightly to both wild type and V804M RET kinase domain in a type-2 binding
mode.
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In addition, NPA101.3, at a concentration of 100 nM, was subjected to a
kinome scan against a 96 kinases panel representing major kinome clusters
(Supporting Information, Table S2). The compound featured good selectivity,
displaying strong binding activity (>90% bound) for only 7 additional kinases: CSF1R
(colony stimulating factor 1 receptor), FRK (fyn related Src family tyrosine kinase),
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HCK (HCK proto-oncogene, Src family tyrosine kinase), LYN (lyn proto-oncogene,
Src family tyrosine kinase), MKNK2 (MAPK interacting serine/threonine kinase 2),
TRKA (neurotrophic receptor tyrosine kinase 1) and TRKC (neurotrophic receptor
tyrosine kinase 3) and weak binding (>10-35% bound) for another 6 kinases
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(Supporting information Table S2 and Fig. S2). It is important to note that
measurement of binding affinity, that is based on control-ligand displacement (Kd), is
more sensitive than the in vitro kinase assay. Among the 7 kinases with >90%
binding, TRKA and CSF1R were selected for an in vitro kinase assay. NPA101.3 was
active against TRKA and CSF1R with IC50 of 32 nM and 46 nM, respectively
(Supporting information, Fig. S3). Finally, we verified the ability of NPA101.3 to
interfere with hERG (potassium voltage-gated channel subfamily H member 2)
conductibility via patch-clamp assay. As shown in Supporting information, Table S4,
the IC dose of NPA101.3 for hERG was 7.57 M (while it was 0.027 M in the case
50
of the positive control E-4031), thereby more than 7000-fold higher than that for RET
(1 nM) (Supporting information, Table S3).
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NPA101.3-mediated inhibition of RET and VEGFR2 phosphorylation and signalling.
Fibroblasts transfected with RET/C634R were treated with increasing doses of
NPA101.3, ranging from 0.1 to 10.0 nM. RET autophosphorylation was assessed by
Western blotting using two different antibodies able to recognize phosphorylated
Y905, located in the RET kinase activation loop, or Y1062, a multidocking site
involved in RET downstream signalling (41). As shown in Figure 4A, RET
phosphorylation started to be inhibited at 0.3 nM and was almost completely blocked
at 3.0 nM. NPA101.3 also inhibited other MTC-associated RET oncogenic point
mutants, including RET/M918T, RET/A883F, and RET/V804L/M (Fig. 4B).
Rearranged RET oncoproteins (CCDC6-RET, NCOA4-RET, FGFR1OP-RET) were
highly sensitive to NPA101.3 inhibition, as well (Fig. 4C). Finally, 1.0 nM NPA101.3
reduced ligand-induced VEGFR2 autophosphorylation; inhibition was virtually total at
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10.0 nM, indicating that NPA101.3 has similar inhibitory activity for VEGFR2 and RET
(Fig. 4D).
NPA101.3 inhibited RET autophosphorylation and signalling along the
SHC/MAPK pathway in human cancer cell lines endogenously expressing RET
oncogenic variants, including MTC cell lines, TT (RET/C634W) and MZ-CRC-1
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(
RET/M918T), PTC cell line TPC-1 (CCDC6-RET); in the lung adenocarcinoma cell
line Lc-2/ad (CCDC6-RET) only RET and SHC (but not MAPK) de-phosphorylation
was detectable (Supporting information, Fig. S4). Virtually no effect on SHC/MAPK
signalling was identified in control cell lines lacking RET oncogenes, including Nthy-
ori-3.1 thyroid follicular cells, BCPAP and 8505-C, derived from thyroid cancers
negative for RET oncogenes, and PC-9, A549 and CALU-1, derived from lung
adenocarcinomas negative for RET oncogenes (Supporting information, Fig. S5).
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NPA101.3-mediated inhibition of RET-driven cell proliferation. The murine pro–B cell
line Ba/F3 requires IL-3 (interleukin 3) for proliferation and survival; this dependency
is bypassed by active tyrosine kinases. Therefore, the Ba/F3 cell line is a standard
cell culture model to determine activity and drug-mediated inhibition of tyrosine
kinases (42). Accordingly, stable transfection of RET/C634R, RET/M918T and
CCDC6-RET oncoproteins promotes IL-3-independent growth (Fig. 5). Treatment
with NPA101.3 blunted RET-driven (IC of 1.6-3.1 nM), but not IL-3-driven parental
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Ba/F3 cell proliferation (Fig. 5), parallel to inhibition of RET phosphorylation
(Supporting information Fig. S6).
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NPA101.3 inhibited proliferation of RET mutant TT, MZ-CRC-1, TPC-1 and Lc-
/ad human cancer cells with an IC of 0.67-3.6 nM (Supporting information, Fig. S7
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and Table S4). The IC dose for all human RET-negative cells was greater than
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100.0 nM, confirming compound selectivity (Supporting information, Fig. S8 and
Table S4).
NPA101.3 inhibits tumorigenicity of RET-transformed cells. The IC50 dose for
NPA101.3-mediated NIH3T3 RET/C634Y cell proliferation inhibition was 4.17 nM;
virtually no effect was observed on proliferation of NIH3T3 HRAS (Harvey rat
sarcoma virus oncogene)/G12V cells up to 100.0 nM (Supporting information Fig.
S9). At 10.0 nM, the compound almost completely inhibited phosphorylation of RET
and SHC, and attenuated phosphorylation of MAPK, in NIH3T3 RET/C634Y cells,
while virtually no effect was detectable in NIH3T3 HRAS/G12V cells up to a dose of
100.0 nM (Supporting information Fig. S10).
In vivo target (RET and VEGFR2) inhibition was studied by treating animals
grafted with NIH3T3 RET/C634Y cells at different doses (0.3, 1 or 3 mg/kg/day p.o.)
of NPA101.3 for two days and then performing Western blot analysis on protein
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extracts. As shown in Supporting information (Fig. S11) 3 mg/kg/day dose of
NPA101.3 strongly inhibited RET autophosphorylation and signalling as well as
VEGFR2 phosphorylation. Then, in order to determine anti-tumorigenic activity and
to better distinguish RET- and VEGFR2-mediated effects, we tested NPA101.3 in
nude mice transplanted with NIH3T3 cells transformed either by RET/C634Y or
HRAS/G12V. Before tumours appeared, animals were treated daily with NPA101.3
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(1.0, 3.0 or 10.0 mg/kg/day) or left untreated. NPA101.3 preferentially inhibited RET
compared to RAS-driven tumours. While 10.0 mg/kg of compound completely
prevented tumor formation induced by oncogenic RET, the treatment reduced, but
did not abrogate, formation of tumours driven by RAS; moreover, at lower doses the
compound significantly reduced growth of RET-, but not RAS-, driven tumors (Fig. 6).
At 1.0 mg/kg, NPA101.3 exhibited strong RET phosphorylation and signalling
inhibition in NIH3T3 RET/C634Y tumours (Supporting information Fig. S12); in
contrast, virtually no effect on RAS signalling (MAPK phosphorylation) was detected
in NIH3T3 HRAS/G12V tumours. Still, VEGFR2 inhibition was detected also in RAS-
driven tumours, an effect that may explain the, albeit reduced, drug effect on their
growth (Supporting information Fig. S13). Importantly, as predicted, no trace of
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NPA101.3 de-methyl metabolites was detected after 1 or 4 hours from oral dosing in
mice (Supporting information Fig. S14). Moreover, in terms of potential toxicity, no
significant change in standard blood markers of organ damage was detected upon 7
days of oral dosing (10 mg/Kg/day) of NPA101.3, indicating that at this dose the
compound had no general toxicity (Supporting information Fig. S15).
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DISCUSSION AND CONCLUSIONS
Drug efficacy of a clinical agent is determined by target inhibition and ADMET
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(adsorption, distribution, metabolism, excretion, and toxicity) profiles (32). During the
IND study of the clinical TKI candidate Pz-1, we identified a major, metabolic pathway
initiated by Phase 1 demethylation of the Pz-1 methylpyrazole substituent. Here, in
order to generate secondary candidates with similar pharmacological profiles to Pz-
1
but no propensity to demethylation, we replaced methylpyrazole with the
metabolically-resistant bioisostere (methylsulfonyl)benzene. This led to NPA101.3, a
demethylation-resistant
polypharmacology.
clinical
candidate
optimized
for
single-agent
Molecular modelling indicated that, similar to Pz-1, NPA101.3 binds the DFG-
out ‘inactive’ conformation of RET and VEGFR2, thus functioning as a bona fide type-
2
TKI (31). This is supported experimentally by the relatively large thermal shift
observed for the RET kinase-NPA101.3 complex. NPA101.3 exhibited an IC50 of
0.001 µM for RET and 0.003 µM for VEGFR2. Noteworthy, similar to Pz-1, NPA101.3
was able to bind tightly to and inhibit (IC50 of 0.008 µM) the RET mutant at the
gatekeeper site (V804M), which is refractory to the clinical inhibitors Vandetanib and
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Cabozantinib (31). NPA101.3 featured excellent selectivity for RET and VEGFR2,
exhibiting affinity only for few other kinases such as TRKs (TRKA and TRKC),
CSF1R, FRK, HCK, LYN and MKNK2. Among them, TRKA and TRKC are well known
driver oncogenes, which expands the potential therapeutic application of NPA101.3
beyond RET-driven cancers (43).
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In cell-based assays, NPA101.3 inhibited RET autophosphorylation and
displayed a potent (IC50 between 0.67 and 4.17 nM) growth inhibitory effect on
different types of RET (either transfected or endogenously expressing) mutant cells
with no detectable effect at doses up to 100 nM in cells negative for RET oncogenes.
Finally, NPA101.3 featured efficacy already at 1 mg/kg/day in RET-driven tumours in
nude mice. At higher doses (10 mg/kg/day), NPA101.3 displayed activity also against
RAS-driven tumours, likely mediated by VEGFR2 inhibition. Therefore, though in a
therapeutic setting toxicity may be increased by hitting multiple targets, it is feasible
that activity of the NPA101.3 against RET-driven tumours, may take advantage from
a combination of cell autonomous (on tumour cells growth mediated by RET
inhibition) and non-cell autonomous (on blood vessels formation mediated by
VEGFR2 inhibition) effects, potentiating its effect and reducing the chances of
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resistance development. Several TKIs with anti-RET activity are available and some
are already registered for treatment of different types of thyroid cancers. Lesson
learned from the use of TKIs in various cancer types has shown that second line
inhibitors are clinically useful when first line treatments are hampered by toxicity
and/or resistance. Based on potency, selectivity, and balanced RET/VEGFR2
inhibition, we posit that NPA101.3 may represent a promising, demethylation-
resistant type-2 TKI for the treatment of RET-driven tumours. Studies regarding PK
() profile and metabolic stability will clarify in the future how NPA101.3 is metabolized
in vivo. This notion will be crucial to propose NPA101.3 as a clinical candidate.
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EXPERIMENTAL SECTION
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General chemistry procedures and synthesis of NPA101.3. Molecular formula Smiles
String:(O=C(NC1=NOC(C(C)(C)C)=C1)CC(C=C2)=CC=C2N(C=N3)C4=C3C=C(C5
=
CC=C(S(=O)(C)=O)C=C5)C=C4) All solvents were reagent grade or HPLC grade
and all starting materials were obtained from commercial sources and used without
further purification. Purity of final compounds was assessed using a Shimadzu ultra-
high throughput LC/MS system (SIL-20A, LC-20AD, LC-MS 2020, Phenomenex®
Onyx Monolithic C-18 Column) at variable wavelengths of 254 nM and 214 nM
(Shimadzu PDA Detector, SPD-MN20A) and was >95%, unless otherwise noted. The
HPLC mobile phase consisted of a water-acetonitrile gradient buffered with 0.1%
formic acid. 1H NMR spectra were recorded at 400 MHz and 13C spectra were
recorded at 100 MHz, both completed on a Varian 400 MHz instrument (Model#
4001S41ASP). All compounds were purified using silica gel (0.035-0.070 mm, 60 Å)
flash chromatography, unless otherwise noted. Microwave assisted reactions were
completed in sealed vessels using a Biotage Initiator microwave synthesizer.
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In vitro kinase assays. Kinase activity was measured by a microfluidic assay that
monitors the separation of the phosphorylated product from substrate (31). The assay
was run using a 12-sipper chip on a Caliper EZ Reader II (PerkinElmer®, Walthman,
USA) with separation buffer (100 mM HEPES, 10 mM EDTA, 0.015% Brij-35, 0.1%
CR-3 PerkinElmer®). In 96-well polypropylene plates (Greiner, Frickenhausen,
Germany), compound stocks (20 mM in DMSO) were diluted into kinase buffer (50
mM HEPES, 0.075% Brij-35, 0.1 % Tween 20, 2 mM DTT, 10 mM MgCl2, and 0.02%
NaN3) in 12-point ½log dilutions (2 mM–6.32 nM). After, 1 μL was transferred into a
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384-well polypropylene assay plate (Greiner). The enzymes (RET, RET/V804M,
VEGFR2, TRKA, CSF1R) (Invitrogen™, Grand Island, USA) were diluted in kinase
buffer to a concentration of 2 nM and 5 L of the enzyme mixture was transferred to
the assay plate. Kinases were pre-incubated with the TKI or control buffer, with gentle
shaking for 60 min to allow the inhibitor to trap the DFG-out conformation; indeed, it
has been reported for the type-2 (DFG-out) p38 inhibitor, BIRB-796, that an increase
in incubation time increases activity (44). A substrate mix was prepared containing
ATP (Ambresco®, Solon, USA) and substrate peptide dissolved in kinase buffer, and
5
L of the substrate mix was added to the assay plate. Running concentrations were
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as follows: ATP (190 M), peptide (1.5 M), compound 12-point ½log dilutions (0.2
mM–0.632 nM). For positive control, no inhibitor was added. For negative control, no
enzyme was added. The plate was run until 10-20% conversion, based on the positive
control wells. The following separation conditions were utilized: upstream voltage -
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500V; downstream voltage, -1900V; chip pressure -0.8. Percent inhibition was
measured for each well comparing starting peptide to phosphorylated product peaks
relative to the baseline. Dose response curves, spanning the IC50 dose, were
generated in GraphPad Prism® 7 and fit to an exponential one-phase decay line; IC50
values were obtained from the half-life value of the curve. IC values were generated
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in triplicate.
Thermal shift assay. Wild type and V804M mutant RET core kinase domain proteins
were expressed in SF21 cells and purified using a GST affinity tag as previously
described (45). Subsequently, both proteins were purified and phosphorylated. The
affinity tag was removed using HRV 3C protease. To determine the protein thermal
shifts, 3M recombinant proteins were incubated with DMSO (vehicle control),
NPA101.3, PP1 or Sorafenib (in 100% DMSO), with a final drug concentration of
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0M, and a final DMSO concentration of 1% (v/v). Sypro-Orange dye (Life
Technologies) was added to each drug treatment, and the thermal shift was
measured in a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems)
over a temperature range of 25–90ꢀ°C. Subsequent analysis was performed using
Protein Thermal Shift Software v1.2 (Applied Biosystems).
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Cell cultures. NPA101.3 was dissolved in dimethyl sulfoxide (DMSO) at 50 mM
concentration and stored at -80°C. Final dosing solution was prepared on the day of
use by dilution of the stock solution in cell growth media. RAT1 cells transformed by
the various RET mutants (46) were a kind gift of M. Billaud and were cultured in
DMEM with 10% foetal calf serum, 2 mM L-glutamine and 100 units/ml penicillin-
streptomycin (GIBCO). NIH3T3 cells transformed by RET/C634Y, HRAS/G12V,
CCDC6-RET, NCOA4-RET and FGFR1OP-RET were cultured in DMEM with 5% calf
serum, 2 mM L-glutamine and 100 units/mL penicillin-streptomycin (GIBCO).
Parental NIH3T3 cells were grown in DMEM with 10% calf serum, 2 mM L-glutamine
and 100 units/mL penicillin-streptomycin (GIBCO). All the RET constructs expressed
in RAT1 or NIH3T3 cells encoded the short isoform of the RET protein (RET-9). Ba/F3
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murine pro-B cells stably expressing NCOA4-RET, RET/C634R and RET/M918T
mutant proteins (all cloned in the long RET-51 isoform) were generated by
electroporation. Parental and transformed Ba/F3 cells were cultured in RPMI
supplemented with 10% foetal calf serum, 2 mM L-glutamine and 100 units/ml
penicillin-streptomycin (GIBCO), and, in the case of parental cells, supplemented with
10 ng/ml IL-3. CALU-1, derived from a human lung adenocarcinoma, were grown in
EMEM with 10% foetal calf serum, 2 mM L-glutamine and 100 units/mL penicillin-
streptomycin (GIBCO). PC-9, derived from a human lung adenocarcinoma, were
grown in RPMI with 10% foetal calf serum, 2 mM L-glutamine and 100 units/mL
penicillin-streptomycin. Nthy-ori-3-1, derived from normal thyroid follicular tissue and
immortalized by SV40 Large T, TPC-1, derived from a human PTC harbouring
CCDC6-RET (47), BCPAP, derived from human PTC, 8505-C, derived from a human
undifferentiated thyroid cancer, and A549, derived from a human lung
adenocarcinoma, were cultured in DMEM with 10% foetal calf serum, 2 mM L-
glutamine and 100 units/mL penicillin-streptomycin. TT, from a human MTC
harbouring RET/C634W (48), and MZ-CRC-1, from a human MTC harbouring
RET/M918T (a kind gift of R. F. Gagel), were cultured in RPMI with 20% foetal calf
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serum, 2 mM L-glutamine and 100 units/mL penicillin-streptomycin (GIBCO). Lc-2/ad,
derived from human lung adenocarcinoma harbouring CCDC6-RET (49), were grown
in RPMI 1640/Ham's F12 (1:1) with 10% foetal calf serum, 2 mM L-glutamine and 100
units/mL penicillin-streptomycin (GIBCO). All the human cell lines were SNP
authenticated in 2017.
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Immunoblotting. Protein lysates were prepared according to standard procedures.
Briefly, cells were lysed in a buffer containing 50 mM N-2-hydroxyethylpiperazine-N'-
2-ethanesulfonic acid (HEPES; pH 7.5), 1% (vol/vol) Triton X-100, 150 mM NaCl, 5
mM EGTA, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium vanadate, 2
mM phenylmethylsulphonyl fluoride (PMSF) and 1 g/mL aprotinin. Lysates were
clarified by centrifugation at 10,000 Xg for 15 min. Lysates containing comparable
amounts of proteins, estimated by a modified Bradford assay (Bio-Rad, Munich,
Germany), were subjected to direct Western blot. Immune complexes were detected
with the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little
Chalfort, UK). Anti-phospho-SHC (#Y317), that recognizes SHC protein when
phosphorylated on Y317, was from Upstate Biotechnology Inc. (Lake Placid, NY).
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Anti-SHC (H-108) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAPK
(#9101) and anti-phospho-MAPK (#9102), specific for p42/44MAPK (ERK1/2)
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phosphorylated on Thr202/Tyr204, were from Cell Signalling (Danvers, MA, USA).
Anti-phospho-VEGFR2/KDR (#2478), specific for VEGFR2/KDR phosphorylated on
Tyr1175 and anti-VEGFR2/KDR (#2479) were from Cell Signalling Technologies
(Danvers, MA, USA). Anti-phospho-p70S6K (#9234), specific for p70S6K
phosphorylated on Thr389 and anti-p70S6K (#2708) were from Cell Signalling
Technologies (Danvers, MA, USA). Anti-RET is a polyclonal antibody raised against
the tyrosine kinase protein fragment of human RET; anti-phospho905 is a phospho-
specific polyclonal antibody recognizing RET proteins phosphorylated at Y905 and
anti-phospho1062 is a phospho-specific polyclonal antibody recognizing RET
proteins phosphorylated at Y1062 (50). Secondary antibodies coupled to horseradish
peroxidase were from Santa Cruz Biotechnology.
Cell growth curves. Nthy-ory-3-1 (10,000/well), TPC-1 (10,000/well), MZ-CRC-1
(100,000/well), TT (200,000/well), Lc-2/ad (100,000/well), BCPAP (10,000/well),
8505-C (10,000/well), PC-9 (10,000/well), A549 (10,000/well), CALU-1 (10,000/well),
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NIH3T3 RET/C634Y (10,000/well) and NIH3T3 HRAS/G12V (10,000/well) were
seeded in 6-well tissue culture plates. Cells were kept in 2% (TPC-1), 5% (Nthy-ori-
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3-1), or 10% (BCPAP, 8505-C, CALU-1, A549, PC-9, TT, MZ-CRC-1 and Lc-2/ad)
foetal calf serum or in 2% (NIH3T3 RET/C634Y and HRAS/G12V) calf serum. Ba/F3
cells (200,000/well in 2ml) were seeded in 6-well tissue culture plates and kept in
10% foetal calf serum. The day after plating, different concentrations of drug or
vehicle were added to the medium and changed every 2-3 days. Cells were counted
every day (Ba/F3), 2 days (fibroblasts) or 2-3 days (human cell lines). To compare
cell growth, we performed unpaired Student’s t test using the Instat software program
(Graphpad Software Inc). All P values were two-sided, and differences were
considered statistically significant at P <.02. IC doses were calculated through a
50
curve fitting analysis from last day of growth curves using the PRISM software
program (Graphpad Software Inc).
Mouse xenograft experiments. NPA101.3 was dissolved in 80% H O, 19.875%
2
Tween 20, 0.125% Xanthan gum. The formulation was stored at room temperature
and vortexed prior to administration. NIH3T3 RET/C634Y (200,000 cells) or NIH3T3
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HRAS/G12V (50,000 cells) were inoculated subcutaneously into dorsal portion (both
sides) of 6-week-old female BALB/c nu/nu mice (n. 32 mice/cell line) (Jackson
Laboratories, Bar Harbor, Maine). After 4 days, before tumours had appeared,
animals were randomly assigned to receive NPA101.3 (1.0, 3.0 or 10 mg/kg daily) (8
mice/group) or vehicle control (8 mice) by oral gavage. Tumour diameters were
measured with caliper every 2-3 days. Tumour volumes (V) were calculated by the
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rotational ellipsoid formula: V= A x B /2 (A=axial diameter; B= rotational diameter).
No mouse showed signs of wasting or other signs of toxicity. Animals were fed ad
libitum on an autoclaved diet and tap water and were maintained at the Dipartimento
di Medicina Molecolare e Biotecnologie Mediche Animal Facility. All manipulations
were performed while the animals were under isoflurane gas anaesthesia. Animal
studies were conducted in accordance with Italian regulations for experimentation on
animals and approved by the Italian Ministry of health (Authorization n. 1023/2015-
PR). To compare tumour growth the unpaired Student’s t test (InStat program,
GraphPad software) was used. P values were statistically significant at P <0.05.
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