A Novel Covalent mTOR Inhibitor, DHM25, Shows in Vivo Antitumor Activity against Triple-Negative Breast Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.5b00991

Constitutive activation of the PI3K/mTOR signaling pathway contributes to carcinogenesis and metastasis in most, if not all, breast cancers. From a chromene backbone reported to inhibit class I PI3K catalytic subunits, several rounds of chemical syntheses led to the generation of a new collection of chromologues that showed enhanced ability to kill PI3K-addicted cancer cells and to inhibit Akt phosphorylation at serine 473, a hallmark of PI3K/mTOR activation. This initial screen uncovered a chromene designated DHM25 that exerted potent antitumor activity against breast tumor cell lines. Strikingly, DHM25 was shown to be a selective and covalent inhibitor of mTOR using biochemical and cellular analyses, modeling, and a large panel of kinase activity assays spanning the human kinome (243 kinases). Finally, in vivo, this novel drug was an efficient inhibitor of growth and metastasis of triple-negative breast cancer cells, paving the way for its clinical application in oncology. (Figure Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00991

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GRAPHICAL ABSTRACT
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OH
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A novel covalent mTOR inhibitor, DHM25, shows in vivo anti-tumor activity against
triple-negative breast cancer cells
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,2,3
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Amélie Fouqué,
Olivier Delalande, Mickael Jean, Rémy Castellano, Emmanuelle
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Josselin, Marine Malleter, Kenji F. Shoji, Mac Dinh Hung, Hariniaina Rampanarivo,
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Yves Collette, Pierre van de Weghe,
and Patrick Legembre
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Université de Rennes-1, 2 avenue du Prof. Leon Bernard, 35043 Rennes, France
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CLCC Eugène Marquis, INSERM ER440 Oncogenesis, Stress & Signaling, rue bataille
Flandres Dunkerque, 35042 Rennes, France
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Equipe Labellisée Ligue Contre le Cancer, rue bataille Flandres Dunkerque, Rennes
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IGDR, CNRS UMR 6290, UFR des Sciences Biologiques et Pharmaceutiques, 2 Av. du
Professeur Léon Bernard, 35043 Rennes, France
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UMR6226, CNRS, Institut des Sciences Chimiques de Rennes, Equipe Produits Naturels,
Synthèses, Chimie Médicinale, UFR des Sciences Biologiques et Pharmaceutiques, 2 Av. du
Professeur Léon Bernard, 35043 Rennes, France
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CRCM, INSERM, U1068; Institut Paoli-Calmettes; Aix-Marseille Université; CNRS, UMR
7258, Marseille, France
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INSERM, U1085, 2 avenue du Prof. Leon Bernard, 35043 Rennes, France
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Faculty of Chemistry, National Vietnam University, 19 Le Thanh Tong, Hanoi, Vietnam
Running title: Generation of a new mTOR inhibitor
Key Words: PI3K, mTOR, chromene, breast cancer, chemotherapy
The authors disclose no potential conflicts of interest.
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ABSTRACT
Constitutive activation of the PI3K/mTOR signaling pathway contributes to carcinogenesis
and metastasis in most, if not all, breast cancers. From a chromene backbone reported to
inhibit class I PI3K catalytic subunits, several rounds of chemical syntheses led to the
generation of a new collection of chromologs that showed enhanced ability to kill PI3K-
addicted cancer cells and to inhibit Akt phosphorylation at serine 473, a hallmark of
PI3K/mTOR activation. This initial screen uncovered a chromene designated DHM25 that
exerted potent anti-tumor activity against breast tumor cell lines. Strikingly, DHM25 was
shown to be a selective and covalent inhibitor of mTOR using biochemical and cellular
analyses, modeling, and a large panel of kinase activity assays spanning the human kinome
(243 kinases). Finally, in vivo, this novel drug was an efficient inhibitor of growth and
metastasis of triple-negative breast cancer cells, paving the way for its clinical application in
oncology.
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INTRODUCTION
The PI3K/mTOR signaling pathway controls cell survival, growth, proliferation,
differentiation, and motility. Therefore, it is not surprising to find this signaling pathway
deregulated in human cancers. PI3K phosphorylates the D3 position on phosphoinositide to
yield phosphatidylinositol-3,4,5-trisphosphate (PIP3). This phosphorylated lipid can then
interact with several proteins containing a pleckstrin homology (PH) domain. Negative
regulation of this pathway is mainly conferred by phosphatase and tensin homolog deleted on
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chromosome 10 (PTEN)
and inositol polyphosphate-4-phosphatase (INPP4B), which
dephosphorylate PIP3 and PIP2, respectively. The serine/threonine kinase Akt possesses a PH
domain, and its recruitment to the plasma membrane induces its activation through
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phosphorylation at threonine 308 by PDK1 and at serine 473 by mammalian target of
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rapamycin complex 2 (mTORC2). Once activated, Akt phosphorylates and thereby
inactivates tuberous sclerosis complex-2 (TSC2), a GTPase-activating protein (GAP) that
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inhibits the GTPase Rheb (RAS homolog expressed in brain). The release of the TSC2-
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driven Rheb blockage leads to the activation of the serine-threonine kinase mTOR. mTOR
belongs to the PI3K-related kinase family and forms at least two complexes whose exhaustive
composition, cellular distribution, and function remain to be defined. While mTOR complex 1
(mTORC1) is instrumental in translation and cell growth regulation via phosphorylation of
substrates such as eukaryotic initiation factor eIF4E binding protein (4E-BP1), mTOR
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complex 2 (mTORC2) regulates a different set of substrates, including Akt. Of interest,
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mTORC1 exerts a negative feedback loop on both PI3K and mTORC2
and thereby
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downregulates Akt activity. Rapamycin, a macrolide interacting with FKBP12, selectively
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inhibits mTORC1.
Despite promising results in animal models of cancer, rapamycin and
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rapalogs were generally disappointing in human trials (reviewed in ). One explanation for
these disappointing results is that by eliminating the mTORC1-dependent negative feedback
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program, rapalogs lead to prolonged activation of the mTORC2/Akt signaling pathway,
which may promote the occurrence of new cancers during chronic treatment. To overcome
this limitation, ATP-competitive inhibitors that potently block both mTORC1 and mTORC2
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are sought as anticancer agents. Beyond rapalogs that show limited clinical benefit in
cancers, few N-heterocyclic derivatives have been identified as mTOR inhibitors. These small
molecules, AZD8055, NVP-BEZ235, OSI-027, INK128 and Torin1, inhibit both mTORC1
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and mTORC2 and are currently in clinical trials. Although these ATP-competitive inhibitors
exert an anti-cancer activity, only NVP-BEZ235 presents some interest to treat breast cancer
patients and may be used in combination with other chemotherapeutic drugs to improve
clinical outcomes in this pathology (tested in phase I and II clinical trials, see on
clinicaltrials.gov/). This deficiency of small inhibitors of the mTOR activity for the treatment
of breast cancer reveals the urgent need for the development of new and potent compounds.
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The most common cancers are colorectal cancers (13.6%) and breast cancers (13.1%). Based
on their gene expression profiles, breast cancers are categorized as luminal A/B, HER-2+, and
basal-like, with a close relationship between this classification and the clinical outcome of the
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tumors. Basal-like breast cancers are closely related to triple-negative breast cancers
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(
TNBCs). Representing 10–20% of all breast cancers, TNBC/basal-like tumors account for a
disproportionate number of mortalities, in part due to their aggressiveness and the lack of
effective therapeutic regimens. Indeed, while the estrogen receptor (ER)-positive cancers
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closely related to luminal A/B cancers receive hormone therapy and HER2-amplified
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cancers are a clinical success because of neutralizing antibodies targeting HER2, TNBCs
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can only be treated with chemotherapy. A recent large-scale study of 50 luminal breast
cancer genomes sequenced at high coverage identified over 1700 genic mutations, but only
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three of these genes were mutated at frequencies that approached or exceeded 10%. Among
these genes, PIK3CA, which codes for the p110α catalytic sub-unit of PI3K, harbored
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mutations in 43% of the analyzed tumors. Although PIK3CA mutations remain less frequent
in TNBCs as compared to non-TNBCs, loss-of-function mutations or reduced expression of
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PTEN or INPP4B are detected in 67% and 53% of TNBCs, respectively, confirming that
deregulation of the PI3K/mTOR signaling pathway is a common event in breast
carcinogenesis and supporting the need to develop potent and selective inhibitors of the
PI3K/mTOR signaling pathway to treat this pathology.
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From a chromene derivative showing a weak affinity for PI3K (IC of 10–50 μM for the
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different p110 catalytic subunits of PI3K), we used cell-based, enzyme, and biochemical
assays to generate a potent and irreversible mTOR inhibitor designated DHM25 [6,8-
dibromo-2-(4-bromophenyl)-3-nitro-2H-chromene] that efficiently kills breast cancer cells.
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RESULTS
Synthesis of chromene derivatives.
To develop new and potent PI3K inhibitors, a chromene backbone designated
S14161/pichromene was selected for its ability to kill malignant cell lines by inhibiting all
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class I PI3K catalytic subunits. Accordingly, a number of synthetic strategies have been
described for the preparation of chromene derivatives. Among them, we sought to use an
approach based on the addition of salicylaldehydes to α,β-unsaturated compounds promoted
by organic bases. The utilization of small chiral organic molecules as metal-free catalysts,
especially in the synthesis of pharmaceuticals was chosen because of the “green” advantage of
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not contaminating the final product with traces of heavy metals.
The chromene derivatives
were synthesized using pipecolic acid or tetramethylguanidine as catalyst to promote the
addition of various substituted salicylaldehydes to several α,β-unsaturated derivatives
(
Scheme 1). By varying first the west part of the pichromene and second the aryl group of the
east part, several structural modifications were progressively undertaken until the generation
of the two chromene derivatives, DHM9 and DHM25 (see below).
Selection of a potent PI3K inhibitor.
By modifying first the west part of the scaffold, new compounds were synthesized, and their
ability to kill two PI3K-dependent leukemic T cell lines (CEM and Jurkat) was tested (Fig.
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S1A).
To focus on compounds that were potentially selective for the PI3K signaling
pathway, chromologs were selected that exerted more cytotoxic activity on CEM/Jurkat cells
than on the leukemic T cell line H9, whose survival relies to a lesser extent on PI3K signaling
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(
Fig. 1B). This screening test highlighted that although pichromene was more efficient at
killing CEM and Jurkat cells than the well-established and potent PI3K inhibitor wortmannin
(
Fig. S1A), it probably did not exert its cytotoxic effect by inhibiting the PI3K signaling
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pathway, because CEM/Jurkat and H9 cells were killed to a similar extent (Fig. 1B-C).
Although PWPHT45, PWPHT47, PWPHT88, PWPHT89, and PWPHT90 showed increased
cytotoxic activity compared to pichromene (Fig. S1A), only PWPHT45 and PWPHT89 killed
more efficiently CEM/Jurkat cells than H9 cells (Fig. 1B-C), indicating that these two
chromene derivatives could exert their cytotoxic effect by selectively inhibiting the PI3K
signaling pathway. To confirm this assumption, the ability of these chemicals to inhibit the
PI3K signaling pathway in CEM cells (Fig. 1D) and in Jurkat cells (Fig. S1B) was next
analyzed by assessing their impact on the phosphorylation of Akt, a downstream PI3K
effector. Confirming the cell-based assay, incubation of CEM or Jurkat cells for 1 or 2 hours
with pichromene (10 μM) did not reduce the level of Akt phosphorylation at serine 473 in
(Fig. 1D and S1B), indicating that this compound was not a direct inhibitor of PI3K and that
its cytotoxic activity on CEM and Jurkat cells likely occurred through a PI3K-independent
mechanism. By contrast, chromene derivatives PWPHT33, PWPHT45, PWPHT47,
PWPHT54, PWPHT52, PWPHT88, and PWPHT89 showed an inhibitory effect on Akt
phosphorylation (Fig. 1D). PWPHT45 and PWPHT89 were, therefore, the most interesting
backbones from this first set of chromologs and were subsequently used to design a second
collection of derivatives (Fig. S2). The aryl substituents were modified in order to study the
influence of various compounds with different electronic or lipophilic properties and steric
hindrance on the cytotoxic activity of our lead compound. Among this second library, five
derivatives displayed better cytotoxic activity toward the leukemic T cell line CEM than
PWPHT45 and PWPHT89 (Fig. 1E). Next, DHM9, DHM10, and DHM25 were selected for
their ability to inhibit Akt phosphorylation at serine 473 (Fig. 1F-I). Of note, DHM9 and
DHM25 corresponded to PWPHT89 and PWPHT45 backbones, respectively, in which the
fluoride atom was replaced by bromine. Finally, these three compounds exhibited an in
cellulo IC of between 0.376 and 2.744 μM, whereas the PI3K inhibitor LY294002, which
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shares some structural similarities with chromologs, showed an IC close to 100 μM in CEM
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cells (Fig. 1H-I). In summary, from a weak PI3K inhibitor (pichromene) and two cell-
based/biochemical assays, two rounds of synthesis led to the identification of two novel
chromologs (DHM9 and DHM25) showing dramatically improved PI3K inhibitory activity as
compared to S14161/pichromene.
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DHM9 and DHM25 efficiently kill breast tumor cells.
Because carcinogenesis in breast epithelial cells relies on the constitutive activation of the
PI3K/mTOR signaling pathway, which occurs through the deregulation of various molecular
mechanisms including down-regulation of PTEN activity and/or expression (see Fig. S3),
constitutive activation of PI3K (gain-of-function mutations in PIK3CA or PIK3R1 indicated in
Fig.2A), or induction of the Ras signaling pathway, we next evaluated the cytotoxic effect of
our leads on nine breast tumor cell lines covering all breast cancer sub-types and exhibiting
deregulation of PI3K signaling through various molecular mechanisms (Fig. 2A).
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Independently of their histological classification (TNBC, non-TNBC, or HER2 ), all breast
cancer cell lines exposed for 24 hours to the selected chromologs underwent a cell death
program (Fig. 2A-B). Among the molecules tested, DHM25 and, to a lesser extent, DHM9
showed the most efficient cytotoxic activity (Fig. 2A). Because all breast malignant cells were
sensitive to DHM25 treatment regardless of mutations in the PI3K signaling pathway, either
these mutations did not affect the inhibitory capacity of DHM25, or DHM25 targeted a
downstream protein in the PI3K signaling pathway.
DHM25 is a selective mTOR inhibitor.
To identify the molecular targets of DHM9 and DHM25, their inhibitory activity against 243
human protein kinases was evaluated using KinaseProfiler assay™. Strikingly, despite
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structural similarities between DHM25 and DHM9 (only one additional bromine in position 8
of DHM25 as compared to DHM9), the KinaseProfiler assay™ revealed that different sets of
kinases were targeted by DHM9 and DHM25 (Fig. 3A). More importantly, no PI3Ks were
inhibited by DHM25 or DHM9. Indeed, while 1 μM DHM9 abrogated the activity of tyrosine
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(
c-yes) and serine/threonine kinases (p70S6K, LOK, BrSK1, TSSK1, and NLK), the same
concentration of DHM25 inhibited only mTOR activity (Fig. 3A). It is noteworthy that Akt
phosphorylation at serine 473, the biochemical marker that we followed to determine the
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PI3K inhibitory effect of chromologs, is the direct target of mTORC2.
To confirm that
DHM25 was an mTOR inhibitor, its ability to inhibit the phosphorylation of mTORC1 and
C2 substrates was evaluated. To this end, the phosphorylation status of 4E-BP1 (a direct
substrate of mTORC1) and S6 (a substrate of p70S6K, another target of mTORC1) was
monitored (Fig. 3B-C). DHM25 activity was also compared with that of rapamycin, an
mTORC1 kinase inhibitor that exerts its activity by preventing its binding to FK506-binding
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protein of 12 kDa (FKBP12) while sparing mTORC2 activity.
While DHM25 exposure
blocked Akt phosphorylation at serine 473 (IC = 1.5 μM), rapamycin treatment did not
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affect the intensity of phosphorylation of this mTORC2 substrate (Fig. 3B). By densitometry
analysis, DHM25 exhibited a similar capacity to inhibit the phosphorylation of 4E-BP1 (IC50
=
8.3 μM) and S6 (IC = 1 μM). By contrast, 4E-BP1 phosphorylation was relatively resistant
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to rapamycin treatment (IC = 9.5 μM) compared to S6 phosphorylation (IC = 0.3 nM). A
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recent study demonstrated that the difference in rapamycin sensitivity between these two
mTORC1 substrates was dependent on their different affinities for the mTORC1 kinase
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domain. Indeed, p70S6K activity was more sensitive to rapamycin treatment because of its
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weaker affinity for mTORC1 as compared to 4E-BP1. The differential inhibitory effects of
rapamycin on 4E-BP1 and S6 contrasted with those of ATP-competitive inhibitors that
blocked the phosphorylation of mTORC1 substrates regardless of their affinity for the
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kinase. Accordingly, these results strongly suggested that DHM25 was a competitive mTOR
inhibitor (Fig. 3B-C).
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The (S)-DHM25 enantiomer kills cancer cells and inhibits mTOR more efficiently than
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R)-DHM25.
More than half of drugs are chiral compounds, and 90% of these molecules are marketed as
racemic mixtures. Because enantiomers do not show the same spatial arrangement, their
biological, pharmacodynamic, and pharmacokinetic properties can differ. In addition, the US
Food and Drug Administration (FDA) recommends that the activity of each enantiomer be
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evaluated for racemic drugs and that chiral drugs be developed as single enantiomers.
DHM25 is a chiral molecule due to its stereogenic carbon atom, C2 (Fig. 4A). To evaluate the
therapeutic activity of both DHM25 stereoisomers, the (R)- and (S)- enantiomers were
separated by HPLC, and the absolute configuration of their chiral centers was determined
using circular dichroism (Fig. S4). Next, the biological effects of (R)- and (S)-DHM25 were
evaluated on Jurkat and CEM leukemic T cells (Fig. 4B-C). Although the difference was not
large, (S)-DHM25 killed leukemic T cells more efficiently than its isomer (Fig. 4B), and this
increased cytotoxic effect correlated with a greater inhibition of mTOR activity (Fig. 4C). In
summary, (S)-DHM25 showed a slightly better mTOR inhibitory effect than its enantiomer,
but this difference was not substantial enough to merit separating the two enantiomers in
subsequent experiments.
Molecular simulations were next undertaken to understand why (S)-DHM25 targeted mTOR
while (S)-DHM9 did not (Fig. S5A). Structural models of DHM9 and DHM25 within the
ATP pocket of mTOR provided the first basis for understanding the binding modes and
structure-activity relationships (SAR) of these compounds. First, the presence of the
additional bromine in DHM25 compared to DHM9 influenced its position inside the ATP
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pocket of mTOR (Fig. S5A-B). While DHM9 seemed to be preferentially positioned in the
adenine pocket, DHM25 overlapped both the adenine pocket and the affinity pocket, notably
through interactions with hinge residues Ile2237, Trp2239, and Val2240 at one side and with
P-loop residues Ile2163, Ser2165, and Gln2167 at the other side, which are all also ATP-
contact amino acids (Fig. S5C). Because interactions between chromene derivatives and the
mTOR catalytic pocket were mainly driven through hydrophobic contacts, the dynamics of
the ligand-free diffusion into the ATP pocket were further investigated. A contact frequency
analysis over simulated trajectories confirmed that DHM25 interacted with more key amino
acid residues (about 80% of ATP experimental contacts) in comparison to DHM9 (only 60%
of ATP contacts, Fig. S5C). Models highlighted that unlike DHM9, DHM25 interacted inside
the ATP-binding pocket of mTOR with amino acid residues of the P-loop (Fig. 4D). It is
noteworthy that despite the fact that these P-loop basic amino acids showed a relatively high
level of conservation among the PI3KK family members (Fig. S6A), this stretch of positively
charged amino acids was lost in the sequences of PI3K family members (Fig. S6B),
potentially explaining why DHM25 exhibited a selectivity for mTOR.
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DHM25 is an irreversible mTOR inhibitor.
Computer modeling was next used to design and synthesize two molecular tools in order to
confirm that this novel drug interacted with mTOR. Based on the most stable position of both
the (R) and (S) enantiomers of DHM25 inside the catalytic pocket of mTOR, DHM25 was
conjugated with a probe (biotin or FITC, Scheme 2) at the C6 position (solvent front position,
Fig. 5A). Strikingly, confocal microscopy revealed that breast cancer cells treated with
DHM25-FITC exhibited a dot-like distribution pattern (Fig. 5B) that co-localized with mTOR
labeled with an anti-mTOR mAb (yellow color in the merge panel, Pearson's coefficient =
0
.899, Fig. 5B). In agreement with modeling analyses, conjugation of biotin to C6 of DHM25
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did not alter its inhibitory effect on mTORC2 activity (Fig. 5C). More importantly, a pull-
down assay using streptavidin beads confirmed that DHM25-biotin interacted with mTOR
(Fig. 5D). In addition, the affinity of DHM25-biotin for mTOR was stronger than that of
DHM9-biotin (Fig. 5E). Overall, these findings confirmed that DHM25 was an mTOR
inhibitor.
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Modeling analysis also suggested that the electrophilic C4 in DHM25 was close enough to the
lysine at position 2187 (K2187; see Fig. 6A and S5) in the mTOR catalytic domain to undergo
a covalent link through a Michael reaction (Fig. 6A). In agreement with this observation, the
DHM25-biotin/mTOR
interaction
was
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lysis
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denaturing/reducing/boiling conditions (Fig. 5D-E). In addition, whereas extensive washing
after 2 hours of incubation with the reversible mTOR inhibitor AZD8055 led to a rapid
restoration of Akt phosphorylation at serine 473, identical treatment with DHM25 did not,
suggesting an irreversible association with the kinase (Fig. 6B). To confirm this possibility,
two types of DHM25 modifications were performed. First, a DHM25 derivative was
synthesized in which the C3-C4 double bond was reduced (Scheme 3 and Fig. 6C).
Conversion of the C3-C4 double bond into a single C-C bond removes the electrophilic
property of the carbon at position C4 and its ability to form a covalent bond with the nitrogen
atom at K2187. Of note, DHM25-H2 showed a dramatic reduction in cytotoxicity toward
leukemic (Jurkat and CEM) and breast cancer cells (Fig. 6D) as compared to DHM25, and
also lost its ability to inhibit phosphorylation of mTORC1 (4E-BP1) and mTORC2 (Akt-
S473) substrates (Fig. 6E). Next, NO , a strong electron-withdrawing group (EWG) in
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DHM25 was replaced by various chemical groups to reduce the capacity of C4 to act as an
electron acceptor. To this end, four chromologs were prepared in which the NO group was
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replaced by aldehyde (LR20), amide (LR28), ester (LR29), or carboxylic acid (LR24)
functional groups showing an electrophilic strength gradient (Scheme 4). Except for LR29,
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the lower the withdrawing capacity of the substituted group, the weaker the cytotoxic activity
of the resulting compound (Fig. 6F). In agreement with this observation, the decreased
cytotoxic activity of LR20 (replacement of NO by an aldehyde) and LR28 (replacement of
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NO by an amide functional group) toward CEM and Jurkat cells (Fig. 6F) was associated
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with their inability to inhibit Akt phosphorylation at Ser473 (Fig. 6G). These chemical
modifications of DHM25 provided some insights into how carbon 4 could serve as a Michael
acceptor to covalently bind lysine at position 2187 inside the ATP pocket of mTOR. Overall,
these findings revealed that DHM25 could act as a covalent inhibitor of mTOR.
DHM25 inhibits CD95-mediated cell migration and prevents in vivo tumor progression
of TNBC cells.
CD95L (also known as FasL) belongs to the TNF (tumor necrosis factor) family and is the
ligand for the “death receptor” CD95 (Fas/APO1). This transmembrane receptor can be
4
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cleaved by metalloproteases to produce a soluble ligand. This naturally processed CD95L
(
cl-CD95L) is increased in patients affected by TNBC and enhances the risk of metastatic
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dissemination in these patients. Unlike membrane-bound CD95L, cl-CD95L fails to induce
apoptosis and instead promotes the formation of an atypical receptosome that we designated
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the motility-inducing signaling complex (MISC). MISC formation leads to the induction of
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the pro-oncogenic PI3K/mTOR signaling pathway.
The ability of DHM25 to prevent this
CD95-mediated cell migration was assessed in TNBC cells. To address this question, the
highest dose of DHM25 that did not cause cell death in two TNBC cell lines (BT549 and
MDA-MB-231) was determined (Fig. 7A). In Boyden chamber assays, although 1 μM
DHM25 did not exhibit any cytotoxicity (Fig. 7A), it inhibited the CD95-mediated
PI3K/mTOR activation (data not shown) and abrogated CD95-mediated cell migration in
TNBC cells (Fig. 7B).
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Finally, the in vivo therapeutic activity of DHM25 against TNBCs was assessed. In
immunodeficient NOD/SCID/γc (NSG) mice, the maximum intravenous tolerated dose of
DHM25 was 10 mg/kg (Fig. S7A-B). Human MDA-MB-231 TNBC cells were orthotopically
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(
mammary fat pad) transplanted in NSG mice, and when tumors were palpable, mice received
repeated intravenous injections of DHM25 (10 mg/kg) or vehicle. DHM25 treatment
significantly and markedly reduced primary tumor growth (primary site, Fig. 7C) and
prevented distant metastasis to lungs (classical secondary tumor site) (Fig. 7C). In conclusion,
we generated an irreversible and competitive mTOR inhibitor that exhibited in vivo anti-
tumor activity against TNBC cells.
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DISCUSSION & CONCLUSIONS
Using different tumor cell lines characterized for their high or low reliance on the
PI3K/mTOR signaling pathway for survival, classical biochemical analysis (Akt
phosphorylation), large-scale kinase profiling, and several rounds of chemical synthesis, we
developed a novel irreversible inhibitor of mTOR showing in vitro and in vivo cytotoxic
activity against breast cancer cells.
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Inactivation of the DHM25 electrophilic warhead reduces or abrogates its ability to inhibit
mTOR activity, indicating that its mechanism of inhibition occurs mainly through its capacity
to covalently interact with a nucleophilic amino acid inside the ATP pocket. In agreement
with this, DHM25 analogs with an inactivated warhead (DHM25-H2) or a reduced
electrophilic C4, which were generated by replacing the strong EWG NO , indicate that the
2
carbon at position 4 undergoes a nucleophilic attack, probably by K2187 in the mTOR
catalytic pocket. Also, washout experiments show that whereas transient incubation with the
reversible mTOR inhibitor AZD8055 allows for the rapid recovery of mTOR activity, similar
treatment with DHM25 does not. This latter experiment highlights one of the advantages
expected in vivo with mTOR covalent inhibitors, namely, prolonged action even when the
drug has been systemically eliminated. Indeed, the overall pharmacological effect in vivo will
be largely dependent on the de novo protein synthesis rate (the half-life of mTOR is
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approximately 18 hours ). Of note, PI3K lipid kinases and PI3KK members such as mTOR
46
share motifs in their catalytic domain, and K2187 in mTOR is a highly conserved motif
among all PI3K members (Fig. S6B), which suggested that DHM25 might also be an inhibitor
of PI3Ks. Nonetheless, our study ruled out this possibility. Also, although DHM25 and
DHM9 differ only by one bromine, they inhibit a completely different set of kinases (Fig.
3A), illustrating that although the covalent bond in the ATP pocket of mTOR is instrumental
in the ability of DHM25 to inhibit mTOR, the electrophilic warheads of DHM25, DHM9, and
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S14161 must be properly oriented for a prolonged time within the targeted protein to
efficiently undergo the appropriate nucleophilic Michael addition. Our hypothesis is that by
interacting with mTOR P-loop residues, DHM25 has better positioning and stronger stability
than DHM9 inside the ATP pocket, favoring its nucleophilic attack by K2187.
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It is noteworthy that some covalent drugs have been evaluated in clinical trials and been
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approved by the US FDA.
Although the risks of covalent inhibitors are known
(
compounds with reactive functionality and potentially increased off-target interactions), the
sustained duration of inhibition of covalent inhibitors yields a number of potential advantages,
including improved inhibitory activity, as competition with endogenous substrates is reduced.
Another advantage is the dissociation of pharmacokinetics from pharmacodynamics (PD), as
PD relies on protein resynthesis. This means that covalent drugs can be clinically useful even
if their half-lives are short, as only a short exposure would be required for the drug to reach its
molecular target and inhibit it for a prolonged period of time.
There is no approved targeted therapy for TNBC, and TNBC patients are treated with
traditional chemotherapy alone. In the first 3 years after diagnosis, TNBC patients account for
a disproportionate number of early deaths not only due to its aggressive nature but also
49
because of the lack of targeted therapeutic treatment options. Therefore, there is a crucial
need for targeted therapy for this pathology. We recently demonstrated that after cleavage by
metalloprotease, soluble CD95L promotes metastatic dissemination of TNBC cells by
50
activating an atypical PI3K/mTOR signaling pathway. The current study demonstrates that
DHM25 is not only able to kill TNBC cells in vitro and in vivo, but it can also block cell
migration of TNBC cells exposed to soluble CD95L at non-cytotoxic (low) concentrations.
These data clearly suggest that DHM25, alone or in combination with classical chemotherapy,
may be an attractive therapeutic option not only as a primary therapy for TNBC patients but
also to reduce/prevent the risk of metastatic occurrence in TNBC patients with high
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concentrations of serum CD95L.
Among 52 ongoing clinical trials exploring the therapeutic effect of mTOR inhibitors on
breast cancer patients, only four are being conducted in metastatic TNBCs using derivatives
of the mTORC1 inhibitor rapamycin (e.g., temsirolimus or everolimus) or an
imidazoquinoline derivative called BEZ235 (ClinicalTrials.gov). Nonetheless, preliminary
results from these studies confirmed those obtained with temsirolimus and everolimus in
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individuals with metastatic breast cancer.
Indeed, rapalogs are primarily cytostatic, not
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cytotoxic, and clinical efficacy largely reflects disease stabilization rather than regression.
One reason for the compromised clinical benefit of rapalogs is that by only targeting
mTORC1, they release the negative feedback circuits of this molecular complex, driving
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mitogenic signaling by activating the upstream PI3K pathway. Indeed, mTORC1-driven
activation of p70S6K induces insulin receptor substrate degradation, which in turn reduces
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PI3K activation. Also, p70S6K phosphorylates Rictor to inhibit mTORC2 activity
and
thereby reduces mTORC2-mediated Akt activation. Consequently, everolimus-mediated
1
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mTORC1 inhibition leads to Akt activation in tumor biopsies of colon cancers. Overall, this
evidence suggests that competitive inhibitors of mTOR that target both mTORC1 and
mTORC2 may represent attractive opportunities to treat PI3K-addicted malignant tumors
such as TNBC.
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EXPERIMENTAL SECTION
Chemistry
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General information. All reagents and solvents were used as purchased from commercial
suppliers or were purified/dried according to Armarego W. L. F. and Chai C. L. L.
th
1
13
(
Purification of Laboratory Chemicals, 6 edition, Elsevier). H and C-NMR spectra were
recorded on a Bruker Avance 300 or 500 instrument using TMS and CDCl respectively as
3
internal standard. Chemical shifts (δ values) are given in parts per million (ppm), coupling
constants (J values) are given in Hertz (Hz), and multiplicity of signals is reported as usual.
HRMS analyzes were obtained using a Waters Q-TOF 2 or a Bruker MicrO-TOF Q II
instrument for ESI. Optical rotations were recorded on a Perkin Elmer Model 341 polarimeter.
The purity of final tested compounds was typically determined to be ⩾95% by HPLC,
conducted on a Shimadzu Prominence system using a Grace Prevail C18 column and diode
array detector. Chiral HPLC was performed using a Daicel Chiralpak IA column. Elementary
analysis of the injected DHM25 was performed on a Thermo-fisher ICAP 6500 Duo
instrument. Melting points were obtained on a hot bench. VCD spectra were measured on a
Bruker Optics Vertex 70 spectrometer and ECD spectra using a Jasco J815 spectrometer. TLC
analyzes were performed using precoated Merck TLC Silica Gel 60 F254 plates. Purifications
by column chromatography on silica gel were performed using Grace Davisil Silica Gel 60
(70-200 mesh and 40-60 mesh) and purifications by preparative thin layer chromatography on
silica gel using Merck Silica Gel 60 PF254. Petroleum ether (PE) used for purifications was the
low boiling point fraction (40-60 °C).
General procedure for the synthesis of chromenes. The mixture of Michael acceptor
derivative (1.0 mmol), salicylaldehyde compound (1.0 mmol, 1.0 eq.) and organocatalyst (0.2
mmol, 0.2 eq.) in 1.5 mL of dry toluene was heated at 80-100 °C for 48-72 h under inert
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atmosphere (conversion followed by TLC). After cooling to room temperature, the mixture
was charged directly on the silica gel column for purification.
Description of the title compounds: 6,8-Dibromo-2-(4-bromophenyl)-3-nitro-2H-chromene (DHM25)
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Mp = 150-152 °C. H NMR (300 MHz, CDCl ) δ 6.67 (s, 1H), 7.24 (d, J = 8.5, 2H), 7.40 (d, J =
3
13
2
.2, 1H), 7.48 (d, J =8.5, 2H), 7.67 (d, J = 2.2, 1H), 7.95 (s, 1H). C NMR (75 MHz, CDCl ) δ 74.1,
3
1
12.6, 115.0, 120.7, 124.3, 127.7, 128.6, 131.7, 132.3, 134.8, 139.3, 142.4, 149.4. HRMS
+
[M+H] calculated for C H NO Br 487.8132, found 487.8133. Anal. calculated for
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C H NO Br : C, 36.77; H, 1.65, N, 2.86. found: C, 36.29, H, 1.56, N, 2.73. (S)-DHM25: [α]
D
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= +154.6 (c = 1, CHCl ); (R)-DHM25: [α] = -157.7 (c = 1, CHCl ).
3
D
3
Cell Lines
All cells were obtained from ATCC (LGC Standards, Molsheim, France). The leukemic T-
cell lines CEM, Jurkat and H9 were cultured in RPMI1640 supplemented with 8% heat-
inactivated FCS and 2 mM L-glutamine in a 5% CO incubator at 37 °C. All breast tumor
2
cells were cultured in DMEM supplemented with 8% heat-inactivated FCS and 2 mM L-
glutamine in a 5% CO incubator at 37 °C.
2
Antibodies, plasmids and other reagents.
LY294002 and Wortmannin were obtained from Calbiochem (Merck Chemicals Ltd.,
Nottingham, UK). AZD8055 was from Selleckchem (Munich, Germany). The chromene and
chromene derivatives are solubilized in DMSO (20 mM). Anti-β-actin was purchased from
S473
Sigma (L’Isle-d’Abeau-Chesnes, France). Anti-Akt, anti-Akt-P , anti-4E-BP1, anti-4E-
Thr37/46
Ser240/244
, anti-S6, anti-S6K-P
BP1-P
, anti-PTEN and anti-mTOR were from Cell Signaling
Technology (Boston, MA, USA).
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Molecular Modeling
mTOR structural model was based on the X-ray experimental structure provided by the
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Protein Data Bank (PDB ID: 4JSN ) and completed for the missing residues (1814-1867 and
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436-2492) by homology modelling tools distributed by Yasara. DHM compounds were
parametrized for the Yamber3 force field following the automated AutoSMILE procedure,
consisting in AM1 quantum calculation and RESP charge fitting protocol. Initial positions of
DHM compounds have been set by analogy to the quercetin placement in the PI3K catalytic
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site (PDB ID: 1E8W ) because of the similarity in the chemical scaffold of ligands and
catalytic site of proteins. Each molecular complex was simulated in a neutralized explicit
water solvent box, under periodic boundary conditions and at a constant temperature of 298
K. Molecular dynamics (MD) trajectories of 62 nanoseconds were collected at 2 picoseconds
intervals for both molecular systems. Production period used for analysis was set after the MD
simulation reach an equilibrated state (stable root mean square deviation). Clustering analysis
upon trajectories were performed using the Jarvis-Patrick method implemented in Gromacs
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tools.
Pull down assay
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CEM cells (10 ) were pre-incubated with indicated concentrations of DHM25, its biotin-
conjugated counterpart or DHM9-biotin for 2h at 37 °C. Then cells were lysed in a
denaturating buffer (50mM Tris pH 7.4, 2 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.5% Na-
deoxycholate monohydrate, 1% Triton X-100, 0.1% SDS) complemented with protease and
phosphatase inhibitors (Sigma) and biotin-conjugated DHM9 or DHM25 was
immunoprecipitated using streptavidin magnetic beads (Ademtech, Pessac, France) for 1 h at
4 °C. After extensive washing, the immune complex was resuspended in a denaturing and
reducing Laemmli Sample Buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-
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mercaptoethanol, 0.01% bromophenol blue), resolved by SDS/PAGE and revealed by
Western blot.
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Circular dichroism
Both enantiomers were obtained by resolution of racemic DHM25 using chiral HPLC
(Chiralpak IA column, MeOH as eluent, 1.0 mL/min: RTDHM25-E1 = 8.13 min, RTDHM25-E2
=
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0.48 min). The absolute configuration of each DHM25 enantiomer was determined by
vibrational and electronic circular dichroism (VCD and ECD) by comparison of experimental
to predicted spectra. VCD spectra were recorded on a Bruker Vertex70 spectrometer (with a
PMA50 module) and theoretical simulated VCD obtained by the SMD/B3PLYP/6-
311+G(d,p)/Lanl2dz method. ECD analyses were performed on a Jasco J815 spectrometer
and theoretical simulated ECD obtained by the SMD/TD-CAMB3LYP/6-
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1++G(d,p)/Lanl2dz method. Thus DHM25-E1 was identified as the (S) enantiomer and
DHM25-E2 as the (R) one.
Reversibility experiments
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CEM cells (2×10 ) were treated in the presence or absence of DHM25 (5 μM) or AZD8055 (5
μM) for 2 h at 37 °C. Next, cells were extensively washed to eliminate the excess of inhibitor
and cells were resuspended in fresh medium. Restoration of mTOR activity was evaluated by
monitoring at different times the phosphorylation level of Akt at Serine 473.
Detergent Lysis experiments and Western Blot Analysis
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Cell lyses and western blot analyses were performed exactly as previously described.
Measure of cell death
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Cell death was assessed using either the viability MTT assay.
Immunoblot analysis.
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Cells were lysed for 30 min at 4 °C in lysis buffer [25 mM HEPES (pH 7.4), 1% v/v Triton X-
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00, 150 mM NaCl, and 2 mM EGTA supplemented with a mix of protease inhibitors
(Sigma)]. Protein concentration was determined by the bicinchoninic acid method (Pierce,
Rockford, IL, USA). Proteins were separated by SDS-PAGE and transferred to a
nitrocellulose membrane. The membrane was blocked for 30 min with TBST (50 mM Tris,
1
60 mM NaCl, 0.05% v/v Tween 20, pH 7.8) containing 5% w/v dried skimmed milk and
then incubated overnight with primary antibody at 4°C in TBSTM. The membrane was
washed (TBST) and secondary HRP-labeled antibody (SouthernBiotech, US) was added for
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5 min. The proteins were visualized with the enhanced chemiluminescence substrate kit
(Pierce).
Kinase profiler screening
To evaluate the kinases targeted by DHM25 and DHM9, Kinase Profiler Service (Millipore)
was used with 243 available kinases. The screening was performed with 1 μM of compound
and 10 μM ATP and kinase substrates according to Millipore's protocol.
Immunofluorescence imaging.
MDA-MB-231 cells were incubated for 3 hours at 37 °C with DHM25-FITC (10 μM) or
FITC alone. Cells were then washed twice in PBS and fixed for 15 min in PBS containing 4%
w/v paraformaldehyde. The aldehyde groups were quenched for 10 min in PBS supplemented
with 5% FCS. mTOR was visualized by using 5 μg/ml of goat anti-mTOR mAb (N19, Santa-
Cruz, CA, USA) incubated for 20 min at 4 °C, washed and incubated for another 20 min at 4
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C with anti-goat IgG (H+L) coupled with Alexa Fluor 594-conjugated antibody (Life
Technologies, CA, USA). Finally, cells were washed twice with PBS, dried, and mounted
with 20 μl of mounting medium (Sigma, MO, USA). Images were acquired using a laser
scanning confocal system (TCS SP8 model mounted on a DMI 6000 CS inverted microscope,
Leica, Heidelberg, Germany). Images were taken using a 40x oil-immersion objective (1.3,
N.A.) and analysed using the ImageJ software (NIH, Bethesda, Maryland, USA).
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In vitro motility assays.
After membrane hydration of Boyden chambers (Millipore, Molsheim, France) containing 8
5
μm pore membranes, 10 cells were added to the top chamber. The bottom chamber was filled
with low serum (1%)-containing medium in the presence or absence of cl-CD95L (100
ng/ml). Breast cancer cells were incubated for 24 h. To quantify invasion, cells were fixed
with methanol and stained with Giemsa. Stained cells were then removed from the top-side of
the membrane using a cotton-tipped swab and five representative pictures for each insert were
taken of the invading cells from the reverse side. For each experiment, invading cells were
lysed and absorbance at 560 nm was measured.
Mouse experiments.
NOD/SCID/γc null mice (NSG) were obtained from Dr C. Rivers (UK). All experiments were
performed in agreement with the French Guidelines for animal handling and approved by
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local ethics committee. Luciferase-expressing MDA-MB-231 cells (5×10 cells in 50 µL
PBS/Matrigel) were transplanted into mammary fat pads of mice (7 weeks old female). Once
tumors were palpable, intraperitoneal injection of DHM25 resuspended in PBS/Tween-
80/DMSO (8/1/1) or control medium was performed repeatedly 5 days a week until day 46.
Bioluminescence analysis was performed using PhotonIMAGER (Biospace Lab) following of
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endotoxin-free luciferin (30 mg/kg). Tumor volume was calculated using the formula V =
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SUPPORTING INFORMATION AVAILABLE: Full experimental details are given in the
supporting information document. This material is available free of charge via the Internet at
http://pubs.acs.org.
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*
CORRESPONDING AUTHOR INFORMATION: Dr. Patrick Legembre, Phone: (+33)-2-
2
323-7241, Fax: (+33)-2-9925-3164, E-mail: patrick.legembre@inserm.fr. Prof. Pierre van de
Weghe, Phone: (+33)-2-2323-3803, Fax: (+33)-2-2323-4425, E-mail: pierre.van-de-
weghe@univ-rennes1.fr.
8
AUTHOR CONTRIBUTIONS:Authors share corresponding authorship.
ACKNOWLEDGEMENTS:
This work was supported by grants from Ligue Contre le Cancer (Comités d’Ille-et-Vilaine,
du Morbihan, des Côtes d'Armor, du Finistère et du Maine et Loire), INCa PLBIO, Ouest
Valorisation and Fondation ARC. A.F. is supported by INCa. The authors also thank members
of the TrGET and CRCM animal facilities for their technical assistance. TrGET is supported
by Institut Paoli-Calmettes (IPC) and Canceropôle PACA. The authors thank the PRISM
platform (Université Rennes-1) for NMR experiments.
ABBREVIATIONS USED:
mTOR: mammalian target of rapamycin; mTORC1 and C2: mammalian target of rapamycin
complex 1 and 2; PI3K: Phosphatidylinositol-4,5-bisphosphate 3-kinase; PI3KK:
phosphatidylinositol 3-OH-kinase-related kinase; TNBC: triple-negative breast cancers.
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