Enhancing Antiproliferative Activity and Selectivity of a FLT-3 Inhibitor by Proteolysis Targeting Chimera Conversion

Article abstract of DOI:10.1021/jacs.8b10320

The receptor tyrosine kinase FLT-3 is frequently mutated in acute myeloid leukemia; however, current small molecule inhibitors suffer from limited efficacy in the clinic. Conversion of a FLT-3 inhibitor (quizartinib) into a proteolysis targeting chimera (

Full text of DOI:10.1021/jacs.8b10320

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Communication
Enhancing Anti-Proliferative Activity and Selectivity
of a FLT-3 Inhibitor by PROTAC Conversion
George M Burslem, Jayoung Song, Xin Chen, John Hines, and Craig M Crews
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10320 • Publication Date (Web): 14 Nov 2018
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Enhancing Anti-Proliferative Activity and Selectivity of a FLT-3
Inhibitor by PROTAC Conversion
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†
†
‡
†
+†
George M. Burslem, Jayoung Song, Xin Chen, John Hines, and Craig M. Crews *
†Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA,
‡Arvinas Inc., New Haven, CT, USA
+Departments of Chemistry and Pharmacology, Yale University, New Haven, CT, USA
E-mail: Craig.Crews@Yale.edu
Supporting Information Placeholder
mutation and a thus a therapeutic target for AML treatment.8
ABSTRACT: The receptor tyrosine kinase FLT-3 is frequently
mutated in acute myeloid leukemia, however current small
molecule inhibitors suffer from limited efficacy in the clinic.
Conversion of a FLT-3 inhibitor (quizartinib) into a proteolysis
targeting chimera (PROTAC) results in a compound which
induces degradation of FLT-3 ITD mutantprotein degradation at
low nanomolar concentrations. Furthermore, the PROTAC is
capable of inhibiting cell growth more potently than the warhead
alone whilest inhibiting fewer off-target kinases. This enhanced
anti-proliferative activity occurs, despite a slight reduction in the
PROTAC’s kinase inhibitory activity, via an increased level of
apoptosis induction, suggesting non-kinase roles for the FLT-3
ITD protein. Additionally, the PROTAC is capable of inducing
FLT-3 ITD degradation in vivo. These results suggest that
degradation of FLT-3 ITD may provide a useful method for
therapeutic intervention..
Several FLT-3 kinase inhibitors have been assessed in clinical
9
trials but demonstrated only limited benefit. It appears that
maximum, sustained inhibition of FLT3 ITD signaling is essential
9-10
to achieve an effective clinical response,
and achieving such a
complete inhibition can be challenging with small molecule
kinase inhibitors: it often necessitates the use of high doses,
potentially resulting in undesirable off-target effects. In our eyes,
this makes FLT-3 ITD an ideal target for a targeted protein
degradation approach since degradation of the FLT-3 ITD protein
will necessarily result in sustained, maximum inhibition.
Additionally, previous studies have shown that siRNA induced
knockdown of FLT-3 ITD in AML cells sensitized them to
1
1
apoptosis and that FLT-3 ITD can act as a scaffold for Pim
1
2
kinases resulting in FLT-3 ITD inhibitor resistance.
Targeted protein degradation is emerging as a powerful new
1
tool to combat disease. At the forefront of targeted protein
degradation technologies are Proteolysis Targeting Chimeras
2
(PROTACs). This class of heterobifunctional small molecules,
developed in our lab, function via recruitment of the cellular
protein degradation machinery to a target of interest. More
specifically, recruitment of an E3 ligase complex by a PROTAC
to a disease-causing protein results in the ubiquitination and
2a
subsequent proteasomal degradation of that protein target . In
addition to being irreversible, this also results in eradication of all
functional activities of the protein rather than the inhibition of a
single one. This approach has been used to degrade multiple
protein targets using those E3 ligases proven amenable for use in
3
this technology. Crucial to the development of a successful
PROTAC is the careful selection of the protein target. Receptor
Tyrosine Kinases (RTKs) represent ideal targets for the PROTAC
technology as they possess both well-documented scaffolding
4
roles as well as their cognate kinase activities.
An important example of a disease relevant RTK is the FMS-
Like Tyrosine kinase 3 (FLT-3), which is expressed in the
majority of Acute Myeloid Leukemia (AML) cases (41/44
5
6
tested). AML is the most common form of acute leukemia, and
0-30% of newly diagnosed AML patients feature FLT-3
2
7
mutations that are associated with poor prognoses. The most
common mutation is an internal tandem duplication (ITD) in the
juxta-membrane domain, which has been validated as the driving
Figure 1. PROTAC-induced degradation of FLT-3 ITD. A –
Structures of compounds used in this study. B – Western blot
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TM
showing the FLT-3 protein levels in MV4-11 cells treated with
the indicated concentration of each compound.
inhibition assay for FLT-3 wt. C – KinomeScan
data for
TM
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PROTAC at 1 µM. D – KinomeScan data for quizartinib at 1
µM.
Having recently demonstrated that RTKs are amenable to
4a
targeted protein degradation via PROTACs, we sought to extend
Evaluating our compounds in cellular proliferation assays
revealed the distinct advantage of degradation over inhibition with
the PROTAC not only performing significantly better than the
control compound but also better than the clinical candidate
quizartinib in both MV4-11 cells (Figure 3A) and MOLM-14 cells
(See S2). Indeed, the FLT-3 PROTAC is >3.5 fold more potent
than quizartinib, with a sub-nanomolar IC50 (0.6 ± 0.08 nM).
Comparison of the control (IC50 = 2.27 ± 0.2 nM) to quizartinib
(IC50 = 1.87 ± 0.1 nM) in this proliferation assay shows a slight
reduction in potency for the former, which is expected from the
reduced inhibitory activity in the in vitro assay following
PROTAC conversion. This indicates that degradation of FLT-3 is
crucial for the enhanced anti-proliferative effect. This is in
contrast to the previously reported FLT-3 PROTACs which,
although also derived from quizartinib, failed to give an
advantage over the warhead alone; perhaps this may be due to
decreased cell permeability or their less potent induction of
this approach to target FLT-3 ITD. Employing the clinical
1
3
candidate quizartinib (AC220 ) as a recruiting element and
replacing the solubilizing morpholine group with our VHL
1
4
ligand via an optimized linker, we were able to develop a FLT-3
PROTAC (Figure 1A) that potently induces FLT-3 ITD protein
degradation in MV4-11 cells (Figure 1B and Figure S2) and
MOLM-14 cells (Figure S1) at low nanomolar concentrations.
Inversion of the stereocenters in the hydroxyproline VHL binding
element results in an diastereomer of equal FLT-3 binding affinity
and physicochemical properties but unable to induce degradation,
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c, 3a, 4a,
thus providing an excellent control molecule (Figure 1A).
1
4a
Indeed, the binding of the FLT-3 control appears to stabilize
the FLT-3 protein as has been previously reported for other FLT-3
3h, 15
ligands.
This mechanism of resistance to FLT-3 ITD
inhibitors may be the reason for the limited benefit in clinical
trials, further emphasizing the benefits of a FLT-3 PROTAC.
3
h
degradation. In some cases, degradation has proven a viable
approach to combat resistance mechanisms against inhibitors
so we tested the FLT-3 PROTAC against cell lines expressing
two of the most common FLT-3 kinase domain mutations, as well
as the ITD, which impart resistance to quizartinib, D835Y and
With this optimized compound in hand, we sought to
characterize the consequence of PROTAC conversion on the
activity and selectivity of our compound as a kinase inhibitor,
compared to the parent compound, quizartinib. We performed
kinase activity assays to confirm that the PROTAC can indeed
still inhibit both FLT-3 ITD as well as wild type FLT-3. The
PROTAC has slightly reduced ability to inhibit both FLT-3 ITD
3
e, 3f,
1
6
13a, 17
F691L.
In both mutants the PROTAC displayed a similar
response to quizartinib but all compounds showed a similar right-
shift in the dose response curve relative to those for FLT-3 ITD
WT, attributable to reduced binding affinity of the mutants for the
recruiting element (Figure S2S4). Given the potent activity of
these compounds in cell lines bearing a FLT-3 ITD mutation, we
also assessed the anti-proliferative activity against a WT FLT-3
AML cell line (OCI-AML-3) and a CML cell line (K562) to
ensure against general toxicity (Figure S3S5). Quizartinib has
(43 ± 3.2 nM) (Figure 2A) and FLT-3 WT (36 ± 3 nM) (Figure
2B) compared to quizartinib (10 ± 0.3 nM and 7.4 ± 0.3 nM
respectively) but retains low nanomolar activity in both cases.
Quizartinib is a moderately selective kinase inhibitor with a
limited number of targets at 1 µM as shown in Figure 2D.
Profiling our PROTAC in a similar fashion, significantly fewer
hits were observed. Conversion of quizartinib into a PROTAC
retained FLT-3 binding but significantly reduced binding affinity
for the other members of the kinase super-family that had
beenwere inhibited (Figure 2C). Furthermore, we investigated the
effect of the PROTAC on the degradation of other kinases to
which it binds and found no reduction in protein levels (Figure
S3). This is consistent with previous observations, from our lab
and others, that replacement of supposed solubilizing groups
during PROTAC conversion can have profound effects on
moderate antiproliferative activity in OCI-AML-3 cells (IC50
73 ± 74 nM) but in this case the PROTAC is comparatively less
–
7
potent (IC50 >2800 nM). This may be due to more restricted
kinase engagement/enhanced selectivity of the PROTAC
compared to quizartinib (Figure 2C/D); however, the results
suggest that only in the case of FLT-3 ITD driven cells is
degradation better than inhibition. The PROTAC showed no anti-
proliferative activity up to 5 µM in K562 cells revealing a >8000-
fold selectivity window against non-AML cells.
3c, 3h
compound promiscuity.
Figure 2. In Vitro Profiling of the FLT-3 PROTAC A – In vitro
kinase inhibition assay for FLT-3 ITD. B – In vitro kinase
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Figure 4. Apoptosis Induction in MV4-11 Cells. Caspase 3/7
activation in MV4-11 cells treated with PROTAC, control or
quizartinib measured by CaspaseGlo assay
Finally, we sought to explore the in vivo activity of our
compounds. Initial assessment of the pharmacokinetic properties
of the FLT-3 PROTAC revealed acceptable pharmacological
properties: a single 10 mg/kg IP injection followed by plasma
level quantitation over the following 24 hours revealed a
maximum concentration of just over 1 µM at 2 hours post
injection and a half-life of just over 2.3 hours (n=3) (Figure 5A).
Importantly, the plasma concentration remains above 5 nM, a
concentration at which significant degradation is observed in
cellulo, for approximately 22 hours post-injection. Based on these
favorable pharmacokinetic properties, the PROTAC and the
control compound were progressed into
a
small-scale
pharmacodynamics study using a previously reported model
system in which 9 athymic mice were injected subcutaneously
1
8
with MV4-11 cells and tumors allowed to develop. Once the
3
tumors had reached approximately 200 mm , the mice were
Figure 3. Cellular Evaluation of FLT-3 PROTACs. A – Cell
Proliferation assay in MV4-11 cells. B – Analysis of downstream
signaling by immunoblotting.
randomly assigned into three treatment groups; vehicle, PROTAC
or control. The mice were treated once every 24 hours for 3 days
with 30 mg/kg compound (to ensure sustained plasma levels >5
nM over 24 hours) or vehicle equivalent before euthanasia 5 hours
following the final dose. No adverse effects were observed during
this period. Tumor samples were collected and probed for FLT-3
levels as well as phospho-STAT-5 (Figure 5B). Under this
treatment regime, both PROTAC and diastereomeric control are
capable of inhibiting downstream signaling of FLT-3 ITD but, as
expected, only the PROTAC is capable of inducing the
degradation of FLT-3. Quantitation by densitometry and
Given the enhanced anti-proliferative activity associated with
FLT-3 ITD degradation, we sought to assess the effect of our
PROTAC on signaling downstream of the kinase. MV4-11 cells
were treated with either quizartinib or FLT-3 PROTAC in serum
free media for 24 hours before lysis and immunoblotting for
phosphorylation events downstream of FLT-3 (Figure 3B).
Curiously, the PROTAC seems to be slightly less able to inhibit
phosphorylation of STAT-5, MEK and ERK than quizartinib in
MV4-11 cells despite its enhanced anti-proliferative effects
averaging of western blots reveals
a significant decrease
(approximately 60%) in total FLT-3 protein (normalized to
(Figure 3A).
tubulin as a loading control) in tumors from mice treated with
PROTAC but no significant change in control treated animals
Seeking to further understand what may be providing the
apparent disconnect between signaling and proliferation, the
levels of apoptosis in MV4-11 cells treated with PROTAC,
control and quizartinib were assessed by CaspaseGlo assay. At
concentrations as low as 0.1 nM, a significant level of apoptosis
induction (i.e. caspase activation) can be observed with PROTAC
but not with quizartinib or the inactive control compound (Figure
4). Indeed, at all doses of PROTAC below 10 nM, caspase
activation was significantly higher better than that following
inhibition with either quizartinib or the inactive control. The
greater potency of the PROTAC to cause caspase 3 activation
(Figure 5C).
(cleavage) was also confirmed by western blot (Figure S4). This
suggests that full FLT-3 ITD-driven AML cell proliferation may
depend on the presence of the protein itself and not just the
increased activity of the kinase domain. This could partially
explain the limited response of traditional inhibitors in the clinic.
Further studies are required to elucidate the FLT-3 ITD kinase-
independent functions and the corresponding pathway(s) through
which degradation of FLT-3 ITD induces apoptosis.
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Supporting Information
Supplemental Figures 1-4, Scheme S1, experimental procedures,
and details on the synthesis of compounds discussed above are
included
in
the
supporting
information.
(PDF)
The Supporting Information is available free of charge on the
ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
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*
Craig.Crews@Yale.edu
Notes
C.M.C. is founder of, consultant to and shareholder in Arvinas
Inc., which partially supports research in his lab. X.C. is an
employee of Arvinas Inc.
ACKNOWLEDGMENT
G.M.B is a Fellow of The Leukemia & Lymphoma Society. J.S.
acknowledges the Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education (NRF-2016R1A6A3A03012989). J.H.
gratefully acknowledges the US National Cancer Institute for their
support (R50CA211252). C.M.C. gratefully acknowledges the US
National Institutes of Health for their support (R35CA197589).
We thank members of the Crews Laboratory for useful
discussions. The MOLM-14 cell line was a gift from David
Scadden, Harvard University. The MOLM-14 mutant FLT-3 ITD
cell lines were a gift from Neil Shah, University of California, San
Francisco.
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more potent inhibitor of FLT-3 ITD driven proliferation than
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itself, which appears to accelerate the induction of apoptosis. This
study demonstrates that conversion of a kinase inhibitor into a
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also a compound with enhanced selectivity, further revealing the
power of this approach. Conceptually, this demonstrates a shift
from occupancy- driven pharmacology to event- driven
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the elucidation of the mechanism of apoptosis induction and
assessment of in vivo efficacy compared to quizartinib.
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H
H
N
N
O
N
O
O
N
N
N
S
O
Quizartinib
PROTAC
Conversion
H
H
N
N
OH
O
N
O
O
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N
N
N
O
O
N
S
O
H
O
O
HN
More Selective
Enhanced Antiproliferative Activity
Enhanced Apoptosis Induction
S
N
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