A cascade screening approach for the identification of Bcr-Abl myristate pocket binders active against wild type and T315I mutant

Article abstract of DOI:10.1016/j.bmcl.2016.06.051

The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.

Full text of DOI:10.1016/j.bmcl.2016.06.051

Accepted Manuscript
A Cascade Screening Approach for the Identification of Bcr-Abl Myristate
Pocket Binders active against Wild Type and T315I Mutant
Marco Radi, Ralf Schneider, Anna Lucia Fallacara, Lorenzo Botta, Emmanuele
Crespan, Cristina Tintori, Giovanni Maga, Miroslava Kissova, Alessia Calgani,
André Richters, Franesca Musumeci, Daniel Rauh, Silvia Schenone
PII:
S0960-894X(16)30662-X
http://dx.doi.org/10.1016/j.bmcl.2016.06.051
BMCL 24004
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 April 2016
16 June 2016
18 June 2016
Please cite this article as: Radi, M., Schneider, R., Fallacara, A.L., Botta, L., Crespan, E., Tintori, C., Maga, G.,
Kissova, M., Calgani, A., Richters, A., Musumeci, F., Rauh, D., Schenone, S., A Cascade Screening Approach for
the Identification of Bcr-Abl Myristate Pocket Binders active against Wild Type and T315I Mutant, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.051
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Bioorganic & Medicinal Chemistry Letters
A Cascade Screening Approach for the Identification of Bcr-Abl Myristate
Pocket Binders active against Wild Type and T315I Mutant
Marco Radi,a,b* Ralf Schneider,c Anna Lucia Fallacara,a, Lorenzo Botta,d Emmanuele Crespan,e Cristina
Tintori,a Giovanni Maga,e Miroslava Kissova,e Alessia Calgani,f André Richters,c Franesca Musumeci,g
Daniel Rauhc and Silvia Schenoneg*
a
degli Studi di Siena, Via A. De Gasperi 2, I-53100 Siena, Italy
b P4T Group, Dipartimento di Farmacia, Università degli Studi di Parma, Viale delle Scienze, 27/A, 43124 Parma, Italy.
c Department of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany
d
f P y y f N “ d II” V . Montesano 49, 80131 Naples, Italy
e Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
f Dipartim d d d d ila, Via Vetoio, 67100 Coppito, L I y
g
d d d d , Viale Benedetto XV, 3, 16132 Genova, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently
represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first
and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition
of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates
have been identified in the last few years. Among these, myristate pocket (MP) binders
discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be
active against the T315I mutant only in combination with first and second generation ATP-
competitive inhibitors. Here we used a cascade screening appraoch based on sequential
fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-
enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has
been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing
Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.
Available online
Keywords:
Bcr-Abl
T315I
Allosteric
Myristate
Kinase
Leukemia
Resistance
2009 Elsevier Ltd. All rights reserved.
The discovery of proteins directly involved in the
pathogenesis of many tumor types has open the way to the
development of selective drugs for specific cancers.1 One of the
first examples of this successful approach is represented by the
use of tyrosine kinase inhibitors (TKIs) for the treatment of
chronic myeloid leukemia (CML), with imatinib mesylate (IM,
Gleevec™) being the first Bcr-Abl targeting drug approved by
the FDA in 2001.2 Despite the high rate of complete cytogenic
response (CCyR) from patients treated each year with IM in
frontline therapy, around 4-13% of these patients can develop
resistance or tolerance to this drug, leading to therapeutic failure.
The most common mechanism of IM resistance is represented by
point mutation in the kinase domain of Bcr-Abl and, in such
cases, second generation TKIs (Dasatinib, Nilotinib, Bosutinib)
can be used in second line therapy.3
15-20% of clinically observed mutations and is frequently
selected on sequential inhibitor therapy. Unfortunately, only one
drug (Ponatinib, Iclusig™) has recently achieved accelerated
approval for the treatment of this resistant CML form, but severe
side effects have limited its clinical application.4 As
a
consequence, the identification of novel Bcr-Abl inhibitors active
on the T315I mutant is one of the top priorities in CML research
and only one additional promising candidate (Axitinib) has been
recently found by Pfizer in a drug-repurposing study (Figure 1).21
Despite the fact that all the Bcr-Abl inhibitors approved so far
for CML treatment target the conserved ATP binding pocket and
share an ATP-competitive mode of action and considering the
poor results obtained in the inhibition of the Bcr-Abl T315I
mutant with these inhibitors, many efforts are currently devoted
to the development of allosteric inhibitors of Bcr-Abl and,
especially, of its T315I mutant.5 Targeting sites outside the ATP-
binding cleft could in fact present several advantages over the
However, the major clinical challenge is currently represented
by the cross-resistant Bcr-Abl T315I mutant which accounts for
———
*
Corresponding authors: Silvia Schenone, (Phone: +39-010-3538362; E-mail: schensil@unige.it); Marco Radi (Phone: +39-0521-906080; E-
mail: marco.radi@unipr.it)

common ATP-competitive strategies: i) minimize the side-effects
caused by the limited selectivity of competitive inhibitors; ii)
overcome the drug-resistance induced by ATP-competitive
inhibitors; iii) lower doses of the allosteric drugs could be used
since these compounds do not have to compete with the high
intracellular concentration of ATP; iv) allow to finely modulate
the kinase activity without affecting the basal activity in order to
avoid some toxic effects which can occur due to the complete
abrogation of the enzyme functions. A few different classes of
allosteric inhibitors active on the T315I mutant have been
discovered in the last few years (Figure 1).5,20,21
site (both in Abl WT and T315I mutant) with functional
consequences on the catalytic machinery.9,10 All these data
confirmed the existence of a cross-talk between the MP and
ATP-binding site but were unable to explain why myristate-
binders GNF-2/5 alone were able to inhibit only wild type Bcr-
Abl and not the T315I mutant.
As a continuation of our efforts in the discovery of novel
compounds active against the T315I mutant,11-14 we became
interested in the identification of compounds that could bind the
allosteric site of Abl: such compounds could mimic the
synergistic effect of GNF-derivatives and ATP-competitive
inhibitors but could also switch from the catalytic pocket to the
MP depending on the nature of the gatekeeper residue (T315 or
I315). To identify compounds with the above behavior, we
planned to submit our internal collection of ATP mimetics11,15-16
to the following cascade screening approach: 1) high-throughput
fluorescence polarization (FP) screening on Abl WT (kinase
domain and full-length); 2) docking and molecular dynamics
(MD) studies on the MP pocket; 3) kinetic-enzymatic studies on
Abl WT and T315I; 4) assays on WT and T315I expressing cells.
Around 200 compounds were initially analyzed for their ability
to displace a GNF-2 based tracer (cmpd 1, Figure 2) from the MP
using GNF-2 as positive control and C4 N-methyl GNF-2 as
negative control17 via FP assay (experimental details are reported
in the Supporting Information). To eliminate any possible inter-
domain interaction effect on the displacement of the tracer 1, FP
screening (at 10 μM of tracer and test cmpds) was conducted on
both the kinase domain and full-length Abl WT. Only
compounds able to displace the tracer (>30% displacement at 2h)
from both kinase domain and full-length Abl were considered as
positive hits and submitted to further investigation (cmpds 2-6,
Figure 2). However, Lebakken and colleagues observed that a
tracer binding to the ATP site of Abl could be displaced by GNF-
2 as a consequence of the cross-talk between the MP and the
catalytic site.18 It is therefore reasonable to assume that a similar
behavior could be observed in the displacement of the tracer 1 by
ATP-competitive inhibitors, leading to the identification of false
positive hits.
N
NH
N
N
N
NH
N
S
O
N
H
O
Axitinib
Ponatinib
F3C
N
N
ATP-COMPETITIVE INHIBITORS
N
MeO
OMe
OMe
N
S
N
O
O
NH
MeO
NH
O
F
HN
CO2H
O
HN
ON012380
DCC-2036
N
O
F3CO
F3CO
NH
N
NH
N
O
N
O
OH
N
N
NH2
H
GNF-2
GNF-5
ALLOSTERIC INHIBITORS
Figure 1. Representative ATP-competitive and allosteric Bcr-Abl T315I
inhibitors
F3CO
NH
N
O
O
Deciphera Pharmaceuticals has developed T315I inhibitors
(DCC-2036 and DCC-2157) that bind to structural pockets used
by Abl to switch between the inactive and active conformations
(switch-pocket inhibitors).6 Onconova Therapeutics has
developed a substrate competitive inhibitor (ON012380) active
against 100% of IM-resistant forms of Bcr-Abl, including the
T315I mutant.7 The family of myristate pocket binders,
discovered by Novartis,8 holds a promising future. Indeed, a
major issue of ATP-competitive inhibitors is represented by the
easiness of mutation development due to the evolutionary
pressure, that is less severe in the myristate-binding pocket (MP).
For this reason MP is an ideal target for the development of
allosteric drugs. The lead myristate-binder GNF-2 and its
optimized derivative GNF-5 proved to be active against the
T315I Bcr-Abl mutant only in combination with first and second
generation ATP-competitive inhibitors (Imatinib, Dasatinib,
Nilotinib), giving complete remission in a T315I Bcr-Abl mutant
murine bone-marrow transplantation model.9 Solution NMR, X-
ray crystallography, mutagenesis and hydrogen exchange mass
spectrometry experiments confirmed that GNF-2/5 binds to the
MP of Abl and showed how this binding led to changes in the
structural dynamics of both the MP and the distant ATP-binding
O
O
OH
O
N
N
H
N
3
H
1
O
OH
F
NH
NH
F
N
N
N
N
N
N
N
N
3
2
Cl
Cl
NH
N
NH
Cl
Cl
N
N
N
N
N
N
N
N
N
N
N
S
N
5
6
4
Cl
Figure 2. GNF-2 based fluorescent probe (1) and compounds selected from
FP assay (2-6).

Figure 3. A) From left to right: superimposition of GNF-2 (mangenta) binding mode (3K5V crystal structure) and 5 (blue) in the MP of Abl wild type (the same
binding mode was found in the MP of T315I) from the last frame of MD simulation; variation of the reaction velocity of Abl WT and Abl T315I as function of
ATP concentration at different fixed concentrations of compound 5. Error bars represent ± S.D. B) From left to right: superimposition of GNF-2 (magenta)
binding mode (3K5V crystal structure) and 6 (orange) in the MP of Abl wild type (the same binding mode as in the MP of T315I) from the last frame of MD
simulation; variation of the reaction velocity of Abl wt and Abl T315I as function of ATP concentration at different fixed concentrations of compound 6. Values
are the means of three independent experiments.
For this reason, additional in silico and in vitro experiments
were conducted to validate the allosteric mechanism of action of
the selected hits. The interaction between compounds 2-6 and
Abl (WT and T315I mutant) was initially analyzed in silico:
molecular docking, molecular dynamics (MD) simulations
together with MM-GBSA analysis were applied according to the
protocol previously described by us.19 Docking results showed
that only compounds 5 and 6 were able to fill in the MPt (Figure
3A and 3B) with a binding mode comparable to the one of GNF-
2 (PDB code: 3K5V).9 Indeed, the styryl group of 5 and 6
perfectly superimposed the p-trifluoromethoxyl group of GNF-2
oriented toward the bottom of the pocket (Figure 3).
maintained a high ΔG also in the MP of the T315I mutant (-35
and -39 kcal/mol respectively). These results led to speculate an
allosteric mechanism of action not only against the wild type but
also against the T315I mutant. The binding free energy calculated
was decomposed into the contribution of each residue (Table S1).
Both 5 and 6 were able to establish contacts with Tyr454 and
Cys483 identified as hot spots in our previous analysis. Weak
contacts (lower than 1 kcal/mol) were detected with Arg351.
Furthermore, analysis of the binding free energy of interaction
between SH2 and kinase domains showed a considerable ability
of 6 to stabilize the compact conformation when it is bound to the
T315I MP rather than to the WT MP (-59 and -50 kcal/mol
respectively). By comparison, 5 was characterized by a weaker
stabilization of the SH2/kinase domain interaction both on WT
and T315I mutant, showing lower ΔG values calculated at the
interaction interface (-42 kcal/mol and -50 kcal/mol
respectively). From our analysis what emerged was an interesting
Furthermore, hydrophobic interactions were found with
residues Leu395, Val357, Val358, Ala356, Leu359, Leu360,
Ala452 and Val487 while a hydrogen bond occurred between the
amino group of 5 and the backbone of Glu481. Compounds 5
and 6 were then submitted to a more detailed MD and MM-
GBSA analysis (a detailed description of the protocol has been
provided in the Supporting Info file) to investigate their effect on
the stabilization of the SH2-SH3/kinase domain interaction,
which is responsible for the allosteric inhibition of Bcr-Abl (both
WT and T315I).
and high capability of
6 to stabilize the autoinhibited
conformation by allowing the formation of strong interactions at
SH2/kinase interface. Hot spots residues were identified in
Arg189, Tyr158, Tyr361, Glu526 and Asp523, which were the
same amino acids detected in the interaction of GNF2 with the
MP. Features like the π-π interactions involving Tyr158 and
Tyr361 were detected with a high energy associated to these two
residues. Noteworthy, hydrogen bonds between Arg189 and
Asp523 and Glu526 were more stable in the case of 6 bound to
MP of T315I rather than 5 (Tables S3 and S4). All these results
suggested an allosteric mechanism of action for both 5 and 6
against Abl wild type and T315I mutant with a higher potency of
6 against both forms of the enzyme.
MD simulations of 30 ns were performed and RMSD (Root
Main Square Deviation) for α carbons was calculated in order to
evaluate the stability of the system (Figure S2). The production
stages of all simulations were used for extracting 150 frames
from the dynamics. RMSFs (Root Main Square Fluctuations) for
all residues were also calculated (Figure S3) for finding high
fluctuation areas during the simulations. Surprisingly, compound
6 showed a stable RMSFs profile in particular considering the
T315I mutant. Lower fluctuations were found for 6, in
comparison to 5, which seemed to be less able to stabilize the
assembled conformation. High values of binding free energy for
both ligands in the MP of Abl WT were found (-35 kcal/mol, -40
kcal/mol for 5 and 6 respectively). Unlike GNF-2,19 5 and 6
In order to confirm our results, enzymatic studies were
conducted to evaluate the mechanisms of action of compounds 5
and 6. In vitro kinase inhibition assays were performed using
recombinant enzymes, obtaining IC50 values of 50 μM and 20 μM
against T315I by 5 and 6 respectively. We also analyzed the
reaction velocity as a function of the ATP concentration, in the

presence of different fixed amounts of inhibitors 5 and 6 (Figure
3). Kinetic parameters for 5 and 6 are summarized in Table 1: as
it can be observed, both compounds decreased Vmaxapp either
towards Abl WT or T315I mutant but did not affect the apparent
mechanism of action of both compounds not only against the WT
Abl enzyme but, most importantly, against the T315I mutant. The
pyrazolo[3,4-d]pyrimidine 6, which showed the higher potency,
represents a promising candidate able to allosterically inhibit the
Abl T315I mutant with no need of combination with an ATP-
competitive inhibitors (differently from GNF-2/5). Our work
open the way to the discovery of a new class of MP binders
which could be further optimized to provide new drug candidates
to fight the T315I mutant form of drug-resistant leukemia.
Table 1. Kinetic parameters of 5 and 6 inhibition toward Abl WT and T315I
(mean ± S.D.)
Wild Type (WT)
5 [M]
0
25
50
100
48.18
±5.91
47.52
±5.73
53.81±
4.47
54.69
±5.87
Kmapp [μM]
Vmaxapp
17.70
±0.94
13.14
±1.06
8.57
±0.84
5.83
±0.62
[pmol/min-1]
T315I
3.48
±0.53
4.11
±0.70
2.9 ±
0.69
4.08
±0.29
Kmapp [μM]
Vmaxapp
22.97
±1.50
14.96
±1.34
8.06
±0.82
4.95
±0.15
[pmol/min-1]
Wild Type (WT)
6 [M]
0
25
50
100
46.11
±5.59
55.3
±5.85
50.09
±4.79
35.31
±4.75
Kmapp [μM]
Vmaxapp
16.35
±1.27
11.52
±1.03
7.53
±1.00
3.66
±0.45
[pmol/min-1]
T315I
3.168
±0.46
3.29
±0.52
4.30
±0.67
4.9
±0.8
Kmapp [μM]
Vmaxapp
25.27
±1.39
16.11
±1.22
10.28
±0.84
6.30
±1.3
[pmol/min-1]
affinity towards ATP (Kmapp) as expected for a non-
competitive inhibitor. Overall, these results confirm the capacity
of 5 and 6 to bind the MP and inhibit the kinase activity with an
allosteric mechanism of action.
Finally, compounds 5 and 6 were evaluated for their cytotoxic
effect on murine 32D parental myeloid cell line and both the wild
type Bcr-Abl (p210) and the T315I mutant expressing cells.
Compounds 5 and 6 have been tested for 48 h at increasing
concentration (0.5-50 µM) and their efficacy was calculated
considering the number of viable cells in respect to the control
(Figure 4A). Notably, both molecules showed no cytotoxicity on
parental 32D cells while compound 6 displayed a higher
cytotoxic activity (1.2 μM and 13.8 μM against IM-sensitive and
IM-resistant cell lines respectively) compared to 5 (8.5 μM and
25.1 μM) in the leukemic cell lines. Surprisingly, 5 and 6 showed
higher activity in cells rather than on isolated enzymes,
suggesting that inhibition of additional targets may be
responsible for the observed cytotoxic effect. Moreover the
phosphorylation levels of the Bcr-Abl substrate CrkL protein
have been analyzed by western blot (Figure 4B). It was observed
a reduction of the phosphorylation on CrkL on Tyr207 after
treatment with our compounds 5 and 6, confirming their efficacy
in inhibiting Bcr-Abl.
In summary,
a cascade screening approach has been
successfully applied to our internal collection of ATP mimetics
leading to the identification of a hit compound (6) acting with an
allosteric mechanism of action against both Abl WT and T315I
mutant. A fast FP screening protocol followed by computational
studies (combining docking and dynamics simulations) have
been applied for the initial identification of two MP binders (5
and 6). Kinetic enzymatic studies confirmed the allosteric
Figure 4. A) Cell death in response to treatment with compounds 5 and 6
for 48 h. Assays were conducted on murine leukemia 32D expressing Bcr-
Abl (WT or T315I mutated).; B) Protein levels of CrkL and p-CrkL (Tyr207)

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2010, 463, 501.
were analyzed by western blot in 32D-T315I mutated cells treated for 24h
with 1µM compounds 5 and 6. As loading control β-actin was utilized.
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Acknowledgments
11. Radi, M.; Tintori, C.; Musumeci, F.; Brullo, C.; Zamperini, C.;
Dreassi, E.; Fallacara, A. L.; Vignaroli, G.; Crespan, E.; Zanoli,
S.; Laurenzana, I.; Filippi, I.; Maga, G.; Schenone, S.; Angelucci,
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2010, 5, 1226.
This project was supported by the National Interest Research
Project PRIN_2010_5YY2HL, the German Federal Ministry of
Education and Research (BMBF) within the framework of
the e:Med research and funding concept (grant # 01ZX1303C)
and the Italian Association for Cancer Research AIRC IG2014-
15868 grant. Lead Discovery Siena s.r.l. is also acknowledged
for financial support.
13. Crespan, E.; Radi, M.; Zanoli, S.; Schenone, S.; Botta, M.; Maga,
G., Bioorg. Med. Chem. 2010, 18, 3999.
14. Santucci, M. A.; Corradi, V.; Mancini, M.; Manetti, F.; Radi, M.;
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Supplementary Material
9.
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Graphical Abstract
A Cascade Screening Approach for the
Identification of Bcr-Abl Myristate Pocket
Binders Active Against Wild Type and T315I
Mutant
Leave this area blank for abstract info.
Marco Radi,* Ralf Schneider, Anna Lucia Fallacara, Lorenzo Botta, Cristina Tintori, Emmanuele Crespan,
Giovanni Maga, Miroslava Kissova, Alessia Calgani, André Richters, Francesca Musumeci, Daniel Rauh and
Silvia Schenone*