M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 9 6 8 e9 7 5

available at www.sciencedirect.com

www.elsevier.com/locate/molonc

Through the open door: Preferential binding of dasatinib

to the active form of BCR-ABL unveiled by in silico
experiments

Erik Laurinia, Paola Posoccoa, Maurizio Fermegliaa, Don L. Gibbonsb,c,

Alfonso Quinta  s-Cardamad, Sabrina Pricla,e,*
a
Molecular Simulations Engineering (MOSE) Laboratory e DEA, University of Trieste, Piazzale Europa 1, 34127
Trieste, Italy
b
Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center,
Houston, USA
c
Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA
d
Department of Leukemia, University of Texas MD Anderson Cancer Center, Houston, USA
e
National Interuniversity Consortium for Material Science and Technology (INSTM), Research Unit MOSE-DEA,
University of Trieste, Italy

A R T I C L E I N F O A B S T R A C T

Article history: Dasatinib is a second-generation BCR-ABL inhibitor approved for the treatment of patients
Received 29 April 2013 with chronic myeloid leukemia, both in the frontline and in the imatinib-resistant/
Received in revised form intolerant settings. The high affinity of dasatinib for the protein is currently assumed to
31 May 2013 result from its ability to bind both the active and inactive conformations of the BCR-ABL
Accepted 4 June 2013 kinase. In the present work, using state of the art molecular simulation techniques we
Available online 15 June 2013 prove that dasatinib exhibits a highly selective preference for the active (open) BCR-ABL
conformation. By using three different BCR-ABL conformations (active, inactive,
Keywords: and intermediate inactive) we show that, from a thermodynamic standpoint, the affinity
BCR-ABL of dasatinib for BCR-ABL drastically decreases in the order: active > alternative
Dasatinib inactive > inactive, as a result of differential contributions from the single residues lining
Binding mode the kinase binding pocket and the concomitant stabilization/destabilization of the kinase
Hydrophobic-spine hydrophobic spine. Molecule-pulling experiments also corroborate this trend as signifi-
Resistance cantly lower forces and smaller times are required to extract dasatinib from its inactive
BCR-ABL complexes with respect to the active complex counterparts. Importantly, our re-
sults support recent NMR solution results demonstrating no evidence of dasatinib bound to
the inactive form of BCR-ABL.
ª 2013 Federation of European Biochemical Societies.
Published by Elsevier B.V. All rights reserved.

1. Introduction evolves in 3 different phases: chronic phase (CP), accelerated

phase (AP), and blast crisis (BC). The BCR-ABL oncogene, a
Chronic myelogenous leukemia (CML) accounts for 15% of leu- product of the reciprocal chromosomal translocation t(9;22)
kemias in adults (Jabbour and Kantarjian, 2012), and typically (q34;q11), is the main pathogenetic driver. The protein product

* Corresponding author. MOSE e DEA, Piazzale Europa 1, 34127 Trieste, Italy. Tel.: þ39 (0)405583750; fax: þ39 (0)40569823.
E-mail address: sabrina.pricl@di3.units.it (S. Pricl).
1574-7891/$ e see front matter ª 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molonc.2013.06.001
 M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 9 6 8 e9 7 5
is the cytoplasmic BCR-ABL tyrosine kinase (TK) whose activ-
ity regulates survival, proliferation, and adhesion of CML cells
(Kurzrock et al., 1988).
The discovery of the role of BCR-ABL in the pathogenesis of
CML provided the rationale for the design of the tyrosine kinase
inhibitor (TKI) imatinib (Gleevec, Novartis Pharmaceuticals,
Basel, Switzerland), an inhibitor that binds with high affinity
the inactive conformation of BCR-ABL (Nagar et al., 2002;
Deininger et al., 2005). Imatinib showed remarkable activity as
a single agent in CML particularly among patients in CP
(Cortes and Kantarjian, 2012). Unfortunately, resistance to ima-
tinib in patients with CML occurs in a significant number of
patients failing imatinib therapy (Druker et al., 2006). Of all
possible mechanisms mediating imatinib resistance (Ohanian
et al., 2012; Yeung and Hughes, 2012), the selection of subclones
bearing BCR-ABL point mutations is the most frequent. At the
protein level, these point mutations result in a distorted confor-
mation of the TK/Imatinib interface, which leads to BCR-ABL
being unable to adopt the tailor-fit inactive (close) conformation
to which imatinib binds (Gibbons et al., 2012).
Dasatinib (Sprycel, Bristol Meyer, New York, NY, USA) is a
second generation BCR-ABL inhibitor able to inhibit the
in vitro and in vivo kinase activity of most imatinib-resistant
BCR-ABL mutants, with the exception of the “gatekeeper” mu-
tation T315I (Shah et al., 2004). Noteworthy, in vitro experi-
ments have shown that dasatinib is 325-fold more potent
than imatinib in cells expressing wild-type BCR-ABL
(Martinelli et al., 2005).
The solved crystal structure of dasatinib in complex with
BCR-ABL (Tokarski et al., 2006) clearly showed that, at vari-
ance with imatinib, this TKI is able to target the active
(open) form of the TK. Notwithstanding, in the same work
Tokarski et al. (2006) hypothesized that the reason for the
greater BCR-ABL affinity of dasatinib towardtoward the BCR-
ABL kinase with respect to imatinib was due, at least in part,
to its ability to bind the inactive conformation of the BCR-
ABL kinase. Indeed, these authors claimed - in a narrative
fashion based on “the modeling of dasatinib in the imatinib-
bound and PD173855-bound forms of the kinase” (not reported
in the paper) e that no “major sterical clashes would preclude
dasatinib from also binding the inactive conformations found
for in these other ABL structures”. In support of this assertion,
Gambacorti-Passerini et al. (2005) predicted that dasatinib
could not only bind to the inactive form of the kinase but
also to another BCR-ABL conformation, termed the intermedi-
ate conformation e in which the kinase activation loop
(A-loop) assumes a conformation utterly similar to the one
found in the active enzyme. According to the binding mode
of Gambacorti-Passerini et al. (2005), dasatinib was likely to
be more active against the inactive and intermediate forms
of BCR-ABL than versus the active open A-loop form, although
no numerical data were offered to sustain these claims. To
support these basic modeling results, these authors just
invoked the lack of involvement of T315I in binding ATP; how-
ever, the importance of the T315I mutation in dasatinib resis-
tance is against the inactive and intermediate forms rather
than the active kinase, because dasatinib does not act as a
true inhibitor of ATP binding.
Herein we performed a systematic in silico investigation
based on extensive and state-of-the art molecular simulations
969
and ultimately verified that dasatinib is unfit to bind different
conformational states of the BCR-ABL kinase. Furthermore,
dasatinib shows a remarkable, almost exclusive preference
towards the kinase open form, in full agreement with recent
relevant NMR solution studies (Vajpai et al., 2008).
2. Methods
2.1. PDB files
The following X-ray structures were employed in all simula-
tions: i) 1IEP: BCR-ABL/imatinib complex (Nagar et al., 2002), in
which the TK is in its inactive form (IF), with the activation
loop in its closed (i.e., DFG-out) conformation and the Tyr-393
residue is unphosphorylated; ii) 1M52: BCR-ABL/PD173955 com-
plex (Nagar et al., 2002), in which the TK is an alternative inac-
tive form (IFa), with the activation loop in a closed (i.e., DFG-out)
conformation, although different from that found in 1IEP, and
the Tyr-393 residue unphosphorylated; iii) 2GQG (chain A):
BCR-ABL/dasatinib complex (Tokarski et al., 2006), in which
the kinase is in its active form (AFA), with the activation loop
in its open (i.e., DFG-in) conformation, and the Tyr-393 residue
is phosphorylated; iv) 2GQG (chain B): BCR-ABL/dasatinib com-
plex (Tokarski et al., 2006), in which the kinase is its active form
(AFB), with the activation loop in its open (i.e., DFG-in) confor-
mation, and the Tyr-393 residue is unphosphorylated.
2.2. Computational details
All classical, parallel molecular dynamics (MD) simulations
and the relevant analyses were performed the Amber 11 suites
of programs (Case et al., 2010) running on the PLX-GPU and
FERMI supercomputers at the CINECA supercomputer center
(Bologna, Italy). Each BCR-ABL/TKI complex was then opti-
mized in a box of TIP3P water molecules (Jorgensen et al.,
1983) in the presence of 0.15 M NaCl. After each system equil-
ibration and heating, 50 ns of data collection MD runs, neces-
sary for the estimation of the free energy of binding (vide infra),
were performed (see SI for full details). For the calculation of
the binding free energy between BCR-ABL and each inhibitor
in water, a total of 50,000 snapshots were saved during the
MD data collection period described above.
The binding free energy DGbind of each BCR-ABL1/TKI com-
plex in water was calculated according to the procedure
termed Molecular Mechanic/Poisson-Boltzmann Surface
Area (MM/PBSA), and originally proposed by Srinivasan et al.
(1998). The theoretical background of this methodology is
described in details in the original papers by Kollman et al.
(2000), and has been successfully employed by our group in
related studies (see SI) (Pierotti et al., 2011). Briefly, an MD
simulation (typically in explicit solvent) is carried out which
yields a representative ensemble of structures, from which
the average total free energy of binding between each drug
and the protein receptor DGbind can then be calculated. The
corresponding IC50 values were calculated from the binding
free energies using the following relationship (Wang et al.,
2001): DGbind ¼ RT ln Kdiss y RT ln IC50.
The contribution of each kinase residue belonging to the
so-called “hydrophobic spine” (Azam et al., 2008) to the
970 M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 9 6 8 e9 7 5

stabilization/destabilization of the active/inactive protein
conformation and its eventual relation to drug resistance was
investigated by means of component analysis (Gohlke et al.,
2003). Accordingly, a per residue binding free energy decomposi-
tion was performed exploiting the MD trajectory of each given in-
hibitor/kinase complex. This analysis was carried out using the
MM/GBSA approach (Tsui and Case, 2000), and was based on
the same snapshots used in the binding free energy calculation.
The entire MM/PBSA computational procedure was opti-
mized by integrating AMBER 11 in modeFRONTIER, a multidis-
ciplinary and multi-objective optimization and design
environment (http://www.esteco.com/home/mode_frontier/
mode_frontier.html).
The steered molecular dynamics (SMD) simulations
(Isralewitz et al., 2001) were performed using snapshots
randomly taken from the MD equilibrated runs as initial struc-
tures. With the adopted settings (see SI), 2.5 nm were covered
in 500 ps of SMD simulation.
3. Results
3.1. Differences between 3D models available for BCR-
ABL kinase
Panel A in Figure 1 compares the crystallographic structures
of the active (AFA) and inactive (IF) BCR-ABL conformations.
The most notable differences between the two kinase conforma-
tions are the opposite orientation of the activation loop (A-loop)
and of the relevant DFG catalytic triad. When the open, active
form of BCR-ABL (AFA) is compared with the alternative, inactive
form of the kinase (IFa), the A-loop in the latter assumes an open
conformation but the catalytic triad is still positioned in a DFG-
out (i.e., inactive) orientation (Figure 1, panel B). Lastly, panel C
in Figure 1 compares the two active chains found in the unit crys-
tal cell of BCR-ABL in complex with dasatinib (AFA and AFB).
Interestingly, chain A is phosphorylated on Try393 whilst chain
B, although still complexed with the TKI in the original crystal, is
unphosphorylated at the same residue. However, the two struc-
tures are almost entirely superimposable.
3.2. Docking and binding affinity
In order to evaluate the possible interactions of dasatinib with
the inactive (IF) and the alternative inactive (IFa) form of BCR-
ABL, the three-dimensional (3D) model of the TKI was posi-
tioned within the two protein binding sites according to an
established procedure (see also SI) (Pierotti et al., 2010). The
validity of the adopted docking protocol was further assessed
by reproducing the available orientations of imatinib and
dasatinib as previously determined on crystal structure (see
SI for details).
In contrast to previous reports (Gambacorti-Passerini et al.,
2005), the mere docking results relative to dasatinib binding to

Figure 1 e Comparison of crystallographic structures available for inactive, active and intermediate state of the TK. (A) Superposition of the active
(AFA, orange) and inactive (IF, cyan) structure of BCR-ABL. (B) Superposition of the active (AFA, orange) and alternative, inactive (IFa, forest green)
structure of BCR-ABL. (C) Superposition of the two active structures of BCR-ABL found in the same crystal structure (AFA, orange, and AFB, blue).
All images are zoomed view of the TK A-loop region. In all structures, the DFG motif and the Tyr393 residue are highlighted as sticks-and-balls.
 M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 9 6 8 e9 7 5
the different forms of the BCR-ABL kinase are not sufficient to
determine whether this TKI shows any real preference for the
active, inactive or intermediate state of the enzyme. Indeed, a
visual inspection of the binding modes of dasatinib in the bind-
ing site of the inactive (IF) and the alternative inactive (IFa)
form of the protein does not reveal evident steric clashes or
substantial difference/distortion of the binding site conforma-
tions when compared to those of the two active kinase chain
conformations (AFA and AFB). Accordingly, all 4 complexes
(IF/dasatinib, IFa/dasatinib, AFA/dasatinib, and AFB/dasatinib)
were subjected to extensive molecular dynamics (MD) simula-
tions in the MM/PBSA framework of theory (Kollman et al.,
2000; Pierotti et al., 2011) to estimate the free energy of binding
of dasatinib (DGbind) e and the relevant IC50 e for each complex
TKI/TK assembly. These results are shown in Table 1.
Regarding dasatinib in complex with the two open forms of
BCR-ABL (AFA and AFAB, differing only by the presence/
absence of a phosphate group on the Tyr393 respectively),
no significant differences in the corresponding binding free
energy values were observed. Specifically, for both chain A
and B bound to dasatinib the largely negative values of DGbind
in Table 1 show the highly favorable interaction energy of the
TKI with the open/active state of the TK. Importantly, the
calculated IC50 is in concert with experimental data, thus indi-
cating an affinity of the drug for the protein in the nanomolar
range (Martinelli et al., 2005).
In the case of the inactive (IF, A-loop in close conformation
and DFG-out conformation) and alternative inactive (IFa, A-
loop in open conformation but DFG-out conformation) forms
of BCR-ABL in complex with dasatinib, the estimated DGbind
values sit above (i.e., are less negative than) 10 kcal/mol,
indicating a net decrease of affinity of the drug for the kinase.
As mentioned above, the major difference between these two
BCR-ABL/TKI complexes resides in the small alteration of the
conformation of the catalytic triad DFG, which in both cases is
in the out conformation. Although these residues are not
proximal to the drug binding site, this difference reflects in a
structural rearrangement of the protein which, in turn, influ-
ences the dasatinib binding site, as detailed in Figure 2.
3.3. Per-residue deconvolution of the free energy of
binding
In order to gain further insight into the binding modes of
dasatinib to the different forms of the BCR-ABL kinase, we
Table 1 e Molecular dynamics free energy of binding (DGbind) and
IC50 values for dasatinib in complex with different active and
inactive forms of BCR-ABL.
BCR-ABL DGbind (kcal/mol) IC50 (nM)a
AFA 12.41  0.01 0.8
AFB 12.29  0.02 1.0
IFa 9.40  0.02 130
IF 8.89  0.01 306
Data are reported with standard errors of the mean.
a DGbind and IC50 (i.e., the concentration of dasatinib that inhibits
the kinase activity by 50%) are related by the following funda-
mental equation: DGbind ¼ RT ln 1/IC50.
971
calculated the energetic contribution for those residues which
afford a substantial contribution to the binding. As shown in
Table 2, in the case of the dasatinib/AFA and AFB complexes,
no meaningful differences were observed at the individual res-
idue binding level. Conversely, in the case of both dasatinib/IF
and IFa assemblies, the overall reduction of drug affinity cannot
be attributed either to a single residue or a particular cluster of
binding site residues. Instead, a general decrease of the binding
energy for all residues involved is predicted. In other words, the
different protein conformations (inactive or alternative inactive
conformation) do not result in a marked decrease of the binding
energy localized in a specific region of the protein binding
pocket but, on the contrary, they exert a sort of domino effect
that affects the entire protein binding site.
This analysis confirms how the conformation adopted by
the kinase in the open forms AFA and AFB is a key factor for
the drug high stability within its protein binding site with
respect to the other two kinase conformations (IF and IFa)
considered. Indeed, the sums of all individual residue contri-
bution to binding in the first two complexes (dasatinib/AFA
and dasatinib/AFB, equal to 10.18 and 9.91 kcal/mol,
respectively) are more favorable (by approximately 2 kcal/mol)
than those calculated for the dasatinib/IF (8.04 kcal/mol) and
dasatinib/IFa (8.25 kcal/mol).
3.4. Hydrophobic spine analysis
The hydrophobic spine is a highly conserved network of hy-
drophobic interactions characteristic of the active kinase
conformation which is stabilized by the gatekeeper mutation
T315I in BCR-ABL (Azam et al., 2008). Mutations at spine resi-
dues may disrupt the hydrophobic connectivity and inactivate
the kinase. In the BCR-ABL protein, the residues making up
the hydrophobic spine are M290, L301, H361, and F382, this
last amino acid further belonging to the catalytic triad DFG
involved in kinase activation.
To describe and quantify the role of the hydrophobic spine
upon dasatinib binding to the 4 different BCR-ABL conforma-
tions, we evaluated the free energy of binding deconvolution
analysis considering the BCR-ABL residues involved in the
spine. A pictorial view of the results is shown in Figure 3.
The different conformational state of the residue F382 in the
DFG-in and DFG-out configuration influences the spine stabili-
zation process. More importantly, both in the case of dasatinib/
IF and dasatinib/IFa complexes (in which the kinase features
the DFG-out conformation), the spine stabilization is much
less effective than for the two alternative complexes (AFA
and AFB) in which the kinase has an open, active conformation
(DFG-in), as quantified in Table 3. In fact, the contribution
afforded by the hydrophobic spine stabilization is quite larger
in the case of the active conformation for which, in turn, the
binding of dasatinib is more favorable. In those BCR-ABL/
dasatinib complexes where the hydrophobic spine is less stabi-
lized, the interaction between M290 and L301 is preserved, but
the interaction energy between M290 and F382 becomes less
favorable (from 0.60 kcal/mol for the AFA and 0.20
and 0.26 kcal/mol for the IF and IFa, respectively), and espe-
cially the stabilizing energies between H361 and F382 become
remarkably weaker (from 1.7 kcal/mol for the AFA to 0.15
and 0.11 kcal/mol for IF and IFa, respectively).
972 M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 9 6 8 e9 7 5

Figure 2 e Molecular dynamics snapshots of dasatinib in complex with active and inactive conformations of BCR-ABL. (A) Equilibrated snapshot
from MD simulation of the AFA kinase form (Y393 phosphorylated, DFG-in conformation) in complex with dasatinib. The TKI is in an atom-
colored stick- and-ball representation (C, gray; O, red; N, blue; yellow, S; green, Cl). The main residues involved in the binding are highlighted as
labeled sticks. Hydrogen atoms, ions and counterions, and water molecules are omitted for clarity. (B) Equilibrated snapshot from MD simulations
of (top left) dasatinib/AFA (kinase open form, Y393 phosphorylated, DFG-in conformation), vs. dasatinib/AFB (kinase open form, Y393
unphosphorylated, DFG-in conformation); (top right) dasatinib/AFA vs. dasatinib/IF (kinase closed form, DFG-out conformation); (bottom left)
dasatinib/AFA vs. dasatinib/IFa (kinase alternative inactive form, DFG-out conformation); (bottom right) dasatinib/IF (kinase inactive form,
DFG-out conformation) vs. dasatinib/IFa (kinase alternative inactive form, DFG-out conformation). In the first case, the two binding sites are
very similar, as is the binding mode and conformation of the inhibitor in the two protein binding pockets. In the other cases, the differences in the
activation loops and binding site are evident. Dasatinib/AFA (orange); dasatinib/IF (cyan); dasatinib/IFa (forest green); dasatinib/AFB (blue).

3.5. Steered molecular dynamics and dasatinib excellent agreement with experimental data. Usually, such
unbinding process technique can yield reliable information both on the main
qualitative and quantitative aspects of drug binding; on the
MM/PBSA is a well-known and validated methodology to esti- other hand, however, it deals only with the thermodynamic
mate the free energy of e.g., drug/protein binding in fair to aspect of the association of a given ligand with its target pro-
tein. Thus, further confirmation of MM/PBSA-based results
concerning the remarkable preference of dasatinib toward
the open form of the BCR-ABL kinase was pursued by applying
Table 2 e Per-residue free-energy decomposition for AFA, AFB, IF a steered molecular dynamics (SMD) protocol. Such computa-
and IFa in complex with dasatinib. tional experiments allow to estimate the force that is required
to pull dasatinib out of its protein binding site, and in the pre-
Residue AFA AFB IF IFa
sent work it was applied for the first time to all 4 Dasatinib/
DGbs
bind DGbs
bind DGbs
bind DGbs
bind BCR-ABL complexes discussed above. As a result, also some
kinetic aspects of the reversible drug binding (as is the present
L248 0.92 0.95 0.78 0.76
M290 1.15 1.02 0.93 0.91 case) are accounted for in the analysis.
V299 1.29 1.08 0.95 1.10 Figure 4 shows a prototypical smoothed force (F) vs. time (t)
I313 1.32 1.41 1.15 1.12 diagram obtained from the SMD runs for the 4 complexes of
T315 1.21 0.92 0.75 0.83 dasatinib with BCR-ABL. As we can see, all curves present an
E316 0.54 0.49 0.35 0.40 initial phase in which the force increases up to a maximum,
F317 1.68 1.87 1.37 1.45
which represents the force value that needs to be applied to
M318 0.56 0.61 0.42 0.36
T319 1.51 1.56 1.34 1.32
detach the drug from its binding site. In the case of the AFA/
P and AFB/dasatinib complexes, the TKI/TK complex rupture
DGbs
bind (kcal/mol) 10.18 9.91 8.04 8.25
force is around 1200 pN, and this unbounded configuration
All values are in kcal/mol. Data standard deviations are in the range
is attained after approximately 300 ps. For these two
0.01 O 0.05.
complexes, the corresponding F vs. t profiles are very similar,
 M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 9 6 8 e9 7 5 973

Figure 3 e Conformation of BCR-ABL hydrophobic spine residues for active and inactive forms of BCR-ABL in complex with dasatinib.
Equilibrated molecular dynamics snapshots of dasatinib in complex with the two BCR-ABL active forms (top left, AFA; top right, AFB), with the
inactive form (bottom left), and with the alternative inactive form IFa (bottom right). All residues belonging to the hydrophobic spine are
highlighted as red sticks, and labeled. Dasatinib is portrayed as atom-colored balls-and-sticks (C, gray; O, red; N, blue; S, yellow; Cl, green).
Hydrogen atoms, ions and counterions, and water molecules are omitted for clarity.

suggesting a similar unbinding process event, as expected. On to the unphosphorylated form of the kinases was detected
the contrary, and in line with all evidence gained from the in solution.
MM/PBSA simulations discussed above, for the IF/and IFa/
dasatinib complexes not only the force at rupture has a
notably lower peak value (approx. 800 pN) but this peak is 4. Discussion
reached quite sooner, i.e., after approx. 180 ps. These SMD ex-
periments ineluctably confirm that dasatinib is characterized In silico analyses performed in this work - based on a combina-
not only by a remarkable lower affinity for both inactive forms tion of MD/SMD simulations e revealed that dasatinib shows a
of BCR-ABL (as quantified by the relevant DGbind values) but marked preference for binding to the active state of the BCR-
also by a kinetically different protein unbinding process ABL kinase. The binding affinity of this TKI for the open
among the different kinase conformations. In other words, form of its target enzyme is not only due to the highly favor-
should dasatinib be able to weakly bind to one of the inactive able DGbind (12.41 kcal/mol), which translates into an IC50
form of the BCR-ABL, notably lower force and time values of 0.8 nM, thus ranking dasatinib as one of more potent small
would be required to detach it from its binding site. These
new findings are in full agreement with Vajpai et al. (2008),
who used solution NMR to study dasatinib binding to phos-
phorylated and unphosphorylated forms of the BCR-ABL ki-
nase. According to their studies, no trace of dasatinib bound

Table 3 e Hydrophobic spine per residue free-energy decomposition

for AFA, AFB, IF and IFa in complex with dasatinib.
AFA L301 H361 F382 AFB L301 H361 F382

M290 1.6 0.02 0.60 M290 1.5 0.07 0.56

L301 0.02 0.01 L301 0.00 0.01
H361 1.7 H361 1.8
P spine P spine
DGbind 4.0 DGbind 3.9

IF L301 H361 F382 IFa L301 H361 F382

Figure 4 e Average force profile of dasatinib unbinding from its
M290 1.6 0.04 0.20 M290 1.6 0.06 0.26
complex with the different active and inactive forms of BCR-ABL.
L301 0.02 0.03 L301 0.01 0.05
H361 0.15 H361 0.11
Comparison of the unbinding force profiles of dasatinib from the IF
P spine P spine (cyan), IFa (forest green), AFA (orange), and AFB (blue) forms of the
DGbind 2.0 DGbind 2.1
BCR-ABL TK. For each protein form, the plots show the resulting
All values are in kcal/mol. Data standard deviations are in the range
mean values from averaging the force profiles from five different
0.01 O 0.05.
SMD runs.
974 M O L E C U L A R O N C O L O G Y 7 ( 2 0 1 3 ) 9 6 8 e9 7 5
molecule inhibitors of wild-type BCR-ABL, both in biochemical
and cellular assays (Martinelli et al., 2005) e but also from a ki-
netic point of view, in terms of reduced ability of escaping
from the protein binding site when bound to its active
conformation.
A per residue deconvolution of the free energy of binding
shows that, in a sort of domino effect, the efficiency of the
amino acid lining the kinase binding pocket in binding dasati-
nib decreases of w2 kcal/mol (that is nearly 1.5 orders of
magnitude) in passing from the open to the close form of
the kinase. The same analysis applied to the protein residues
making up the kinase hydrophobic spine also indicate that,
correctly, this network of hydrophobic interaction is progres-
sively disrupted when dasatinib is bound to the alternative
inactive and the inactive form of the protein. Further, SMD
simulations mimicking molecule-pulling experiments also
corroborate the MM/PBSA-based dasatinib/BCR-ABL affinity
trend, as significantly lower forces and smaller times are
required to extract dasatinib from its inactive BCR-ABL com-
plexes with respect to the active complex counterparts.
Our results explain why, by considering the stabilization/
destabilization of the BCR-ABL hydrophobic spine in the
open/close TK forms, the gatekeeper mutation T315I is highly
resistant to dasatinib. Indeed, it has been experimentally veri-
fied (Young et al., 2006) that the T315I BCR-ABL variant
induced equivalent levels of total cellular phosphotyrosine
in cell lysates as the wild-type counterpart. Also, since the
gatekeeper residue T315 is situated near the tip of the hydro-
phobic spine, the substitution of a bulkier hydrophobic resi-
due such as isoleucine at this position should result, as
suggested (Young et al., 2006), in stabilization of the active
state, to which dasatinib binds, by strengthening the hydro-
phobic spine. At the same time, however, the change in
conformation of the TK binding pocket leads to an overall
rearrangement of dasatinib within the protein cavity, with
an overall loss of several stabilizing protein/drug interactions.
Notwithstanding the stabilization of the BCR-ABL hydropho-
bic spine and, hence, of its active conformation, the affinity
of dasatinib for the TK is drastically decreased by virtue of a
substantial penalty in the free energy paid by the TKI upon
binding. The altered binding site conformation also results
in a greater facility for dasatinib to escape from T315I BCR-
ABL; interestingly the SMD corresponding forceetime profile
is utterly similar to that predicted for the dasatinib/IF complex
(see SI for details).
Current structural understanding of kinases is largely
based on x-ray crystallographic studies, whereas very little
data exist on the conformations and dynamics that kinases
adopt in the solution state. Recently, it has been reported
the first characterization of ABL TK in complex with three
“ib” TKIs (imatinib, nilotinib, and dasatinib) by solution NMR
techniques (Vajpai et al., 2008). While imatinib and nilotinib
in complex with the TK feature the A-loop in the close
(inactive) conformation, the BCR-ABL/dasatinib complex
preserves the active conformation, in stark contrast with pre-
vious predictions based upon molecular docking concepts
(Gambacorti-Passerini et al., 2005), but in full agreement
with the present results. Interestingly, these NMR experi-
ments were performed with non-phosphorylated protein, a
technical choice to ensure that the protein might adopt the
inactive DFG-out conformation (Vajpai et al., 2008). Since the
presence of a phosphotyrosine residue at position 393
(PTR393, see Figure 1) in the A-loop concurs to stabilize the
active conformation of the protein by forming interactions
with neighboring side chains (Young et al., 2006), the authors
anticipated that this occurrence would further reduce the
flexibility of the BCR-ABL/dasatinib complex, thereby narrow-
ing the conformational space available to the DFG-in configu-
ration. For the same reason, any propensity of the BCR-ABL/
dasatinib complex to adopt the inactive DFG-out conforma-
tion would be even more reduced. Our results fully corrobo-
rate this hypothesis, and concur in ascertaining the highly
selective preference of dasatinib for binding the active, open
conformation of the BCR-ABL kinase.
Authorship
Contribution: E.L. and P.P. designed and performed research
and analyzed data; M.F., A.Q.C. and D.L.G. analyzed data and
wrote the paper; S.P. performed research, analyzed data and
wrote the paper.
Conflict of interest
The authors declare no competing financial interests.
Acknowledgments
E.L., P.P., M.F., and S.P. acknowledge the financial support
from ESTECO through the project DDOS. Part of this work
was carried out in the framework of the HPC-Europa 2 project
MONALISA (CINECA Supercomputing Center, Bologna, Italy),
funded by the European Commission e DG Research in the
7th Framework Program (Grant agreement n 228398).
Appendix A.
Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.molonc.2013.06.001.
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