Technical advance Related Commentary, page 3650

An anticancer C-Kit kinase inhibitor

is reengineered to make it more active
and less cardiotoxic
Ariel Fernández,1,2,3 Angela Sanguino,3 Zhenghong Peng,4 Eylem Ozturk,3,5
Jianping Chen,2 Alejandro Crespo,1 Sarah Wulf,1 Aleksander Shavrin,4
Chaoping Qin,6 Jianpeng Ma,1,6,7 Jonathan Trent,8 Yvonne Lin,9 Hee-Dong Han,9
Lingegowda S. Mangala,9 James A. Bankson,10 Juri Gelovani,4 Allen Samarel,11
William Bornmann,4 Anil K. Sood,9,12 and Gabriel Lopez-Berestein3
1Department of Bioengineering and 2Applied Physics Division, Rice Quantum Institute, Rice University, Houston, Texas, USA.
3Departmentof Experimental Therapeutics and 4Department of Experimental Diagnostic Imaging, Chemistry Section,
University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 5Department of Chemistry, Hacettepe University, Ankara, Turkey.
6Graduate Program of Structural and Computational Biology and Molecular Biophysics and 7Department of Biochemistry and Molecular Biology,

Baylor College of Medicine, Houston, Texas, USA. 8Division of Cancer Medicine, Department of Sarcoma Medical Oncology,
9Department of Gynecologic Oncology, and 10Department of Imaging Physics, University of Texas MD Anderson Cancer Center,

Houston, Texas, USA. 11Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois, USA.
12Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

Targeting kinases is central to drug-based cancer therapy but remains challenging because the drugs often
lack specificity, which may cause toxic side effects. Modulating side effects is difficult because kinases are
evolutionarily and hence structurally related. The lack of specificity of the anticancer drug imatinib enables
it to be used to treat chronic myeloid leukemia, where its target is the Bcr-Abl kinase, as well as a proportion
of gastrointestinal stromal tumors (GISTs), where its target is the C-Kit kinase. However, imatinib also has
cardiotoxic effects traceable to its impact on the C-Abl kinase. Motivated by this finding, we made a modifi-
cation to imatinib that hampers Bcr-Abl inhibition; refocuses the impact on the C-Kit kinase; and promotes
inhibition of an additional target, JNK, a change that is required to reinforce prevention of cardiotoxicity. We
established the molecular blueprint for target discrimination in vitro using spectrophotometric and colori-
metric assays and through a phage-displayed kinase screening library. We demonstrated controlled inhibi-
tory impact on C-Kit kinase in human cell lines and established the therapeutic impact of the engineered
compound in a novel GIST mouse model, revealing a marked reduction of cardiotoxicity. These findings
identify the reengineered imatinib as an agent to treat GISTs with curbed side effects and reveal a bottom-up
approach to control drug specificity.

Introduction design concept, we modified imatinib to significantly reduce its

Protein kinases, the signal transducers of the cell, are paradigmatic impact on Bcr-Abl, prima facie curbing cardiotoxicity, while retain-
targets for drug-based cancer therapy (1–13). However, their evolu- ing anticancer activity on other primary targets.
tionary — and hence structural — relatedness often leads to unfore- The rationally redirected molecular impact of the prototype is
seen cross-reactivities (13–16), now surfacing through the advent corroborated in vitro and validated through tumor-derived cell
of high-throughput screening technologies (6). Although the rela- lines and in an animal model. Thus, by sculpting into the ligand
tionship between specificity and anticancer activity remains nebu- the de-wetting differences across imatinib targets, we focused the
lous, a lack of specificity often underlies toxic side effects as the inhibitory impact specifically on the C-Kit kinase, a target for
inhibitory impact diffuses away from clinically relevant targets (5, gastrointestinal stromal tumor (GIST) (9–11), while suppressing
14). Unwanted side effects can even be traced to inhibitory impact Bcr-Abl inhibition and promoting JNK inhibition, as needed to
on a primary target, as in the reported cardiotoxicity of imatinib reinforce the prevention of cardiotoxicity (15). The discriminat-
(STI571; Gleevec), likely due to its impact on Bcr-Abl (15), the chi- ing molecular design is reflected in selective anticancer activity on
meric Abelson kinase target in the treatment of chronic myeloid GIST cell lines and a GIST animal model, and a prima facie reduc-
leukemia (CML) (5, 7, 8). Here we report on a rational drug-rede- tion of cardiotoxicity.
sign strategy to control and refocus the inhibitory impact guided
by a structure-based target discriminator. Thus, using a novel Results
Discriminatory ligand design by sculpting unique nonconserved dehydra-
Nonstandard abbreviations used: BNP, brain natriuretic peptide; CML, tion propensities. To engineer the affinity discriminator for C-Kit,
chronic myeloid leukemia; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine;
we compare the patterns of residence times of water molecules
EF, ejection fraction; GIST, gastrointestinal stromal tumor; Lck, lymphocyte-
specific tyrosine kinase; NRVM, neonatal rat ventricular myocyte; TMB, that solvate the aligned interfacial regions of PDB-reported imat-
3,3′,5,5′-tetramethylbenzidene. inib targets Bcr-Abl (PDB.1FPU), C-Kit (PDB.1T46), and lym-
Conflict of interest: The authors have declared that no conflict of interest exists. phocyte-specific tyrosine kinase (Lck; PDB.3LCK) (http://www.
Citation for this article: J. Clin. Invest. 117:4044–4054 (2007). doi:10.1172/JCI32373. rcsb.org/pdb/home/home.do; Figure 1, A–C; additional details

4044 The Journal of Clinical Investigation    http://www.jci.org    Volume 117    Number 12    December 2007
 technical advance

Figure 1
Comparison of de-wetting propensities of major imatinib targets and drug redesign dictated by de-wetting differences. (A) De-wetting propensi-
ties of C-Kit residues in contact with imatinib (PDB.1T46; green bars) and of aligned residues in Bcr-Abl kinase (PDB.1FPU; red bars) and Lck
(PDB.3LCK; black bars). Residue i is in contact with the ligand if an atom of the latter lies within its domain, D(i) (see Methods). The de-wetting
propensity is quantified by the mean residence time of solvating water molecules. Error bars denote Gaussian dispersion over 5 molecular
dynamics runs. (B) Pattern of de-wetting hot spots that arise as backbone hydrogen-bonded residues in C-Kit kinase (ribbon backbone repre-
sentation in light blue; de-wetting hot spots in green), aligned with Bcr-Abl kinase (magenta backbone; de-wetting hot spots in red). The imatinib
methylation site leading to the expulsion of water from C-Kit C673-G676 de-wetting hot spot is highlighted (yellow rectangle). Imatinib is thus
modified to favorably expel interfacial water molecules from the C-Kit microenvironment, a feature not conserved in Bcr-Abl. Hydrogen bonds are
represented as segments joining α-carbons of the paired residues, and those lacking the propensity for dehydration are shown as light gray seg-
ments. Residues from the C-Kit chain are labeled in green, and those from Bcr-Abl are labeled in white. (C) Differences in de-wetting hot spots
upon alignment of C-Kit (light blue backbone; hot spots in green) and Lck kinase (gold backbone; hot spots in black). Labels for Lck residues are
shown in white. (D) Prototype molecule WBZ_4 (N-{5-[4-(4-methyl piperazine methyl)-benzoylamido]-2-methylphenyl}-4-[3-(4-methyl)-pyridyl]-
2-pyrimidine amine). The added methyl group is indicated in red.

are provided in Supplemental Figure A.1–A.3; supplemental mate- (see Methods). Such bonds become energetically enhanced and
rial available online with this article; doi:10.1172/JCI32373DS1). stabilized upon removal of surrounding water and thus consti-
Hydrating molecules with low residence times (Figure 1A) con- tute de-wetting sites.
stitute our blueprint for ligand reengineering, since they signal A de-wetting hot spot in C-Kit not conserved in the alterna-
a local propensity for water removal. The crux of the redesign tive targets is the residue pair C673-G676, which is mapped into
strategy is then the sculpting in the ligand of nonconserved local the M318-G321 pair in Bcr-Abl and the M319-G322 pair in Lck
de-wetting propensities in the aligned targets: the ligand is engi- (Figure 1, A–C). Thus, this local difference in de-wetting propen-
neered to remove interfacial water upon association according sity prompted us to conceive and synthesize a methylated variant
to weaknesses in the target hydration shell. Since this blueprint of imatinib, heretofore named WBZ_4 (synthesis described in Sup-
is typically not conserved across targets (Figure 1A) — although plemental Data) (Figure 1D). Achieving a higher level of favorable
most surface residues in the binding region are — we can modu- and selective interactivity required an exogenous modulation of
late the inhibitory impact of the compound to a certain extent. the target microenvironment, in turn requiring a modification of
The key de-wetting hot spots correspond to amide-carbonyl back- the parental compound. As dictated by the de-wetting blueprint
bone hydrogen bonds pairing backbone-exposed residues (13) (Figure 1, B and C), the added methyl group would promote the

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technical advance

Figure 2
Comparison of de-wetting propensities of C-Kit and JNK. (A) Aligned de-wetting patterns for C-Kit kinase (green) and JNK1 (blue) restricted to the
C-Kit residues in contact with imatinib. (B) De-wetting hot spots arising as backbone hydrogen-bonded residues in C-Kit kinase (backbone in light
blue; de-wetting hot spots in green), aligned with JNK (blue backbone; de-wetting hot spots in yellow). The JNK residues M111 and N114, aligned
with the de-wetting hot spot C673-G676 in C-Kit, are not paired by a hydrogen bond. Yet M111 is a de-wetting hot spot for JNK (Figure 1E),
hence a harnessing spot for the designed imatinib modification.

favored dehydration of the pair C673-G676 upon binding to C-Kit, bond and consequently stabilizes the inhibitor binding. The effect
while it would hamper the association with Bcr-Abl and Lck, since of WBZ_4 is the opposite on Bcr-Abl. Our simulations demon-
the latter kinases favor hydration of the catalytic loop residues strate that, in this case, the imatinib complex has a better stability
aligned with the targeted C-Kit residues C673-G676 (Figure 1A). in the same loop region, while WBZ_4 significantly destabilizes
Reported assays on imatinib-triggered cardiomyocyte damage the loop. This is due to the fact that the M318-G321 backbone
point to a beneficial JNK (JNK1) inhibition to reduce the induced hydrogen bond on the Bcr-Abl loop is well dehydrated intramo-
collapse of mitochondrial membrane potential and preserve sarco-
lemmal integrity (15). For this reason, we tested the molecular har-
nessing of WBZ_4 on the de-wetting pattern of JNK1 (PDB.2G01).
This analysis required alignment of JNK1 with the imatinib–C-Kit
complex (PDB.1T46) (Figure 2, A and B). The high de-wetting pro-
pensity at JNK1 residue M111, the residue that aligns with C673 in
C-Kit, instills confidence in the affinity of WBZ_4 for JNK.
Molecular dynamics analysis. We first probed WBZ_4 in silico by
monitoring the spatiotemporal effect of the specific methylation
on the kinase-inhibitor association through molecular dynamics
(Figure 3, A and B). As C-Kit associates with imatinib, the loop fac-
ing the para-position on the terminal ring (Figure 1B and Figure 3A)
was found to be unstable. In all 5 simulation trajectories, the back-
bone hydrogen bond between C673 and G676 was irreversibly
replaced by water-mediated interactions within 1 ns (Figure 3A).
When WBZ_4 replaced imatinib within the C-Kit complex, the
same loop was found to be stabilized due to the improved dehy-
dration of the hydrogen bond by the added methyl group on the
ligand (Figure 3B). Thus, for C-Kit, WBZ_4 introduces favorable
interactions engaging the solvent-exposed preformed hydrogen

Figure 3
Water exclusion patterns promoted by imatinib and WBZ_4 on the pri-
mary target C-Kit. (A) Snapshot of the SBMD simulation for C-Kit kinase
bound to imatinib at 1 ns. The main-chain hydrogen bond between
C673 and G676 is competitively and irreversibly replaced by hydro-
gen bonding to a water molecule, revealing the instability of the intra-
molecular interaction. (B) Snapshot of the SBMD simulation of C-Kit
kinase in complex with WBZ_4 at 1 ns. The main-chain hydrogen bond
between C673 and G676 is stabilized by the water expulsion promoted
by the added methyl on the inhibitor.

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 technical advance

Figure 4
High-throughput screening at 10 mM for
WBZ_4 (red) and imatinib (STI_571; blue;
control) over a battery of 228 human kinases
displayed in a T7-bacteriophage library (Ambit
Biosciences). Hit values are reported as per-
centage bound kinase.

lecularly and positioned closer to imatinib;

thus this bond becomes well protected (17)
by imatinib, and the addition of the methyl
group clashes sterically with the loop. Thus,
molecular dynamics provides a convincing
rationale for the discriminatory power of
WBZ_4 relative to imatinib.
High-throughput screening: avoiding the c-Abl
kinase while inhibiting JNK. To test our pre-
dictions, WBZ_4 was first screened for
affinity against a T7-bacteriophage library
displaying 228 human kinases (Ambit
Biosciences) (6) (Figure 4), using imatinib
screening as control. The selective affin-
ity of WBZ_4 for C-Kit kinase and JNK1
is noteworthy. Predictably, the affinity of
WBZ_4 for Abl1 is reduced by 75% and
by 95% or more on all other Abl variants,
while, in contrast with imatinib, WBZ_4
shows no detectable affinity for Lck. The
impact of WBZ_4 on all additional imat-
inib targets was comparable, while its con-
trolled specificity is apparent.
In vitro assays: assessing the expected modu-
lation of inhibitory impact. The specificity
of WBZ_4 toward C-Kit was established
first through in vitro assays that dissect
the kinetics of competitive inhibition (8,
18) independently of cancer cell circuitry
(19–22). The inhibitory impact of imatinib
(triangles) and WBZ_4 (squares) on the
rate of phosphorylation was determined
by spectrophotometry, assaying for the
activity of C-Kit and Bcr-Abl (Figure 5, blue
and red plots, respectively) (8). The assay
couples production of ADP, the by-prod-
uct of downstream phosphorylation, with
the concurrent detectable oxidation of
NADH. This oxidation results upon trans-
fer of phosphate from phosphoenolpyru-
vate (PEP) to ADP, followed by the NADH-
mediated reduction of PEP to lactate. Thus,
substrate phosphorylation is monitored by
the decrease in absorbance at 340 nm due
to the oxidative conversion of NADH to
NAD+. These kinetic assays revealed a high
specificity of WBZ_4 for C-Kit, in contrast
with imatinib. WBZ_4 enhances the inhibi-
tion of C-Kit activity even beyond imatinib
levels, revealing a higher competitive affin-
ity of the prototype compound for the ATP-

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technical advance

Figure 5
Kinetic inhibitory impact of compounds WBZ_4 and imatinib deter-
mined by measuring phosphorylation rates through spectrophoto-
metric assays of C-Kit and Bcr-Abl kinase activity. The kinases are
inhibited by WBZ_4 (squares) and by the parental compound (trian-
gles). Phosphorylation rate plots are given for Bcr-Abl (red) and C-Kit
(blue). The open red symbols correspond to inhibition of unphosphory-
lated Bcr-Abl, while the filled red symbols correspond to the Tyr412-
phosphorylated form. Error bars represent dispersion over 5 runs for
each kinetic assay.

for imatinib/Abl is approximately 1 μM, while the WBZ_4/Abl value

is greater than 100 μM (Figure 6A). The active recombinant Abl
kinase and its substrate (Abl-tide) were incubated in the presence
of various WBZ_4 or imatinib concentrations and ATP (100 nM).
Phosphorylation of Abl-tide was detected by spectrophotometry
following incubation with anti-rabbit phospho–Abl-tide antibody
binding pocket [KI (imatinib) ≈ 55 ± 7 nM; KI (WBZ_4) ≈ 43 ± 5 nM]. and subsequently with HRP antibody (see Methods).
On the other hand, the pattern of inhibition for Bcr-Abl is depen- The specificity of the inhibitory impact on C-Kit is significantly
dent on the phosphorylation state of this kinase (20). At 1 μM enhanced as WBZ_4 substitutes imatinib (Figure 6, A and B), while
concentration, imatinib reduces 66% of the activity of Tyr412- C-Kit inhibition is 23% ± 12% greater for WBZ_4 (Figure 6B). The
phosphorylated kinase (8) (KI ≈ 5 ± 1 μM) and approximately 100% impact of our prototype ligand on alternative imatinib targets,
of the activity of the unphosphorylated state (KI ≈ 50 ± 5 nM). By such as the PDGFR kinase, could not be modulated, since such
contrast, 1 μM WBZ_4 reduces by less than 20% the activity of both proteins are not reported in PDB, and thus no de-wetting pattern
states of Bcr-Abl. The prototype compound has reciprocal affinity can be reliably identified (compare high-throughput screening of
constants KI ≈ 18 ± 3 μM and KI ≈ 11 ± 2 μM for phosphorylated WBZ_4 versus imatinib in Figure 4).
and unphosphorylated Bcr-Abl, respectively. In vitro assays for selective anticancer activity. The controlled inhibi-
In vitro colorimetric assays were performed over a 1 pM to 100 μM tory impact was further tested through in vitro models. The
range in ligand concentration to assess the inhibition of phosphor- initial decisive tests entailed treating the GIST882 (12) cell line
ylating activity by antibody recognition of phosphorylated peptide and the CML K562 (7) cell line with WBZ_4 and contrasting its
substrates (see Methods). The IC50 (50% inhibition concentration) inhibitory impact on cell proliferation and in-cell specificity with

Figure 6
In vitro comparison of inhibitory impact of imatinib and WBZ_4 on Abl and C-Kit kinase. (A) In vitro phosphorylation inhibition assay for Abl
enzyme in the presence of WBZ_4 (pink) or imatinib (blue). Active recombinant Abl enzyme (1 μg/ml) and its substrate (Abl-tide; 1 μg/ml) were
incubated for 1 hour at 37°C in the presence of various WBZ_4 or imatinib concentrations. ATP (100 nM) was added to the reaction mixture.
Phosphorylation of Abl-tide peptide was detected by incubation in consecutive order with anti-rabbit phospho–Abl-tide antibody and anti-rab-
bit HRP antibody. TMB was added to initiate the chromophore reaction, and 2 minutes were allowed for color development. The reaction was
terminated by the addition of 1 M H2SO4. Phosphorylation of the substrate was quantified as absorbance units (AU) by spectrophotometry at
450 nm. Values obtained with the enzyme without the inhibitors (WBZ_4 or imatinib) were assumed to represent 100% phosphorylation and
were compared with the values obtained with the addition of the inhibitors. (B) In vitro phosphorylation inhibition assay for C-Kit in the presence
of WBZ_4 (pink) or imatinib (blue). Active recombinant C-Kit kinase (25 ng/ml) and its substrate Poly(Glu4-Tyr) (150 nM) were incubated for
1 hour at 37°C in the presence of various WBZ_4 or imatinib concentrations. ATP (100 nM) was added to the reaction mixture. Phosphorylation
of Poly(Glu4-Tyr) peptide was detected by incubation in consecutive order with anti-phosphotyrosine antibody and anti-rabbit HRP antibody.

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 technical advance

Figure 7
Comparison of inhibitory impact of imatinib and WBZ_4 on cell prolif-
eration. (A) Cell proliferation assay for GIST882 cells. WBZ_4 inhib-
its cell proliferation of C-Kit–positive ST882 cells. GIST cancer cells
ST882 (GIST882) were seeded in 96-well plates at a density of 8 × 103
cells per well. The cells were treated with various concentrations of
WBZ_4 (red) and imatinib (light blue) for an additional 48 hours. Cell
proliferation was determined by Alamar blue assay (see Methods). Cell
proliferation is expressed as the percentage of proliferating cells rela-
tive to untreated cells. The WBZ_4 compound was incorporated into
liposomes (see Methods) to facilitate cellular delivery. (B) Cell prolif-
eration assay for K562 cells. WBZ_4 does not significantly inhibit cell
proliferation of Bcr-Abl–expressing K562 cells. K562 cells were seeded
in 96-well plates at a density of 1 × 104 cells per well in 50 μl of medi-
um. Two hours later, 50 μl medium containing different concentrations
(0.01, 0.1, 1 μM) of liposome-encapsulated WBZ_4 (red) or soluble
imatinib (light blue) were added to the wells to reach a final volume of
100 μl per well. Following 48 hours of exposure, the Alamar blue assay
was performed (see Methods). Plates were read at dual wavelength
(570 and 595 nm) in an ELISA plate reader.

those associated with imatinib. Because of its relative insolubil- activity of both compounds (Figure 7A) is likely to arise from com-
ity, WBZ_4 was incorporated into liposomes to promote cellular parable inhibitory impact (~85%; Figure 8A) on C-Kit phosphory-
delivery (see Methods) (21). The proliferation of C-Kit–expressing lation and the fact that both compounds are nanomolar-affinity
GIST882 cells treated with WBZ_4 was markedly inhibited in a inhibitors of C-Kit (Figure 5).
quantitative dose-dependent manner, similar to what occurred Animal models: toward a selective GIST molecular therapy. To assay
in imatinib-treated cells, with maximum impact at 1 μM bulk WBZ_4 for anticancer activity in vivo, an animal model for GIST
concentration (Figure 7A). By contrast, the inhibitory impact of growth based on female C.B-17/IcrHsd-PrkdcSCID mice was devel-
WBZ_4 on CML K562 cells at the same bulk physiological dose oped de novo (see Methods). The model involved the subcutane-
of 1 μM is almost negligible (cell proliferation less than 10% lower ous injection of GIST882 cells (23). The efficacy of WBZ_4 was
than the proliferation of untreated cells), while 1 μM imatinib found to be comparable to that of imatinib, as determined by
promotes a decrease in cell proliferation of approximately 66% the decrease in tumor volume and weight (Figure 9, A and B). No
(Figure 7B). These results demonstrate the higher specificity in obvious toxicities were observed in the animals during treatment,
anticancer activity of WBZ_4. as determined by behavioral changes such as eating habits and
A Western blot assay on treated cancer-derived cell lines (Figure 8, mobility. Furthermore, mouse weights were not significantly dif-
A and B) was performed to determine the in-cell specificity of ferent among the 3 groups of animals, suggesting that eating and
WBZ_4. The immunoblots revealed specificity toward C-Kit con- drinking were not affected. Selectivity of WBZ_4 in the animal
sistent with the selective anticancer activity on the GIST882 cell model using female C.B-17/IcrHsd-PrkdcSCID mice was corrobo-
line that expresses C-Kit. Thus, the activating phosphorylation of rated by assaying on the xenograft induced by CML cells K562
C-Kit at sites Tyr703 and Tyr721 in ST882 cells
is inhibited by WBZ_4 in a dose-sensitive man-
ner as occurred in cells treated with imatinib
(Figure 8A). By contrast, phosphorylation of
Bcr-Abl at Tyr 412 (22) in K562 cells was not
markedly inhibited (<15%) by WBZ_4, while
densitometry revealed an imatinib-induced
inhibition of approximately 85% (Figure 8B).
Since imatinib is a micromolar inhibitor
of phosphorylated Bcr-Abl (Figure 5), the
attack on CML cells at 1 μM bulk concentra-
tion (growth decrease 63%; Figure 7B) must be
attributed to the inhibition of phosphorylated
Bcr-Abl (Figure 8B) combined with the effective
nanomolar inhibition of the unphosphorylat- Figure 8
ed form (Figure 5). By contrast, WBZ_4 hinders Higher specificity of WBZ_4 compared with imatinib as assayed through Western blots.
the phosphorylation of Bcr-Abl only partially (A) Western blot of C-Kit inhibition. WBZ_4 inhibits phosphorylation of C-Kit kinase in
GIST882 cells. Gel bands from the Western blot assays (see Methods) of C-Kit and its
(Figure 8B), and in any case, it is an ineffective
phosphorylated form in GIST cells treated with WBZ_4 and imatinib. The β-actin assay
(micromolar) inhibitor of both forms of Bcr-Abl was adopted as control. (B) Western blot of Bcr-Abl inhibition. Phosphorylation of Bcr-Abl
(Figure 5). Hence, its antitumor activity is pre- kinase is not significantly inhibited by WBZ_4 in CML K562 cells. Electrophoretic gel bands
dictably minimal on CML cells (Figure 7B). In for Western blots for Bcr-Abl kinase and its phosphorylated form in CML cells treated with
the case of GIST cells, comparable antitumor WBZ_4 and imatinib.

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technical advance

Figure 9
Xenograft models of anticancer activity. (A) Effect of WBZ_4 or imatinib therapy on in vivo GIST growth determined by longitudinal tumor volume
measurements. Mice were randomized to treatment with either control (normal PBS and empty liposomes give indistinguishable results within
experimental uncertainty), imatinib, or liposome-formulated WBZ_4. *P < 0.01. (B) Effect of WBZ_4 or imatinib therapy on in vivo GIST growth
determined by weight measurements. Animals from all groups were sacrificed after 6 weeks of therapy, tumors were excised, and the weight was
recorded. *P < 0.05. (C) Effect of WBZ_4 or imatinib therapy on in vivo CML growth induced through a xenograft of K562 tumor cells, determined
by longitudinal tumor volume measurement. These results corroborate in vivo the in vitro finding of WBZ_4 selectivity.

(Figure 9C). While imatinib is shown to significantly impair with cardiomyocyte apoptosis via collapse of the mitochondrial
tumor growth (P < 0.01), the prototype WBZ_4 has virtually no membrane potential and release of cytochrome c (24). We further
effect, in accordance with its engineered specificity and hence lack confirm that imatinib (and WBZ_4) had little effect on p38MAPK
of inhibitory impact on Bcr-Abl kinase. activation at concentrations below 50 μM and actually increased
Cardiotoxicity in mice. As previously noted, studies of imatinib- ERK activation in these cultured cells. In contrast, WBZ_4
induced cardiotoxicity identified a protective JNK (JNK1) inhi- appeared to reduce levels of pJNK1/2 at each dose tested, suggest-
bition as a means to reduce the collapse of mitochondrial mem- ing that upstream activation of JNKs was inhibited by WBZ_4.
brane potential (15). Thus, WBZ_4 was engineered to inhibit Thus, a direct inhibitory effect on JNK activity would predict that
JNK, a feature preliminarily corroborated in vitro (Figure 4). This WBZ_4 should have reduced cardiotoxicity in vivo. This expecta-
protective role of WBZ_4 was also validated in vivo. Western blot- tion is indeed supported by our observations using the surrogate
ting of cell extracts derived from the same imatinib- and WBZ_4- marker brain natriuretic peptide (BNP), a most sensitive indicator
treated cardiomyocytes (see Methods) (Figure 10A) revealed that of myocardial hypertrophy and cardiac impairment (25).
both drugs increased the phosphorylation state of ERK1/2 and The expected curbing of cardiotoxicity in WBZ_4 anticancer
p38MAPK as compared with untreated cultures. In contrast, cells therapy was confirmed by examining the mRNA levels of BNP in
treated with WBZ_4 showed reduced levels of JNK1/2 activation the left ventricle of mice from the same groups assayed for antican-
at each concentration tested, as compared with untreated cells or cer activity. The BNP mRNA levels were about 58% higher in the
cells treated with imatinib. Our results confirm the recent obser- ventricles from imatinib-treated animals (Figure 10B), while no
vations of Kerkelä et al. (15) demonstrating that imatinib induces significant difference was detected in the WBZ_4-treated animals
the ER stress response and activates JNKs in neonatal rat ventricu- when compared with untreated mice. Finally, the reduced cardio-
lar myocytes (NRVMs). JNK activation has been clearly associated toxicity of WBZ_4 was directly tested in mice by determination of

4050 The Journal of Clinical Investigation    http://www.jci.org    Volume 117    Number 12    December 2007
 technical advance

Figure 10
In vivo assays for cardiotoxicity of imatinib and WBZ_4. (A) Western blot of JNK inhibition in cardiomyocytes. Equal amounts of extracted cel-
lular protein (50 μg per lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Western blots were then probed with
primary antibodies specific to the phosphorylated forms of ERKs, JNKs, and p38MAPK. To ensure equal loading, blots were also probed with an
antibody specific to GAPDH. The position of molecular weight standards is indicated to the left of each blot. (B) Effect of WBZ_4 or imatinib
therapy on mouse heart BNP. The mRNA levels of BNP (a sensitive marker of myocardial hypertrophy and cardiac impairment) were examined
in the left ventricle of mice from the groups in Figure 9A. The BNP mRNA levels were about 58% higher in the ventricles from imatinib-treated
animals (*P = 0.02), but no significant difference was noted in the WBZ_4-treated animals. (C) Comparison of left-ventricular EF after 6 weeks
of control (groups treated with either PBS or empty liposomes), imatinib, or WBZ_4 therapy in mice (doses are described in Methods). *P = 0.02
compared with PBS or empty liposomes; †P < 0.01 compared with WBZ_4.

the percentage ejection fraction (EF) in the left ventricle by MRI of kinase; (b) to reduce the inhibitory impact on Bcr-Abl kinase; and
treated mice subjected to imatinib or WBZ_4 therapy (Figure 10C). (c) to promote JNK inhibition in order to reduce cardiotoxicity. The
Following 6 weeks of therapy, the cardiac EF was significantly 3 basic goals have been achieved, as attested by in vitro and in vivo
lower in the imatinib group compared with controls (P = 0.02), results. The imatinib redesign turned our ligand into a selective
which is consistent with previous findings (15). Remarkably, prototherapeutic agent to treat GIST with a prima facie curbing
WBZ_4 treatment had no effect on cardiac EF despite prolonged of cardiotoxicity. A combination of in silico, in vitro, and cellular
therapy (Figure 10C). assays and animal testing validates our rational design strategy to
refocus inhibitory impact based on the novel target discriminator.
Discussion
We have modulated inhibitory impact by rationally reengineer- Methods
ing imatinib motivated by the need to selectively inhibit the C-Kit Calculation of local dehydration propensities. We introduce a descriptor of
kinase and curb imatinib cardiotoxicity. Since originally reported hydration tightness for soluble proteins defined as the mean residence time
(15), this crucial side effect of imatinib has been a subject of some of hydrating molecules within a domain around each residue on the pro-
controversy (26). Our own in vivo and animal model assays corrob- tein surface. The local mean residence time, <τ>i, of hydrating molecules at
orate the original findings and inspired our reengineering strategy. residue i is defined with respect to a spherical domain, D(i), of 6.2-Å radius
The discrepancy with the alternative study presented in ref. 26 is (approximate width of 3 water layers) (17) centered at the α-carbon of resi-
due to the fact that the latter is not a prospective one, as patients due i. The actual computation of residence times is given in Supplemental
did not have baseline functional studies. Data. The mean residence times were obtained from classical trajectories
Our bottom-up approach is centered in a rational ligand redesign generated by molecular dynamics simulations.
sculpting discriminating molecular features of the targets, i.e., their Backbone exposure for protein targets. The extent of backbone exposure at
nonconserved dehydration propensities. Consequently, the rede- a particular residue was determined by counting the number of nonpo-
sign had 3 basic goals: (a) to refocus the primary impact on C-Kit lar side-chain groups contained within a 6.2-Å radius sphere (approxi-

The Journal of Clinical Investigation    http://www.jci.org    Volume 117    Number 12    December 2007 4051
technical advance

mate thickness of 3 water layers) centered at the α-carbon. The extent of Kinetic spectrophotometric assays. To determine the enhancement of speci-
backbone shielding, η, in structured regions averaged over a nonredun- ficity of WBZ_4 relative to imatinib, kinetic assays of the inhibition of the
dant curated PDB database (1,662 proteins) is η = 14.2, with Gaussian Bcr-Abl and C-Kit kinase were conducted. The rate of phosphorylation due
dispersion of 8.2. Thus, a backbone site is regarded as exposed if the to kinase activity in the presence of inhibitors was spectrophotometrically
region is structurally disordered or η < 6. The statistics vary for desolva- assayed (11, 21): the ADP production was coupled to the NADH oxida-
tion radius in the range 6 Å < r < 7 Å, but the tails of the distribution tion and determined by absorbance reduction at 340 nm. Reactions were
identify the same sites of backbone exposure. The structural integrity of carried out at 35°C in 500 μl of buffer (100 mM Tris-HCl, 10 mM MgCl2,
soluble proteins requires that most backbone amides and carbonyls be 0.75 mM ATP, 1 mM phosphoenol pyruvate, 0.33 mM NADH, 95 U/ml
protected from hydration. Thus, residues with absent backbone coordi- pyruvate kinase, pH 7.5). Autophosphorylation of the kinase is slow, requir-
nates in a PDB entry are regarded as exposed and so are residues from ing traces of hematopoietic cell kinase (Hck) to catalyze phosphorylation at
natively disordered proteins. sites Tyr393 and Tyr412. The reactant concentrations are 10 nM (Bcr-Abl),
Molecular dynamics simulations of protein-ligand interactions. The stochas- 12 nM (C-Kit), [ATP]o = 10 mM, and [phosphorylation substrate]o= 0.5 mM,
tic boundary molecular dynamics (SBMD) method (27) was employed where the subscripted “o” signifies “initial.” The adopted peptide sub-
to reduce simulation times while capturing localized interactions at the strates (Invitrogen) for kinase phosphorylation are: EAIYAAPFAKKK
protein active site. The simulation were performed using the CHARMM for Tyr412-phosphorylated Bcr-Abl (19, 20); AEEEIYGEFEAKKKKG for
program (28). Polar-hydrogen potential function (PARAM19) (29) was unphosphorylated Brc-Abl (8, 19, 20); and KVVEEINGNNYVYIDPTQLPY
adopted for the protein, and a modified TIP3P water model (30) was used for Tyr703/Tyr721-phosphorylated C-Kit (31).
for the solvent. Partial charges on the kinase were computed by ab initio In vitro colorimetric assays for kinase inhibition. Phosphorylation assays of
quantum calculation, and the van der Waals parameters of its atoms were antienzymatic activity of imatinib and WBZ_4 against Abl enzyme and
adopted from amino acid side chains subsumed in the CHARMM force C-Kit kinase were performed. The biotinylated substrate peptide [25 μM
field. The system had a reaction zone and a reservoir region, and the reac- Abl-tide for Abl or 150 nM Poly(Glu4-Tyr) for C-Kit] in 100 mM sodium
tion zone was further separated into a reaction region and a buffer region bicarbonate buffer (pH 8.0) was coated onto 96-well plates by incuba-
(28). Further details are provided in Supplemental Data. tion for 1 hour at room temperature. Subsequently, nonspecific binding
Synthesis of prototype compound. The total synthesis of WBZ_4 (N-{5-[4-(4- sites were blocked by incubation with 3% BSA for 1 hour. Phosphoryla-
methyl piperazine methyl)-benzoylamido]-2-methylphenyl}-4-[3-(4-meth- tion of peptide in 96-well plates was initiated by adding 10 ng Abl or
yl)-pyridyl]-2-pyrimidine amine) is described in Supplemental Data. 25 ng C-Kit in assay buffer (20 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM
Liposomal incorporation of WBZ_4. WBZ_4 is under 10% water soluble. To MnCl2, 0.2 mM ATP, 1 mM dithiothretiol, 25 mM β-glycerol phosphate,
promote cellular delivery, it was incorporated into liposomes that serve as 1 mM sodium orthovanadate, 5 mM EGTA). Different concentrations
drug carriers (21). WBZ_4 dissolved in DMSO was added to 1,2-dimyristoyl- of drug inhibitors were added simultaneously to the wells and incubat-
sn-glycero-3-phosphocholine (DMPC) (Avanti) at 1:10 wt/wt drug/lipid ratio ed for 1 hour at 37°C. Anti–phospho–Abl-tide or anti-phosphotyrosine
in the presence of excess tert-butanol. Tween-20 was added to the mixture and anti-rabbit antibodies (dilution 1:1,000) were incubated, in con-
in a 1:19 ratio of Tween-20 to WBZ_4/DMPC. The mixture was vortexed, secutive order, for 1 hour at room temperature. The chromophore reac-
frozen in an acetone/dry ice bath, and lyophilized. Before adding to cell cul- tion was initiated by adding 3,3′,5,5′-tetramethylbenzidene (TMB), and
tures, this lyophilizate was reconstituted with normal 0.9% NaCl solution. absorbance at 450 nm was measured 10 minutes later. Working volume
Cell proliferation assays. GIST cancer cells ST882 (8 × 103 cells per well) was 100 μl (300 μl for BSA). Washing procedure (5 × 300 μl PBS) was
and CML cells K562 (1 × 104 cells per well) were seeded in 96-well plates in performed after each step. Materials included: anti–phospho–Abl-tide
100 μl of RPMI-1640 medium supplemented with 10% FBS and cultured (rabbit, polyclonal; Upstate), anti-phosphotyrosine (rabbit, polyclonal;
for 24 hours. Cells were treated for 48 hours with 0.01, 0.1, and 1 μM/ml Upstate), and anti-rabbit (HRP conjugate; Upstate) antibodies; Abl, Abl-
WBZ_4 and imatinib. Cell proliferation was determined by alamarBlue tide (biotin conjugate; Upstate), c-Kit, Poly(Glu4-Tyr) (biotin conjugate;
assay (BioSource; Invitrogen). Following 48 hours of exposure, 50 μl of Upstate), TMB (Pierce Biotechnology), 96-well plates (streptavidin-coat-
medium was removed from each well and placed into a new 96-well plate. ed) (Nunc), and plate reader (Tecan).
To reach a final volume of 100 μl per well, 40 μl of fresh medium and Animal models. All animal studies were approved by the Institutional Ani-
10 μl of Alamar blue probe were added. Plates were read at dual wavelength mal Care and Utilization Committee, University of Texas MD Anderson
(570 and 595 nm) in an ELISA plate reader (Kinetic Microplate Reader; Cancer Center. Female C.B-17/IcrHsd-PrkdcSCID mice were purchased from
Molecular Devices Corp.). Harlan Sprague Dawley Inc. and housed in facilities approved by and in
Western blots: inhibition of C-Kit phosphorylation in GIST cells. GIST882 cells accordance with the American Association for Assessment and Accredita-
were plated at 2.5 × 105 cells/well in 6-well plates in RPMI-1640 medium tion of Laboratory Animal Care, the United States Department of Agricul-
supplemented with 10% FBS. After allowing the cells to attach for 24 hours, ture, the United States Department of Health and Human Services, and
the cells were treated with 1 μM of WBZ_4 or imatinib for 6 hours. For the NIH. Mice were used according to institutional guidelines when they
Western blotting, 50 μg of total protein was separated on a SDS-PAGE were 8–12 weeks of age. GIST882 cells (23) were harvested from subconflu-
(Bio-Rad) and blotted onto nitrocellulose membranes (Life Science ent cultures by a brief exposure to 0.25% trypsin-EDTA (Invitrogen). Tryp-
Research; Bio-Rad). Antibodies used were: anti–phospho–c-Kit polyclonal sinization was stopped with medium containing 10% FBS. The cells were
antibody, Tyr703 and Tyr721 (Zymed Laboratories Inc.), anti–c-Kit mono- then washed twice in serum-free medium and resuspended in serum-free
clonal antibody (E-1): sc-17806 (Santa Cruz Biotechnology Inc.), and anti– HBSS (Invitrogen). Single-cell suspensions with greater than 95% viability,
β-actin monoclonal antibody (Sigma-Aldrich). as determined by Trypan blue exclusion, were used for the injections. To
Western blots: inhibition of Bcr-Abl phosphorylation in CML cells. K562 cells produce tumors, 6 × 106 GIST882 cells per 100 μl were injected subcutane-
were plated at 5 × 105 cells/well. Two hours later, the cells were treated for ously into the unilateral flank of each SCID mouse. Five mice per group in
6 hours with 1 μM WBZ_4 or imatinib. Antibodies used were: anti–phos- the vehicle and imatinib groups and 7 mice in the WBZ_4 group were used.
pho–c-Abl (Tyr 412) monoclonal antibody 247C7 (Cell Signaling Technol- Once tumors were palpable (11 weeks from injection), mice were started
ogy Inc.) and anti-Abl monoclonal antibody (Sigma-Aldrich). on therapy with daily intraperitoneal injections of normal saline (vehicle),

4052 The Journal of Clinical Investigation    http://www.jci.org    Volume 117    Number 12    December 2007
 technical advance

imatinib (50 mg/kg), or liposomal WBZ_4 (50 mg/kg). Treatment was con- antibody specific to GAPDH. Primary antibody binding was detected with
tinued for 6 weeks, with weekly 2-dimensional measurements of tumor HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibody,
size. All mice were sacrificed when the tumor size approached 1.5 cm in the and visualized by enhanced chemiluminescence.
control group. Tumors were collected, fixed in formalin, and analyzed by MRI and assessment of cardiac function. Assessment of left-ventricular func-
H&E staining. Representative images were taken from each tumor using a tion was performed as previously described (34). In brief, representative
light microscope at ×40 and ×100 magnification. in vivo axial images of the left ventricle in diastole and systole of control
For the K562 model, tumor cells were collected from subconfluent and test mice were acquired in a 4.7T MR scanner cardiac gating using a
suspensions, as described above. To produce tumors, 10 × 106 K562 cells magnetization-prepared spoiled gradient echo sequence. To assess cardiac
per 100 μl were injected subcutaneously into the unilateral flank of each function, short axis cardiac cine images were acquired using a magneti-
SCID mouse. Once tumors were palpable (2 weeks after injection), the zation-prepared, cardiac-gated spoiled gradient echo sequence (TE/TR
mice were then randomized into the following groups (n = 7 per group): 2.1 ms/~23.5 ms; 16 phases covering 1.5 R-R cycles; in-plane resolution
(a) normal saline daily; (b) empty liposomes daily; (c) imatinib (doses 312 μm × 312 μm; 1.25-mm slice thickness). For assessment of EF, regions
listed above); and (d) WBZ_4 (doses listed above). Treatment continued of interest encompassing the cavity of the left ventricle were drawn. For
until mice in any of the groups developed large tumor burden, at which each animal, the average region of interest of 4 central slices in systole was
point all animals were sacrificed. Tumors were measured weekly during divided by the average region of interest of 4 central slices in diastole taken
treatment and at necropsy. at the same location in the left ventricle to derive the EF.
Liposomal WBZ_4 preparation for in vivo testing. In excess tert-butanol Cardiotoxicity/RT-PCR analysis. Total RNA was extracted from mouse
(Sigma-Aldrich), WBZ_4 and DMPC (Avanti Polar Lipids Inc.) were mixed heart samples using the RNeasy kit (QIAGEN) according to the manufac-
in a 1:10 molar concentration. Tween-20 (Sigma-Aldrich) was also added turer’s instructions. The mRNA levels of BNP were examined in the left
to the mixture. Following a quick freeze in a dry ice–acetone water bath, the ventricle of mice from the GIST animal model groups treated exclusively
mixture was lyophilized for 24 hours. The liposomes were stored at –20°C with either WBZ_4 or imatinib. The RT-PCR was performed as described
until ready to use. Immediately before each treatment, the liposomes were previously (34). The primer sequences were as follows: BNP, 5′-AGCTGCT-
reconstituted in normal saline. GGAGCTGATAAGA-3′ (fwd) and 5′-TTACAGCCCAAACGACTGAC-3′
Cardiomyocyte cell culture experiments: reagents. PC-1 tissue culture medi- (rev) (24); β-actin, 5′-ATCTGGCACCACACCTTCTACA-ATGA-3′ (fwd)
um was from BioWhittaker. DMEM was purchased from GIBCO-BRL and 5′-CGTCATACTCCTGCTTGCTGATCCAC-3′ (rev).
(Invitrogen), type II collagenase from Worthington, and penicillin-strepto- Statistical analysis for in vivo tests. Continuous variables were compared with
mycin from Fisher Scientific/MediaTech. Phospho-specific ERK and JNK either the Student’s t test or ANOVA. Prior to analysis, all distributions
polyclonal antibodies were from Promega, and phospho-specific p38MAPK were examined for outliers and non-normality. If appropriate, nonpara-
polyclonal antibody was from Cell Signaling. GAPDH monoclonal anti- metric tests (Mann-Whitney U test) were utilized to compare differences.
body was from Novus Biologicals. SuperSignal West Pico chemilumines- All statistical analyses were performed with SPSS software. P < 0.05 was
cent substrate was obtained from Pierce Biotechnology. All other reagents considered statistically significant.
were from Sigma-Aldrich or Baxter S/P.
Cardiomyocyte cell culture experiments: NRVM isolation. Animals used in Acknowledgments
these experiments were handled in accordance with the Guide for the care The research of A. Fernández was supported by NIH grant R01
and use of laboratory animals (NIH publication no. 85-23. Revised 1985). GM072614 and by a grant from the Gulf Coast Center for Com-
NRVMs were isolated from the hearts of 2-day-old Sprague-Dawley rat putational Cancer Research. J. Ma acknowledges support from
pups via collagenase digestion as previously described (32). Dissociated the Welch Foundation. G. Lopez-Berestein acknowledges support
cells were preplated for 1 hour in serum-free PC-1 medium to selectively from CA. A.K. Sood acknowledges support from the University
remove non-muscle cells. Myocytes were then plated in PC-1 medium at of Texas MD Anderson Cancer Center SPORE in ovarian cancer
a density of 1,600 cells/mm2 onto collagen-coated, 35-mm plastic dishes (P50CA83639) and a Program Project Development Grant from
and left undisturbed in a 5% CO2 incubator for 24 hours. Unattached cells the Ovarian Cancer Research Fund Inc. The GIST882 cells were
were removed by aspiration, and the attached cells were maintained in a a gift from Jonathan Fletcher, Dana-Farber Cancer Institute–
solution of DMEM/Medium 199 (4:1) containing antibiotic/antimycotic Harvard Medical School.
solution for 24 hours. Thereafter, cells were maintained in control medium
or treated with imatinib or WBZ_4 (1–50 μM; 24 hours). Received for publication April 11, 2007, and accepted in revised
Cardiomyocyte cell culture experiments: Western blotting. NRVMs were homog- form September 5, 2007.
enized in lysis buffer (33). Equal amounts of extracted proteins were sepa-
rated on 10% SDS-polyacrylamide gels with 5% stacking gels. Proteins were Address correspondence to: Ariel Fernández, Department of Bio-
transferred to nitrocellulose membranes and the Western blots probed engeneering, Rice University, 6100 Main, MS 142, Houston, Texas
with antibodies specific to the phosphorylated forms of ERKs, JNKs, or 77005, USA. Phone: (713) 348-3681; Fax: (713) 348-3699; E-mail:
p38MAPK. To ensure equal loading, membranes were also probed with an [email protected].
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4054 The Journal of Clinical Investigation    http://www.jci.org    Volume 117    Number 12    December 2007