Redesigning kinase inhibitors to enhance specificity

Article abstract of DOI:10.1021/jm800453a

Kinases are important targets in molecular cancer therapy. However, the evolutionary relatedness and structural conservation of these proteins often lead to unforeseen cross reactivity, yielding unexpected side effects. Thus, the use of promiscuous drugs is likely to introduce dangerous clinical uncertainties. Here, we show how to rationally redesign two promiscuous kinase inhibitors, staurosporine (7) and EKB-569 (8), with the goal of turning them into more selective ligands. This problem is addressed by exploiting a structure-based selectivity filter for specificity: the pattern of packing defects in the target. These singularities, called dehydrons, are solvent-exposed intramolecular hydrogen bonds that may be protected by drugs upon association and are not conserved across protein families. Our redesigned compounds possess a significantly focused activity, as experimentally corroborated in high-throughput screening assays. Thus, our design strategy proves to be operative to reduce the inhibitory impact of promiscuous kinase ligands, enhancing their safety as therapeutic agents.

Full text of DOI:10.1021/jm800453a

4890
J. Med. Chem. 2008, 51, 4890–4898
Redesigning Kinase Inhibitors to Enhance Specificity
Alejandro Crespo,† Xi Zhang,‡ and Ariel Fernández*,†
Department of Bioengineering, Rice UniVersity, Houston, Texas 77005, Program in Applied Physics, Rice Quantum Institute, Rice UniVersity,
Houston, Texas 77005
ReceiVed April 21, 2008
Kinases are important targets in molecular cancer therapy. However, the evolutionary relatedness and structural
conservation of these proteins often lead to unforeseen cross reactivity, yielding unexpected side effects.
Thus, the use of promiscuous drugs is likely to introduce dangerous clinical uncertainties. Here, we show
how to rationally redesign two promiscuous kinase inhibitors, staurosporine (7) and EKB-569 (8), with the
goal of turning them into more selective ligands. This problem is addressed by exploiting a structure-based
selectivity filter for specificity: the pattern of packing defects in the target. These singularities, called
dehydrons, are solvent-exposed intramolecular hydrogen bonds that may be protected by drugs upon
association and are not conserved across protein families. Our redesigned compounds possess a significantly
focused activity, as experimentally corroborated in high-throughput screening assays. Thus, our design strategy
proves to be operative to reduce the inhibitory impact of promiscuous kinase ligands, enhancing their safety
as therapeutic agents.
Introduction
defects consist of solvent-exposed intramolecular backbone
hydrogen bonds and constitute vulnerabilities arising from
imperfections in side chain packing. These structural singulari-
ties, called dehydrons,25 are targetable-sticky spots because they
promote their own further dehydration as a means to strengthen
and stabilize the underlying amide-carbonyl electrostatic
interaction.23–27 Dehydrons have been turned into an operational
selectivity filter for two reasons: (i) they may be targeted by
drugs that further wrap them (protect from water attack) by
bringing nonpolar groups to their proximity upon association19,28,29
and (ii) they are not conserved across paralogues.6,30 In this
work, we report on a rational redesign of promiscuous inhibitors
to exogenously wrap nonconserved dehydrons with the goal of
enhancing their target-discriminatory power.
The use of small-molecule inhibitors of protein function is
one of the most efficient ways to treat human disease and
malignancy.1–5 In this regard, protein kinases, the essential signal
transducers, have become important targets in molecular cancer
therapy.1–5 However, most protein targets of therapeutic interest
have surviving paralogues, i.e., proteins that share a common
ancestor with the target and have diverged after speciation.6 In
particular, kinases are lumped up into families, which typically
share a very similar fold and specific structural features.6,7 This
structural conservation is often responsible of unexpected cross
reactivities,8,9 yielding uncertain or even life-threatening side
effects.10–12
Although there is no clear correlation between anticancer
activity and specificity, promiscuous inhibitors are obviously
more prone to yield side effects than selective drugs. Even the
most successful anticancer drug imatinib (STI571, 1),3,13 with
an activity profile limited to 5 primary kinases (Abl, C-Kit, Lck,
PDGFR,andCSF1R),8,9 hasshowntobepotentiallycardiotoxic.11,12
Moreover, the more promiscuous anticancer kinase inhibitors8,9
sunitinib (SU11248, 2)14 and sorafenib (Bay 43-9006, 3)15 have
also been found to be cardiotoxic, and to an even larger extent
than imatinib.12 The other commercial kinase inhibitors (Scheme
1) dasatinib (BMS-354825, 4),16 erlotinib (OSI-774, 5),17 and
gefitinib (ZD1839, 6)18 have also a broad activity profile.8,9
The therapeutic use of promiscuous inhibitors may be
potentially hazardous unless a rational strategy to control their
specificity is adopted. Such control may be achieved if we can
identify selectivity filters in the target, i.e., structural features
that are unique to the target, and chemical modifications to the
drug that promote interactions with such unique features. Thus,
much of the cross reactivity may be removed by redesign guided
by the identification of structural features that promote promis-
cuity and selectivity filters that enable target discrimination.19–22
A selectivity filter of broad applicability has been recently
identified: the packing defects of soluble proteins.23–27 These
In principle, most kinase inhibitors can be turned into selective
wrappers of dehydrons through minimal chemical modification
that preserve the generic chemotype. Thus, relevant kinase
inhibitors with considerable cross reactivities such as stauro-
sporine (7)31 (inhibiting 87% out of the 290 kinases screened
with KD < 3 µM),9 sunitinib (57%), dasatinib (28%), EKB-
569 (8)32 (18%), sorafenib (18%), erlotinib (15%), gefitinib
(7%), or imatinib (6%) may in principle be turned into drugs
with enhanced specificity through wrapping redesign. The
generic strategy consists of modifying the parental compound
to turn it into a wrapper of unique dehydrons while also
removing potential sources of cross reactivity. These arise
because ligand groups are often engaged in interactions with
groups on the backbone or on side chains that are invariant
across the target family.
To test the target-discriminatory power of wrapping redesign,
we focus in this work on a major challenge arising thereof: the
re-engineering of promiscuous inhibitors. First, we report on
the redesign of staurosporine (Scheme 2), the most promiscuous
kinase inhibitor known,9 to elicit an inhibitory impact with
enhanced specificity. Our redesign introduces a single wrapping
modification to target one of the least conserved kinase
dehydrons. The resulting ligand 9 (Scheme 2) possesses a
significantly more focused impact than the parental compound,
as corroborated in high-throughput screening assays. We also
report on the successful cleaning of the “dirty” inhibitor 8
* To whom correspondence should be addressed. Phone: (713)348-3681.
Fax: (713)348-3699. E-mail: arifer@rice.edu.
†
Department of Bioengineering, Rice University.
Program in Applied Physics, Rice Quantum Institute, Rice University.
‡
10.1021/jm800453a CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

Redesigning Kinase Inhibitors to Enhance Specificity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4891
Scheme 1. Chemical Structures of Commercial Anticancer Drugs
Scheme 2. Chemical Structures of Compounds 7 and 9
Such levels of cross reactivity make it impossible to envision
staurosporine as a therapeutic agent. Thus, despite its inhibitory
potency, staurosporine is solely regarded as a research
compound.8,9 Staurosporine is a natural competitive inhibitor
that binds to the ATP pocket of almost all kinases in the active
conformation (the activation loop is fully extended and exposed
to solvent).33–35 To illustrate its binding mode, the crystal
structure of the EGFR kinase in complex with staurosporine is
shown in Figure 1 (PDB 2ITW). Staurosporine has a larger
solvent-accessible surface area than ATP (360 Å2 vs 323 Å2,
respectively),36 and it is a more rigid molecule, hence reducing
the entropic penalty upon association. The high promiscuity of
this inhibitor is due to the numerous contacts it makes (it is a
Scheme 3. Chemical Structures of Compounds 8 and 10
(Scheme 3). This is carried out by removing the promiscuity-
promoting elements in the compound while appending a
nonpolar group that wraps a unique dehydron of the primary
target of 8, the epidermal growth factor receptor (EGFR) kinase.
The experimental high-throughput screening assay of the
wrapping compound 10 (Scheme 3) confirms its considerably
focused activity profile.
The solution to the challenging design problems presented
in this work singles out the wrapping redesign as a paradigm
shifter in the engineering of highly selective inhibitors.
Results
Figure 1. Ribbon representation of EGFR kinase (PDB 2ITW, blue)
in complex with staurosporine. Relevant structural features that frame
the ATP-pocket (white circle) are depicted for clarity: nucleotide-
binding loop (red), P-loop (orange), RC-helix (yellow), catalytic loop
(green), and activation loop (pink).
Staurosporine Redesign. Staurosporine is the most cross-
reactive kinase inhibitor known to date.8,9 It binds tightly (KD
< 3 µM) to ∼90% of the 119 (or 290 in the latest assay) human
kinases screened through phage-display ATP-competitive assays.8,9

4892 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Crespo et al.
Figure 3. Selected aligned backbones (ribbon representation) of EGFR
(PDB 2ITW, blue), PDPK1 (PDB 1OKY, red), and CDK2 (PDB 1AQ1,
yellow) kinases complexed with compound 9. The R776-Q791 dehy-
dron in EGFR (green virtual bond joining R-carbons) maps into the
well-wrapped backbone hydrogen bonds (gray virtual bonds joining
R-carbons) K144-E160 in PDPK1 and K65-E81 in CDK2. The methyl
group at the pyrrol N6-position (indicated by the green box) turns the
ligand into a wrapper of the nonconserved dehydron.
Figure 2. Induced-fit conformation of the ATP-binding pocket of
EGFR kinase (PDB 2ITW, blue) generated when crystallized with
staurosporine. The ligand forms two hydrogen bonds with the conserved
backbone atoms of residues in the nucleotide-binding loop (Q791 and
M793) and hydrophobic interactions with residues framing the pocket
(gatekeeper: T790). EGFR kinase has seven dehydrons (green virtual
bond joining R-carbons) within its binding pocket: G721-F723 and
G721-G724 in the P-loop, R776-Q791, M793-G796, P794-G796, and
G796-V845 in the nucleotide-binding loop, and D855-G857 in the
activation loop.
can significantly restrict its inhibitory impact to the 8 kinases
that share the dehydron at the aligned position (Table 1). The
bacteriophage high-throughput screening of 9 is shown in Figure
4: only 26 kinases representing 12% of the 220 kinases assayed
haVe significant affinity for the ligand. This percentage hit
signals a massiVe enhancement in selectiVity when compared
with the 88% of the parental compound. Most significantly, our
structure-based prediction of affinity based on the presence or
absence of the dehydron at the aligned position includes five
hits (ABL1, EGFR, LCK, PTK2, and SRC), only one false
positive (MAP3K5) and not a single false negative over the 18
instances (Table 1) where packing prediction can be contrasted
with experiment. These results reveal ∼94% of accuracy in the
prediction. The other hits of 9 are: AAK1, ABL1 (H396P),
ABL1(T315I),ABL1(Y253F),CAMK2A,CAMK2B,CAMK2D,
CAMK2G, CAMKK1, CAMKK2, CSF1R, FLT3, KIT, KIT
(D816V), LOK, MARK2, PAK6, PDGFRA, PDGFRB, PTK2B,
and SLK. For these cases, we either do not have a crystal
structure of the protein or the protein is crystallized in complex
with a ligand other than staurosporine and hence with a different
induced fit. However, all these hits correspond to staurosporine
targets with high-binding affinity (nM or sub-nM range).9 In
cases of PDB-reported structure with a ligand other than
staurosporine, wrapping predictions can be made but without
the same confidence. For example, the active KIT structure
complexed with ADP (PDB 1PKG) has a dehydron at the
aligned position. Thus, all close KIT paralogues (CSF1R, FLT3,
PDGFRA, PDGFRB with sequence identity >60%) will prob-
ably be targeted by 9, as corroborated in the screening (Figure
4). Furthermore, the LOK kinase (PDB 2J7T) has a dehydron
in such position and represents a hit. However, PAK6 (PDB
2C30) and SLK (PDB 2J51) have a well-wrapped hydrogen
bond, and 9 still binds. Therefore, affinity predictions based on
structures crystallized with ligands other than staurosporine are
less reliable. However, the accuracy of our prediction is still
high (46 out of 56 cases or ∼82%) if we further extend our
structure-based analysis to include the set of 53 kinases with
reported PDB structure that were recently screened (Methods)8
(Table S1, Supporting Information). Nevertheless, the higher
selectivity of 9 is significant, showing high in vitro activity
large and rigid nonpolar molecule) with conserved polar and
hydrophobic groups of protein kinases.33–35 For example, it
makes strong van der Waals interactions with residues framing
the ATP pocket such as L718, G719, F723, V726, A743, K745,
T790, L792, M793, G796, S797, and L844. Staurosporine also
forms two hydrogen bonds with the conserved backbone atoms
of residues in the nucleotide-binding loop: Q791:O-Pyrrol:N6
and M793:N-Pyrrol:O5 (Figure 2). These residues are also
involved in intermolecular hydrogen bonds with ATP. These
extensive interactions with conserved regions make the re-
engineering of staurosporine a challenging problem.
To redesign staurosporine through a wrapping modification,
we compare all 37 PDB structures of kinases in complex with
ligands that share the same indolo[2,3-a]pyrrolo[3,4-c]-carbazole
chemotype (Methods). The EGFR-staurosporine complex has
seven dehydrons within the ATP pocket: G721-F723 and G721-
G724 in the P-loop, R776-Q791, M793-G796, P794-G796, and
G796-V845 in the nucleotide-binding loop, and D855-G857 in
the activation loop (Figure 2). From these seven, dehydrons
R776-Q791, M793-G796, and D855-G857 are potentially more
accessible to a wrapping modification of staurosporine (at least
one R-carbon of the residues paired by the dehydron is within
7 Å from any atom of the ligand). A comparative wrapping
analysis of the 37 aligned structures reveals that dehydron R776-
Q791 is the least conserved and more easily accessible to a
wrapping modification (generally methylation) in this structural
assortment. This dehydron is conserved in 8 out of the 37 kinase
structures, ABL1, EGFR, GSK3ꢀ, LCK, MAP3K5, MAP3K17,
PTK2, and SRC (Figure 3, Table 1), whereas dehydrons M793-
G796 and D855-G857 are conserved in 9 and 12 structures,
respectively. Thus, dehydron R776-Q791 may be targeted by
wrapping it through a specific methylation of staurosporine
(compound 9) at the imide N6-position of the pyrrol ring (Figure
3, Methods).31,33,37 This modification is mostly useful because
will also remove one conserved hydrogen-bond interaction,
increasing specificity. Thus, by redesigning staurosporine to turn
it into a wrapper of the R776-Q791 dehydron in EGFR, we

Redesigning Kinase Inhibitors to Enhance Specificity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4893
Table 1. Wrapping Comparison of the 37 PDB-Reported Kinases in
Complex with Staurosporine or with Ligands that Share the Same
Chemotype at Position Aligned with EGFR Dehydron R776-Q791 (DH
) Dehydron, HB ) Well-Wrapped Hydrogen Bond)a
may be involved in hydrogen bonds with backbone atoms of
residues in the P-loop or in the nucleotide-binding loop. As
shown in Figure 5, one important source of promiscuity of
compound 8 is its terminal acryl group, which plays the role of
electrophile in the irreversible Michael reaction with the
nucleophile-conserved residues Cys or Ser in the nucleotide-
binding loop of EGFR paralogues. The water-solublizing
terminal N-dimethyl group may also accelerate such addition,
serving as an intramolecular base catalyst for Michael reaction
with the Cys or Ser residues due to the spatial proximity.32
Another source of promiscuity of compound 8 is the intermo-
lecular electrostatic interaction between its cyanide group and
the gatekeeper residue (Thr or Met), typically conserved within
the family (Figure 5).
wrapping
PDB classification
hit in
screening
match prediction
experiment
kinase
ABL1
CDK2
CHK1
DAPK1
EGFR (WT)
EGFR (G719S) 2ITQ
EGFR (L858R) 2ITU
FYN
2HZ4
1AQ1
1NVR
1WVY
2ITW
DH
HB
HB
HB
DH
HB
HB
HB
DH
HIT
NO HIT
YES
YES
not screened
not screened
HIT
not screened
not screened
NO HIT
YES
YES
2DQ7
1Q3D
2NRY
2OIC
1SM2
1SNU
1YVJ
1QPD
1QPJ
2CLQ
2GCD
1NXK
2PZY
1R0P
2HW7
2HY8
1OKY
1OKZ
1E8Z
1YHS
2IWI
GSK3ꢀ
not screened
IRAK4
HB
HB
not screened
To validate whether such interactions are responsible for the
promiscuity of compound 8, we establish a correlation between
the affinities of 8 for 53 paralogues of EGFR reported in the
PDB (Methods) and the extent of residue conservation at the
Michael reaction site and at the gatekeeper position. To do so,
we align each paralogue structure with the EGFR structure (PDB
1M17) and examine residues that align with C797 (Michael
reactant) and T790 (gatekeeper) (Methods).43,44 A statistical
model is built to assess such correlation (Methods),45,46 revealing
that the affinity profile of 8 is indeed dictated by these two
sources of promiscuity (P-value ) 0.007). Thus, the terminal
acryl group and the cyanide group of 8 (Figure 5) are the “dirty”
moieties responsible for its promiscuity.
ITK
NO HIT
not screened
HIT
YES
JAK3
LCK
HB
DH
HB
DH
DH
YES
NO
MAP3K5
MAP3K17
NO HIT
not screened
MAPKAPK2
HB
not screened
MET
MKNK2
PAK1
NO HB
HB
HB
NO HIT
NO HIT
NO HIT
YES
YES
YES
PDPK1
HB
NO HIT
YES
PIK3CG
PIM1
PIM2
PKAC-R
PRKCQ
HB
HB
HB
HB
HB
HB
not screened
NO HIT
NO HIT
NO HIT
not screened
YES
YES
YES
We thus remove the sources of promiscuity by introducing
the two following chemical modifications (Figure 6): (i) Replace
the double bond (the Michael acceptor) in the acryl group with
a single bond. (ii) Replace the cyanide group with a methyl to
retain the chemotype while removing the electrostatic interaction
with the gatekeeper.
1STC
1XJD
2J0J
PTK2
2J0K
DH
DH
DH
NO HB
HB
HIT
YES
YES
2J0M
1BYG
2BUJ
1XBC
1U59
SRC
STK16
SYK
HIT
To promote selectivity, we further introduce a wrapping
modification in the drug to target a nonconserved dehydron in
the intended target. When EGFR is crystallized in the induced-
fit conformation generated by an inhibitor (erlotinib) that shares
8′s 4-anilinoquinoline chemotype (PDB 1M17), we now find
only six dehydrons within the binding pocket (Methods): G721-
F723 and G721-G724 in the P-loop, M793-G796, P794-G796,
and G796-V845 in the nucleotide-binding loop, and D855-G857
in the activation loop. From these six, dehydrons M793-G796
and D855-G857 are potentially more accessible to a wrapping
modification of the drug. By examining these two dehydrons
across the 53 EGFR-paralogues, we find that the least conserved
and more accessible to a wrapping modification is dehydron
D855-G857 (Figure 6). Only 12 paralogues retain this dehydron
(Table 2): AURKA, CLK3, EGFR, EPHA3, ERBB2, FYN,
LCK, PAK6, PAK7/PAK5, PIM2, SLK, and STK10, whereas
dehydron M793-G796 is conserved in 17 kinases. Thus, we
choose dehydron D855-G857 as the nonconserved selectivity
feature to be targeted. To do so, we append a methyl group at
position 3 of the terminal benzene ring of 8 that becomes a
wrapper or protector of such feature (Figure 6). The synthesis
of the redesigned compound 10 follows a pathway that
recapitulates the synthesis of the parental compound 8 (Meth-
ods).32 Compound 10 buries a solvent-accessible surface area
similar to 8′s (393 Å2 vs 397 Å2, respectively),36 showing a
similar size and binding orientation and similar entropic penalty
upon association. However, 10 not only lacks 8′s sources of
promiscuity but also introduces a wrapping modification to
promote selectivity.
not screened
NO HIT
NO HIT
YES
YES
ZAP70
HB
a Compound 9 is predicted to bind only to kinases that have a dehydron
at the aligned position, and the prediction is contrasted with its experimental
screening.
toward selected therapeutically relevant targets such as KIT (for
treatment of gastrointestinal stromal tumors),38 PTK2 (involved
in the metastasis of ovarian carcinoma),39 SRC (implicated in
the metastatic/invasive phenotype),40 or EGFR (for treatment
of nonsmall cell lung cancer).41,42 Thus, the wrapping modifica-
tion of staurosporine may potentially be turned into a realistic
clinical opportunity.
Cleaning Inhibitor 8. The irreversible kinase inhibitor 8
developed by Wyeth-Ayerst41 was launched as a major inhibitor
of the EGFR kinase (IC50 ) 38.5 nM). Thus, its therapeutic
interest to treat nonsmall cell lung cancer (NSCLC), colorectal
neoplasia, and other EGFR-dependent solid tumors became
apparent.41,42 Phase I and II trials for such therapeutic applica-
tions are currently in progress and closed to new patients.42
Recent high-throughput screening assays8,9 revealed ∼50 targets
(KD < 3 µM) for 8, making it a promiscuous drug with likely
side effects. Other anti-NSCLC agents such as gefitinib or
erlotinib share the same 4-anilinoquinoline chemotype,32 yet
they are more specific EGFR inhibitors.8,9
The promiscuity of 8 can be traced to its intermolecular
interactions with highly conserved residues within the EGFR
kinase family. Compound 8 has a large solvent-accessible
surface area (397 Å2)36 that may increase the nonspecific van
der Waals interactions with residues framing the ATP pocket.
Moreover, its polar amide group that increases drug solubility
Our structure-based affinity profile prediction for 10 is based
on the conservation of the EGFR D855-G857 dehydron wrapped

4894 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Crespo et al.
Figure 4. Affinity profile of compound 9. High-throughput screening at 10 µM of 9 (red) over a battery of 220 human kinases displayed in a
T7-bacteriophage-expressing library (Ambit Bioscience, San Diego, CA). The screening assay of staurosporine (blue) was used as control.8,9 Hit
values are reported as percentage bound kinase.
Figure 5. Structural alignment of two targets of 8: the EGFR kinase
Figure 6. EGFR kinase structure (same representation as Figure 5)
(PDB 1M17, blue ribbon representation, atoms in balls and sticks) and
complexed with compound 10 (licorice representation). To remove
the paralogue PAK1 kinase (PDB 1YHV, red ribbon representation,
promiscuity, the acrylic double bond (Michael electrophile) and the
atoms licorice), complexed with the drug (licorice). Atoms are depicted
gatekeeper-interacting cyanide group of 8 are replaced by a single bond
following standard color convention (chlorine in green, fluorine in light
and a methyl group, respectively. To selectively target EGFR, another
green). The sources of promiscuity of compound 8 are the terminal
methyl group is added to the terminal benzene ring as a wrapper of
acryl group (electrophile group in the Michael reaction) and its cyanide
the least conserved D855-G857 dehydron (green virtual bond joining
group (involved in intermolecular electrostatic interaction with a Thr
R-carbons).
or Met gatekeeper). EGFR has a poorly conserved D855-G857 dehydron
for those kinases that originally do not bind to 8 because they
are likely to also clash sterically with 10. This is expected
because the binding to 8 implies that no steric hindrance occurs
(the reciprocal does not hold). We thus predict as “hits” only
those kinases that introduce no steric hindrance and possess a
dehydron in the position that aligns with EGFR D855-G857.
Only 8 hits are thus predicted (Table 2): CLK3, EGFR, EPHA3,
ERBB2, FYN, LCK, SLK, and STK10. In cases where the
residues aligning with EGFR D855-G857 are not engaged in a
(green virtual bond joining R-carbons) that may be targeted to achieve
selectivity. PAK1 contains the same two promiscuity-fostering features,
while it has a well-wrapped hydrogen bond aligned at such position
(gray virtual bond joining R-carbons). Targeting such dehydron will
ensure a discriminatory binding of EGFR without hitting PAK1, as
experimentally corroborated.
by 10 (but not by 8) and the existence of steric hindrances with
the targets. In cases where there is a dehydron aligned with the
EGFR D855-G857 dehydron, we predict steric hindrance only

Redesigning Kinase Inhibitors to Enhance Specificity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4895
Scheme 4. Synthetic Pathway of Compound 10a
promote promiscuity. Subsequently, we introduce a chemical
modification that wraps a relatively unique dehydron in the
intended kinase target. This rational redesign makes compound
10 more selective than the parental compound 8: out of the 220
kinases experimentally screened, compound 10 binds strongly
(sub-µM affinity or % of inhibition >10) to 5 kinases, whereas
compound 8 binds to 19 (Figure 7).
Conclusions
This work describes and validates a generic strategy in
molecular design aimed at turning highly cross-reactive kinase
inhibitors into significantly more selective drugs. To demonstrate
the power of this approach, we selected the most challenging
design problems, represented by the cleaning of the highly
promiscuous ligands staurosporine and inhibitor 8. The redesign
of these compounds was guided by a selectivity filter: the pattern
of packing defects, the so-called dehydrons, in the drug target
that are not conserved across paralogues.25,30 Thus, relatively
unique dehydrons have been targeted by the redesigned com-
pounds to further protect them from water attack upon associa-
tion. In this way, we have significantly enhanced specificity in
a highly controllable manner and even for the most promiscuous
kinase inhibitors. Thus, a generic strategy may involve modify-
ing the parental compound to turn it into a wrapper of poorly
conserved dehydrons while removing potential sources of ligand
promiscuity. This strategy may be successfully applied to other
cross-reactive kinase inhibitors such as sunitinib and sorafenib,
documented to entail a risk of side effects.12 The structure-based
affinity predictions for our wrapping prototypes were bench-
marked against experimental screening,8,9 revealing over 90%
accuracy. The reliability of wrapping predictions is contingent
on the availability of target structures. The latter are typically
reported for induced-fit conformations arising in drug/target
complexes. Hence, the wrapping prediction requires that the
PDB-reported complexes share the same ligand chemotype as
the parental compound to ensure induced-fit similarity. Particular
attention to induced-fit diversity is needed in drug design,
especially in wrapping design, due to the conformational
plasticity of kinases. Thus, wrapping design will undoubtedly
benefit from an ever-increasing amount of reported data on
structural adaptation of targets to diverse ligands as well as high-
throughput screening of drug variants, enabling a dissection of
specificity-promoting features. The efficacious solution to the
problem of cleaning promiscuous drugs reported here highlights
the value of the wrapping redesign as a paradigm shifter in the
engineering of drug selectivity.
a (a) Toluene, reflux. (b) Dowtherm, 256 °C. (c) POCl3, reflux. (d) Fe,
NH4Cl, CH3OH, reflux. (e) N,N-diisopropylethylamine, THF, 0 °C. See
Supporting Information for details.
dehydron or in a well-wrapped hydrogen bond, we further
examine whether such a dehydron can be induced upon ligand
binding with a minimal structural adaptation. Four kinases
(ABL1 (H396P), BTK, PTK2, and SYK, out of 6 cases: ABL1
(H396P), BTK, FLT3, PTK2, STK16, and SYK) can induce
this dehydron upon drug binding with no steric hindrance,
representing “possible” hits (Table 2). The experimentally
obtained affinity profile (Table 2, Figure 7) for compound 10
agrees Very well with our predicted profile: it actually binds 6
(CLK3, EGFR, ERBB2, LCK, SLK, and STK10) out of the 8
hits inferred with certainty and two of the possible hits (BLK
and PTK2). It has only 3 false positives (EPHA3, FYN, and
the other possible hit SYK) and not a single false negative (Table
2) for the 50 cases where our prediction can be contrasted with
experiment. These results show ∼94% of accuracy in the
prediction.
Methods
Target Identification. To redesign staurosporine performing a
comparative analysis of nonconserved selectivity features, we
collected all 37 PDB-reported kinase structures complexed with
staurosporine or with ligands that share the same indolo[2,3-
a]pyrrolo[3,4-c]-carbazole chemotype.37 We only selected kinase
structures in complex with such ligands, to avoid a less reliable
identification of dehydrons, arising from different induced fits
generated in presence of other drug/inhibitors.
A similar structural analysis was performed for cleaning 8.
Because its primary intended target is the EGFR kinase, we adopted
a PDB-reported structure of EGFR in complex with an inhibitor
(erlotinib) that shares the same 4-anilinoquinoline chemotype (PDB
1M17).32 A comparative analysis to identify potential sources of
cross reactivity and nonconserved specificity-promoting features
was performed for the 53 EGFR-paralogues with reported PDB
structure that were recently screening using a battery of 119 T7-
phage expressed kinases.8
These results reveal that the wrapping redesign introduces a
significant increase in the selectivity of the “dirty” inhibitor 8.
This is accomplished by first removing the drug features that

4896 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Crespo et al.
Table 2. Wrapping Comparison for the Set of 53 Paralogue Kinases of EGFR with Reported PDB Structurea
wrapping
classification
steric
hindrance
predicted
affinity
experimental
affinity
match prediction
experiment
kinase
ABL1
ABL1 (T315I)
ABL1 (H396P)
AURKA
BTK
CAMK1D
CAMK1G
CDK2
PDB
2GQG
2V7A
2F4J
1MQ4
1K2P
2JC6
2JAM
1AQ1
1UNG
1Z57
2EU9
2CMW
2C47
NO HIT
NO HIT
possible HIT
NO HIT
possible HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
NO HIT
not screened
not screened
NO HIT
HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
NO HIT
NO HIT
HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
YES
possibly induced
DH
possibly induced
NO
YES
NO
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
NO
CDK5
CLK1
CLK3
DH
DH
NO
NO
CNSK1G1
CNSK1G2
DAPK2
DAPK3
EGFR
EPHA2
EPHA3
ERBB2
FGFR1
FGFR2
FLT3
FYN
HCK
INSR
JAK2
JNK1
JNK3
KIT
LCK
2A2A
2J90
1M17
1MQB
2QO9
1OVC
1AGW
1GJO
1RJB
2DQ7
1QCF
1GAG
2B7A
1UKH
1PMN
1PKG
1QPC
2CLQ
2AC3
2JAV
1DI9
NO HIT
HIT
HIT
DH
DH
NO
NO
YES
YES
YES
YES
NO
NO HIT
NO HIT
NO HIT
HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
possibly induced
DH
YES
NO
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
YES
DH
NO
MAP3K5
MKNK2
NEK2
P38-R
P38-γ
1CM8
1YHV
2CDZ
2C30
PAK1
PAK4
PAK6
PAK7/PAK5
PDGFRB
PIM1
PIM2
PKAC-R
PTK2
RPS6KA5
SLK
SRC
STK10
STK16
SYK
TIE2
TNK2
VEGFR2
DH
DH
YES
YES
2F57
1LWP
1YXT
2IWI
2GU8
2ETM
1VZO
2J51
DH
YES
NO
possibly induced
DH
NO HIT
HIT
NO HIT
HIT
NO HIT
possible HIT
NO HIT
NO HIT
NO HIT
NO HIT
HIT
NO HIT
HIT
not screened
NO HIT
NO HIT
NO HIT
NO HIT
NO
2SRC
2J7T
DH
NO
YES
NO
2BUJ
1XBB
1FVR
1U46
2P2H
possibly induced
possibly induced
NO
YES
YES
YES
a Compound 10 is predicted to bind only to kinases with a dehydron or possibly induced dehydron in positions aligning with EGFR D855-G857 dehydron
and with no steric hindrance. The prediction is contrasted with its experimental screening.
Moreover, the comparative wrapping analysis for staurosporine
redesign was further extended to include this set of 53 paralogue
kinases with reported PDB structure (Supporting Information).
For such comparative analysis, we have performed a structural
alignment of all kinases in each set of targets. The alignment was
performed using the DaliLite web-based program for pairwise
structure comparison.43,44 Structure conservation across targets
within kinase families enables such alignment.6
Statistical Analysis. To verify that the sources of promiscuity
of compound 8 are the extent of residue conservation at both the
Michael reaction site and at the gatekeeper position, we performed
a statistical analysis by building a logistic regression model.45,46
Thus, we established a correlation between the affinities of 8 for
the set of 53 EGFR-paralogues reported in PDB and the extent of
residue conservation at such positions. The two “explanatory
variables” are the types of residues aligning with C797 (Michael
nucleophile) and T790 (gatekeeper). Thus, X1 ) 1, if the residue
aligning with C797 is Cys or Ser (possible Michael nucleophiles),
and X2 ) 1, if the residue aligning with T790 is Thr or Met (possible
intermolecular electrostatic interaction with the cyanide group). The
“responding variable” is the affinity of 8 toward the 53 EGFR-
paralogues (Y ) 1, if KD < 3 µM).8 The null hypothesis is the
assumption that there is no correlation between the responding
variable and the explanatory variables.45,46
Dehydron Identification. Dehydrons may be readily identified
from atomic coordinates of proteins with reported structure. Thus,
we first identify all intramolecular backbone hydrogen bonds within
the structure as bonds whose N-O distances are <3.5 Å and
N-H-O angles are >110°. For each hydrogen bond identified,
we then calculate its extent of wrapping, F, by quantifying the
number of the side chain carbonaceous nonpolar groups contained
within a “dehydration domain” around such bond. This domain is

Redesigning Kinase Inhibitors to Enhance Specificity
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4897
methyl group, as previously reported.33 This modification was
achieved by following the short pathway based on intramolecular
Diels-Alder reaction of pyrano[4,3-b]indol-3-one, described in ref
37, page 4399, replacing the first step by treatment of commercial
2-nitrocinnamaldehyde with 1,2-dimethyl hydrazine (for pyrrol N6
methylation) instead of hydrazine. The synthesis and spectroscopic
characterization of 9 is provided as Supporting Information.
4-Dimethylamino-butanoic-acid-[4-(5-chloro-4-fluoro-3-meth-
ylphenylamino)-3-methyl-7-methoxy-quinoline-6-yl]-amide (10).
The synthesis of 10 entails several chemical modifications of the
parental compound 8:32 replacing the double bond (Michael
acceptor) in the acryl group with a single bond, replacing the
cyanide group with a methyl, and appending a methyl group at
position 3 of the terminal benzene ring. Thus, 10 was synthesized
by following a pathway that recapitulates the synthesis of 8, albeit
with different reactants (Scheme 4).32 The synthesis and spectro-
scopic characterization of 10 is provided as Supporting Information.
High-Throughput Screening Assay. A high-throughput screen-
ing assay of compounds 9 and 10 at 10 µM were conducted (Ambit
Biosciences, San Diego, CA) against a bacteriophage library
displaying 220 human kinases. The screening assays of both parental
compounds (staurosporine and 8, respectively) were used as
control.8,9 A rough estimation of the binding constant (Kd-1) for
each assay was provided by the single-hit value in the primary
screen at a single compound concentration. Kinase profiling was
performed using a bacteriophage library displaying fused human
kinases that may attach at the ATP site to a fixed-ligand matrix
that may be competitively displaced from binding by the tested
compound.8,9
Acknowledgment. This research is supported by NIH grant
R01-GM072614 and by an unrestricted grant from Eli Lilly.
We thank Drs. Harry Harlow and Chen Su (Eli Lilly) for their
valuable input.
Supporting Information Available: Wrapping comparison,
predicted affinity profile, and experimental screening of 9 against
56 PDB-reported kinase structures (Methods). Synthesis and
spectroscopic characterization of compounds 9 and 10. This material
is available free of charge via the Internet at http://pubs.acs.org.
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