Discovery of small molecule Mer kinase inhibitors for the treatment of pediatric acute lymphoblastic leukemia

Article abstract of DOI:10.1021/ml200239k

Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at subnanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.

Full text of DOI:10.1021/ml200239k

Letter
pubs.acs.org/acsmedchemlett
Discovery of Small Molecule Mer Kinase Inhibitors for the Treatment
of Pediatric Acute Lymphoblastic Leukemia
Jing Liu,†,∥ Chao Yang,†,∥ Catherine Simpson,† Deborah DeRyckere,⊥ Amy Van Deusen,†
Michael J. Miley,§ Dmitri Kireev,† Jacqueline Norris-Drouin,† Susan Sather,⊥ Debra Hunter,‡
Victoria K. Korboukh,† Hari S. Patel,† William P. Janzen,† Mischa Machius,§ Gary L. Johnson,§,‡
H. Shelton Earp,‡,§ Douglas K. Graham,⊥ Stephen V. Frye,†,‡ and Xiaodong Wang*,†
†Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman
School of Pharmacy, §Department of Pharmacology, ‡Lineberger Comprehensive Cancer Center, Department of Medicine, School of
Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
⊥Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, Colorado 80045, United States
S
* Supporting Information
ABSTRACT: Ectopic Mer expression promotes pro-survival signaling and contributes to
leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL).
Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as
chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have
applied a structure-based design approach to discover novel small molecule Mer kinase
inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at
subnanomolar concentrations. Furthermore, the lead compound shows a promising selectivity
profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also
describe the crystal structure of the complex between the lead compound and Mer, opening new
opportunities for further optimization and new template design.
KEYWORDS: Mer inhibitors, acute lymphoblastic leukemia, pyrazolopyrimidines, chemosensitizer
cute lymphoblastic leukemia (ALL) is the most common
malignancy in children and represents nearly 30% of all
relative to wild-type lymphocytes when treated with dexa-
methasone, a critical component of ALL induction chemo-
therapy. In human ALL cell lines, Mer expression also mediates
resistance to other commonly used ALL chemotherapeutics
(vincristine, 6-mercaptopurine, methotrexate, and doxorubi-
cin).3 Taken together, these data provide a rationale for
development of Mer kinase inhibitors as selective therapeutics
for ALL and other Mer related diseases. In addition, these
inhibitors may mediate synergistic antileukemia activity in
combination with standard chemotherapy, thereby permitting
dose reduction and diminished toxic side effects.
Mer is a member of the TAM (Tyro3, Axl, Mer) RTK family.
Each member of the TAM family contains an extracellular
domain, a transmembrane domain, and a conserved intra-
cellular kinase domain.4,7 Compared with other tyrosine
kinases, the intracellular kinase domain of the TAM family is
quite dissimilar (40% sequence identity). This makes Mer an
excellent candidate for targeting selectively by small molecules.
A
pediatric cancers.1 Although most childhood ALL types are
curable, T-cell ALL, a subtype that accounts for 10−15% of
pediatric ALL cases, has a poorer prognosis, with a 5-year
relapse-free survival rate of 60−75% even with effective
treatment.2 Extensive exposure to current chemotherapeutic
regimens is associated with toxic side effects (growth slowing,
organ damage, and secondary malignancy) and development of
chemoresistance.3
Previous studies have indicated a role for Mer receptor
tyrosine kinase (RTK) in ALL. Mer is ectopically expressed in
at least 50% of pediatric T-cell ALL samples as well as in pre-B
ALL samples, particularly those with a (1;19)(q23;p13)
translocation encoding an E2A/PBX1 fusion protein.1,3−5 In
contrast, Mer is not expressed in normal mouse and human T-
and B-lymphocytes at any stage of development. This tumor
specific expression pattern may confer a wide therapeutic
windowat least in these cell types. In previous studies,
transgenic expression of Mer in mouse lymphocytes resulted in
development of T-cell leukemias and lymphomas.6 In addition,
a Mer expressing human B-ALL cell line was able to produce a
lethal malignancy in immunocompromised mice; development
of leukemia was significantly delayed or completely eliminated
by Mer shRNA knockdown in these cells.3 Mer also contributes
to lymphoblast chemoresistance. For example, Mer transgenic
lymphocytes have a statistically significant survival advantage
To date, only one weak Mer inhibitor, Compound-52 (IC50
=
11 μM) (Figure 1a), has been reported, and the crystal
structure of its complex with Mer has been determined at 2.8
Å.8 Although Compound-52 is not valuable for further drug
development due to its limited potency and lack of selectivity,
Received: October 20, 2011
Accepted: January 9, 2012
Published: January 9, 2012
© 2012 American Chemical Society
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Figure 1. (a) Chemical structure of Compound-52; (b) docking model of 1 in the X-ray structure of Mer; (c) chemical structure of 1; (d) design of
candidate Mer kinase inhibitors based on the pyrazolopyrimidine scaffold.
Scheme 1. Synthetic Routes for the Pyrazolopyrimidine Scaffold
the crystal structure provided an opportunity to develop Mer
inhibitors using structure-based drug design.
relationships (SAR) at the R2 and R3 positions. The three
reactions (Suzuki coupling, alkylation, and oxidation/SNAr
displacement) in path a can be carried out in varying order to
optimally explore SAR at different positions. Accordingly, path
b (3 → 7 → 5 → 6) is more suitable for modifications at the R3
position while path c (3 → 7 → 8 → 6) is preferable when
multiple analogues with variation at the R1 group are required.
To gain initial information on SAR at the R1 and R2
substitution sites, a small compound library with R3 fixed as
the MeNH group was synthesized via path c in Scheme 1.
Inhibition of Mer kinase activity by analogues was tested at the
ATP Km using a microfluidic capillary electrophoresis (MCE)
assay24−26 in which phosphorylated and unphosphorylated
substrate peptides were separated and analyzed through a
LabChip EZ Reader. Representative examples from this library
are shown in Table 1. For Mer activity, the order of preferred
substituents at the R1 position is aryl > alkenyl > alkyl (9, 13,
and 14). For the R2 position, the distance between the pyrazole
ring and the polar group on R2 is important, and a para-
substituted cyclohexylmethyl group was preferred (9−12).
Although trans- and cis-4-hydroxylcyclohexylmethyl analogues
14 and 15 were equally potent for inhibition of Mer kinase
activity, trans-4-aminocyclohexylmethyl analogue 16 was 3-fold
more potent than the cis-analogue 17. Activity against Axl and
Tyro3 was also determined to monitor their selectivity within
the TAM family and develop a pan-TAM SAR. In general, these
analogues showed modest selectivity within the TAM family.
They are generally less potent against Tyro3 (>5-fold) while
there is little difference in the activities between Mer and Axl.
On the basis of the crystal structure of Compound-52 with
Mer, a pyrazolopyrimidine heterocycle (1, Figure 1b, 1c) was
proposed. This molecule occupied the adenine pocket, formed
two hydrogen bonds with the hinge-backbone Met674, and was
stabilized within a lipophilic cage formed by Leu593, Phe673,
and Met730. In addition, its isopropyl group pointed toward
the activation loop. The structural features of 1 also enabled at
least three sites (R1, R2, and R3) (Figure 1d) for facile
modification to optimize the inhibitory potency, selectivity, and
bioavailability. Furthermore, pyrazolopyrimidines are popular
synthetic pharmacophores and have been explored as ATP
competitive inhibitors of kinases.9−20 The substituents at the
R1, R2, and R3 positions were chosen by considering their basic
chemical features. For instance, polar substituents of various
sizes at the R2 substitution site were expected to form an ionic
bond and/or hydrogen bond with Asp678. At the R1 and R3
positions, a variety of groups would be tested.
We have developed an efficient route to prepare
pyrazolopyrimidine analogues (Scheme 1). The key inter-
mediate 3 was synthesized from bromination of the known
compound 2 with phosphoryl tribromide (POBr3) in a sealed
tube.21 In path a, Suzuki coupling between 3 and a boronic acid
provided 4, which was subsequently alkylated at the N1
position of the pyrazole ring to yield 5.22 3-Chlorobenzoper-
oxoic acid (mCPBA) oxidation followed by the displacement of
the methyl sulfinyl/sulfonyl group by amines then provided the
final pyrazolopyrimidine analogue 6.23 This sequence of
reactions (path a) is ideal for exploration of structure−activity
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Table 1. Preliminary SAR at the R1 and R2 Positions
Table 2. SAR Study of R1 and R3
a
Values are the mean of two or more independent assays performed at
b
the ATP Km. Is a racemate.
With 4-aminocylcohexylmethyl selected as an optimized R2
group on the basis of in vitro potency, the effect of substitution
on the phenyl ring at the R1 position was then explored (Table
2). The para-methoxyl substituted analogue 18 was 5-fold more
potent than meta substituted 19 and 56-fold more potent than
ortho substituted 20. In addition, the chemical properties of the
substituents have a significant effect on Mer kinase inhibition.
In general, electron withdrawing groups decreased activity,
while electron donating groups increase activity. For example,
compound 18 (para-methoxyl) was 7-fold more potent than 21
(para-CF3). The introduction of a phenyl group at the para
position (22) slightly decreased the activity compared to that of
18. 3-Pyridyl (23) and 6-F-3-pyridyl (24) as the R1 substituents
were also less active than 16. However, introduction of a
piperidine ring at the 6-position of the 3-pyridyl provided a very
potent compound, 25. Replacement of the pyridine ring with
the more hydrophobic phenyl ring further increased the
potency by 4-fold (26). These results suggest that electron-
donating, para-substituted phenyl or pyridinyl groups are
suitable R1 substitutions.
a
b
Is a 2:1 mixture of cis/trans isomers. Values are the mean of two or
more independent assays performed at the ATP Km.
formed with the hinge region of the protein. Replacement of
the secondary amine with a tertiary amine greatly decreased
activity (27). Extension of the alkyl chain (C3−C5) at the R3
site boosted the potency of the analogues (28−30) while a
branched alkyl chain (31) and cycloalkyl groups (32) were less
favorable. Introducing polar groups into the alkyl chain
decreased the activity of the analogues dramatically (33 and
34). However, a phenyl ring or a simple para-substituted
phenyl ring was well tolerated at this position (35−38).
Varying the R3 group affected not only the analogues’ activity
toward Mer but also the selectivity within the TAM family
members. The best selectivity was achieved by analogue 29,
which has 17-fold selectivity for Mer over Axl and 12-fold
selectivity for Mer over Tyro3.
As shown in Table 2, the secondary amine (NH) at the R3
position was critical for potency due to a hydrogen bond
Upon completion of the SAR studies in Tables 1 and 2, we
next decided to synthesize a small focused library of six
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Table 3. Focused Library
compounds with a “mix and match” combination of the optimal
substituents at the R1 and R3 positions (Table 3). Indeed, this
exercise turned out to be highly productive, as all six
compounds show potent inhibition of Mer kinase activity,
with IC50 values in the range 0.15−3.0 nM.
(94% and 92%, respectively), Ret (79% and 59%, respectively),
and its mutant Ret Y791F (67% and 56%, respectively).
In addition, a cocrystal structure of Mer in complex with 43
was determined at a resolution of 2.69 Å. The structure
highlights an unanticipated binding mode (see Figure 2). The
inhibitor, including its flexible lipophilic substituent R3, is fully
confined to a relatively small adenine pocket instead of
protruding into the lipophilic back-pocket. This binding mode
is probably determined by the following structural features of
Mer. First, Mer has a relatively large gatekeeper residue
(Leu671). It also forms the “gate” to the back-pocket with
another four residues (Ile650, Ala740, Asp741, and Lys619)
(Figure 2). The two residues Leu671 and Ile650 situated on the
loop connecting the αC helix to the β4 strand (right below the
gatekeeper) render the lipophilic back-pocket inaccessible to
inhibitors. It is also noteworthy that one of the gate-forming
residues, Ile650, is not conserved among the TAM family
members. Hence, this pocket area might be instrumental for
Mer inhibitor selectivity toward Axl and Tyro3, which feature
methionine and alanine, respectively, in this position.
These analogues are more active against Mer than Axl and
Tyro3. The inhibition constants (Ki) of these analogues for
Mer were also determined (Table 3). As anticipated, these
compounds were also shown to be ATP competitive inhibitors
(details in the Supporting Information). The most active
compound, 42, and the low molecular weight compound, 43
(UNC569), were also profiled against 72 kinases (details in the
Supporting Information) including the TAM family members
using the same MCE assay. This panel of kinases provides a
general survey of kinase families and coverage of the TAM and
closely related MET family. Tested in duplicate at a
concentration 10-fold higher than its IC50 value against Mer,
both 42 and 43 inhibited only three other kinases by greater
than 50%: FLT3 (82% and 82%, respectively), MAPKAPK2
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Accession Codes
The atomic coordinates for the X-ray crystal structure of 43
have been deposited with the RCSB Protein Data Bank under
the accession code 3TCP.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 919-843-8456. Fax: 919-843-8465. E-mail:
xiaodonw@unc.edu.
Author Contributions
∥These authors contributed equally.
Funding
This work was supported by the University Cancer Research
Fund, School of Pharmacy, Carolina Partnership, the University
Research Council at University of North Carolina, and Federal
Funds from the National Cancer Institute, National Institute of
Health, under Contract No. HHSN261200800001E. The
content of this publication does not necessarily reflect the
views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S.
Government. Use of the Advanced Photon Source was
supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract No.
W-31-109-Eng-38.
Figure 2. X-ray crystal structure of 43 in complex with Mer (kinase
domain). The surface of the adenine pocket is outlined in brown mesh.
The molecular surface of five residues constituting a “gate” to the back-
pocket is shown in cyan mesh.
The R2 substituent occupies the ATP sugar pocket with its
amino group forming a hydrogen bond with the carbonyl of
Arg727. Again, this represents a counterintuitive interaction
given that the ionized amino nitrogen could have formed an
energetically stronger interaction with Asp678, as is the case for
the inhibitor Compound-52.8 Finally, the R1 substituent
interacts mostly with the solvent and can be exploited for
tuning solubility and other pharmacokinetic properties. The
information acquired from this structure opens new oppor-
tunities for further optimization of inhibitory potency and
pharmacokinetic properties of the current series.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Furthermore, the ability of 43 to inhibit Mer autophosphor-
ylation in human B-ALL cells was tested. Human Pre-B
leukemia 697 cells were treated with various concentrations of
43 for 1 h prior to addition of a phosphatase inhibitor to
stabilize phospho-Mer. Mer protein was immunoprecipitated
from lysates, and phospho-Mer was detected by Western blot
using a rabbit polyclonal antibody raised against a triphos-
phorylated Mer peptide. Phospho-Mer levels were quantitated
by densitometry, and IC50 values were determined using
nonlinear regression. In this cell-based assay, the IC50 for
inhibition of phospho-Mer by 43 was 141 nM.
The in vivo pharmacokinetic (PK) properties of 43 were also
assessed in mice via both intravenous (IV) and oral (PO)
administration (details in the Supporting Information). 43 had
low systemic clearance (19.5 mL/min/kg), high volume of
distribution (Vss = 5.83 L/kg), and good oral bioavailability
(57%).
In conclusion, we have successfully developed a robust SAR
and discovered potent small molecule Mer inhibitors within the
pyrazolopyrimidine scaffold. A cocrystal structure of Mer with
the lead compound 43 has also been determined and reveals an
unusual binding mode in the catalytic kinase domain. The
selectivity and DMPK properties of the lead compound are very
promising, suggesting it is a viable lead candidate for
optimization toward Mer inhibitors for clinical applications.
■
We thank Professor Sirano Dhe-Paganon, Structural Genomics
Consortium, University of Toronto, for providing the Mer
kinase domain expression construct and Professor K. H. Lee’s
lab at UNC for HRMS support.
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