Discovery of a potent and selective BCL-XLinhibitor with in vivo activity

Article abstract of DOI:10.1021/ml5001867

A-1155463, a highly potent and selective BCL-X_Linhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-X_L-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-X_Lbiology as well as a productive lead structure for further optimization.

Full text of DOI:10.1021/ml5001867

Letter
pubs.acs.org/acsmedchemlett
Discovery of a Potent and Selective BCL‑XL Inhibitor with in Vivo
Activity
Zhi-Fu Tao,*,† Lisa Hasvold,† Le Wang,† Xilu Wang,† Andrew M. Petros,† Chang H. Park,†
Erwin R. Boghaert,† Nathaniel D. Catron,† Jun Chen,† Peter M. Colman,§,∥ Peter E. Czabotar,§,∥
Kurt Deshayes,‡ Wayne J. Fairbrother,‡ John A. Flygare,‡ Sarah G. Hymowitz,‡ Sha Jin,† Russell A. Judge,†
Michael F. T. Koehler,‡ Peter J. Kovar,† Guillaume Lessene,§,∥,⊥ Michael J. Mitten,† Chudi O. Ndubaku,‡
Paul Nimmer,† Hans E. Purkey,‡ Anatol Oleksijew,† Darren C. Phillips,† Brad E. Sleebs,§,∥
Brian J. Smith,§,∥,# Morey L. Smith,† Stephen K. Tahir,† Keith G. Watson,§,∥ Yu Xiao,† John Xue,†
Haichao Zhang,† Kerry Zobel,‡ Saul H. Rosenberg,† Chris Tse,† Joel D. Leverson,† Steven W. Elmore,†
and Andrew J. Souers†
†AbbVie, Inc., 1 North Waukegan Road, North Chicago, Illinois 60064 United States
‡Genentech, Inc., 1 DNA Way, South San Francisco, California 94080 United States
§The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia
∥Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia
⊥Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, VIC 3010, Australia
S
* Supporting Information
ABSTRACT: A-1155463, a highly potent and selective BCL-
XL inhibitor, was discovered through nuclear magnetic
resonance (NMR) fragment screening and structure-based
design. This compound is substantially more potent against
BCL-XL-dependent cell lines relative to our recently reported
inhibitor, WEHI-539, while possessing none of its inherent
pharmaceutical liabilities. A-1155463 caused a mechanism-
based and reversible thrombocytopenia in mice and inhibited
H146 small cell lung cancer xenograft tumor growth in vivo
following multiple doses. A-1155463 thus represents an
excellent tool molecule for studying BCL-XL biology as well
as a productive lead structure for further optimization.
KEYWORDS: BCL-XL, BCL-2, apoptosis, cancer
bioavailable inhibitor of both BCL-2 and BCL-XL, which has
poptosis is a highly conserved process that is critical for
Acellular homeostasis and development. Dysregulation in demonstrated clinical antitumor activity in lymphoid malig-
nancies that are believed to be dependent upon BCL-2 for
survival.9,10 Consistent with preclinical studies showing the role
of BCL-XL in platelet survival,11,12 thrombocytopenia was the
predominant clinical dose-limiting toxicity of navitoclax when
dosed as a single agent.9 This observation prompted the
generation of the BCL-2 selective inhibitor ABT-199,13 which
has shown antileukemic activity in refractory chronic
lymphocytic leukemia patients without the dose limiting
thrombocytopenia elicited by navitoclax treatment. These
clinical data demonstrate that selective inhibitors of a single
BCL-2 family protein can have particular promise in targeting
specific cancer types.
apoptotic signaling is a common requirement for oncogenesis,
tumor maintenance, and chemoresistance.1 Dynamic binding
interactions between the pro-apoptotic (e.g., BAX, BAK, BAD,
BIM, NOXA) and antiapoptotic (e.g., BCL-2, BCL-XL, MCL-
1) BCL-2 family proteins control commitment to programmed
cell death.2,3 BCL-2 plays a dominant role in the survival of
lymphoid malignancies where it is frequently overexpressed,4
while overexpression of BCL-XL has been correlated with drug
resistance and disease progression of multiple solid tumors and
hematological malignancies.5 Targeting these proteins via small
molecule inhibitors has thus been a longstanding goal in the
oncology community.
Despite the inherent difficulties in targeting protein−protein
interactions, a combination of nuclear magnetic resonance
(NMR)-based screening and structure-based design afforded
ABT-263 (navitoclax).6−8 Navitoclax is a first-in-class, orally
Received: May 8, 2014
Accepted: August 26, 2014
Published: August 26, 2014
© 2014 American Chemical Society
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We previously reported that the synergy observed between
navitoclax and chemotherapies is driven primarily by the
inhibition of BCL-XL in solid tumors.14,15 Additionally, a BCL-
XL selective inhibitor offered the potential for reduced
immunosuppressive effects relative to dual inhibition of BCL-
2 and BCL-XL.9,10 A program to develop selective inhibitors
was thus initiated with the immediate goal of identifying a
compound suitable for key in vivo studies.
Previously, a series of selective BCL-XL inhibitors containing
hydrazinylbenzothiazole cores 1 or 2 (Figure 1) was
reported.16,17 Optimization led to the identification of WEHI-
539 (3), which demonstrated selective and mechanism-based
cell killing in mouse embryo fibroblast (MEF) cells engineered
to be dependent on BCL-XL via loss of MCL-1 (mcl-1−/−).16
WEHI-539 also demonstrated cell killing in the BCL-XL
dependent human small cell lung cancer (SCLC) cell line
relationship (SAR) by NMR fragment-based approach.19 The
crystal structures of the hydrazone (1 and 2) and amide (4)
cores with BCL-XL demonstrated that these molecules bound
the P2 pocket of BCL-XL in very similar fashion and could thus
be used interchangeably as first site ligands.16−18 Consequently,
the slightly higher solubility of core 1 in the aqueous conditions
of the NMR experiment prompted its nomination as the first
site ligand. To identify second site ligands, NMR screening was
undertaken (see Supporting Information) with a library of 875
highly soluble fragments in the presence of 1.
Of the various second site ligands that bound in the presence
of 1, bicyclic heterocycle 6 (Figure 2) demonstrated good
affinity (Kd ≈ 4 mM) along with multiple vectors for
attachment and modification. Six protein−ligand NOEs
observed in a 13C-edited, 12C-filtered 3D NOESY spectrum
were then used to dock 6 into the crystal structure of BCL-XL
bound to 1. This model demonstrated the phenyl group of 6 to
be less than 4 Å away from the picolinic acid (Figure 2),
indicating that a small tether could be used for linking the two
molecules. Furthermore, the proximity of the meta- and para-
positions of the phenyl moiety of 6 suggested these vectors to
be optimal for linkage.
A small set of compounds (7−11, Table 1) with varying
linker lengths and attachment points were thus synthesized to
explore these hypotheses. The combination of para-substitution
and a 4-atom tether clearly emerged as the local optimum with
respect to BCL-XL affinity, as the resultant compound (10)
exhibited a subnanomolar Ki value while preserving excellent
binding selectivity (>4000-fold) over BCL-2. However, as
predicted by the large and hydrophobic nature of the
pharmacophore (MW = 688.8, ClogP = 6.2) and the presence
of a carboxylic acid, the addition of 1% human serum (HS) to
the TR-FRET assay conditions right-shifted the apparent
binding affinities of all analogues by approximately 10- to 30-
fold. Despite this attenuated binding, 10 showed potent cell
killing in mcl-1−/− MEF cells in the presence of 10% fetal
bovine serum (FBS).
Figure 1. First generation hydrazone-based BCL-XL inhibitors and
second generation amide-based analogues.
To further understand the binding mode, an X-ray cocrystal
structure of 10 bound to BCL-XL was obtained (1.8 Å
resolution, Figure 3). The core of compound 10 resided within
the P2 pocket as expected.16−18 The benzothiazole ring
nitrogen and amide proton formed the same key hydrogen
bonds with Leu108 and Ser106, respectively, of the BCL-XL
protein as observed with the hydrazone-based inhibitors.16
Similarly, the carboxylic acid of 10 formed a hydrogen bond
with the side chain of Arg139 within the protein. As predicted
from the NMR-derived model, the tethered phenyl-1H-
pyrazolo[3,4-d]pyrimidine bound within the hydrophobic P4
pocket of BCL-XL, with the phenyl and pyrazole rings lying in
closest proximity to hydrophobic residues Tyr101 and Tyr195,
respectively.
Because the pyrimidine ring of 10 was solvent exposed, the
contribution of this moiety to BCL-XL affinity was unclear. This
prompted the synthesis and evaluation of analogue 12 (Table
1), which retained the pyrazole ring but lacked the pyrimidine
ring. While 12 showed slightly improved binding affinity
relative to 10, it suffered from a substantial diminution in BCL-
XL binding in the presence of 1% HS and a 10-fold decrease in
activity in mcl-1−/− MEF cells compared to pyrazolopyrimidine
10. These data suggested that the pyrimidine ring of the latter
compound was conferring a reduction in protein binding via
increased polarity7 while adding no additional benefit in target
affinity.
Figure 2. Left: Highly soluble fragment 6 identified as a 2nd site
binder by NMR in the presence of core 1. Right: NMR-derived ternary
structure of 1 (green) and 6 (magenta) bound to BCL-XL.
H146 (Figure 1). However, the utility of WEHI-539 as a tool
molecule was limited by the presence of a labile and potentially
toxic hydrazone moiety. Additionally, the poor physicochemical
properties of this compound made in vivo dosing untenable.
Initial efforts to redesign the hydrazone-based cores 1 and 2
afforded an amide-based pharmacophore 4.18 Although 4
showed some loss in BCL-XL affinity relative to the parent
hydrazone it demonstrated acceptable pharmacokinetic (PK)
properties in rats and had no inherent pharmaceutical
liabilities.18 The generation of compound 5 via extension into
the critical “P4” binding pocket demonstrated that analogues
with on-target cellular activity could be obtained;18 however,
the lack of potency in the higher bar human tumor H146 cell
line indicated that further optimization was necessary.
The P4 binding pocket in BCL-XL has previously been
demonstrated to be critical for tight binding of BH3-peptides
and inhibitors.6,7,16 Thus, we targeted this region for our
optimization efforts. In order to facilitate the identification of
productive P4-binding moieties beyond the methylamine
represented in WEHI-539,16 we employed a structure−activity
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a
Table 1. SAR of Fragment-Linked Analogues
a
All assays were run in triplicate.
Table 2. Biological Activity of Alkyne 15 and Halogenated
a
Analogues
BCL-2
H146 10%
HS EC50
(μM)
halogen BCL-XL BCL-XL 1%
Ki
compound
15
position Ki (nM) HS Ki (nM) (nM)
Figure 3. X-ray crystal structure of compound 10 (green) bound to
BCL-XL. Protein is shown as a ribbon diagram where key amino acids
are colored orange, oxygen atoms are red, and nitrogen atoms are blue.
Key hydrogen bonds are shown in red dotted lines. PDB code: 4TUH.
none
2-Cl
2-F
0.02
0.28
0.41
0.10
60
0.60
0.18
16
0.04
280
80
b
17
<0.01
0.065
(A-1155463)
18
19
3-F
0.01
0.03
1.12
0.33
88
90
0.051
0.126
2-F, 5-F
To extend this hypothesis, the pyrimidine ring of 10 was next
utilized as a scaffold for the incorporation of polar groups. In
previous optimization efforts toward dual BCL-2/BCL-XL
inhibitors, human serum albumin (HSA) binding could be
alleviated via the incorporation of amine groups at a specific
position.6,7 Furthermore, the basicity of the amine group could
a
b
All assays were run in triplicate. Geometric mean of over 35 assays
run in triplicate.
be optimized to offset the acidic portion of the pharmacophore
to afford substantial benefits in cellular potency and oral
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beneficial in reducing HSA binding while simultaneously
increasing target affinity, although the reason for the latter is
unclear given the solvent-exposed nature of these moieties.
These observations lead to the design and preparation of
aminoalkyne-linked 15 (Table 2), a simplified analogue that
could both achieve π-stacking interactions as well as reduce
HSA-binding. The affinity of this analogue was encouraging,
albeit slightly lower than that of 13 and 14. However, the
excellent potency of 15 against mcl-1−/− MEF cells (EC50 = 8
nM) suggested a possible increase in permeability, a result that
was ascribed to the lower basicity of the terminal alkynyl amine.
We then tested the ability of 15 to kill H146 cells. Gratifyingly,
in the presence of 10% HS, 15 demonstrated an EC50 of 600
nM (Table 2).
Alkyne analogue 15 was an attractive lead structure given its
straightforward synthesis and decreased molecular weight (MW
= 652) relative to pyrazolopyrimidine analogues 13 (MW =
789) and 14 (MW = 844). The electron-rich 4-alkynyl phenoxy
moiety was thus targeted for additional modification, with the
hypothesis that halogens could create additional interactions in
the hydrophobic P4 pocket while conferring beneficial effects
on metabolic stability.20 To this end, a set of analogues with a
single halogen on either the 2 or 3 position (16−18) were
prepared along with the 2,5-difluorinated analogue 19 (Table
2). In all cases, halogenation of the phenyl ring enhanced the
cytotoxic activity of the corresponding analogues in H146 cells
as compared to 15. The single fluorine analogues 17 (A-
1155463) and 18 in particular showed an approximately 10-fold
boost in cell killing activity.
Figure 4. X-ray crystal structure of A-1155463 (green) bound to BCL-
XL. Key amino acids are colored orange, oxygen atoms are red, and
nitrogen atoms are blue. Dotted contacts are <4 Å. PDB code: 4QVX.
Table 3. Pharmacokinetic Properties of A-1155463 in SCID-
a
Beige Mice
Cmax (μM)
Tmax (hr)
AUC (μM·h)
C12h (μM)
1.8
2
9.19
0.18
a
A-1155463 was dosed at 5 mg/kg IP in a vehicle containing 5%
DMSO, 10% EtOH, 20% Cremaphor ELP, and 65% D5W.
absorption. Inspired by this example, we synthesized a pair of
analogues with tethered amines projecting from the solvent-
exposed pyrimidine moiety (13 and 14, Table 1).
Interestingly, the addition of tethered amines increased the
target affinity of compounds 13 and 14 relative to 10. This was
exemplified by the Ki values to BCL-XL, which were below the
limits of detection (10 picomolar) in the TR-FRET assay and
dramatically improved in the presence of 1% HS. The enhanced
target affinity of 13 and 14 translated into significant gains in
cellular activity, with both analogues showing highly potent
killing of mcl-1−/− MEF cells (Table 1).
The presence of the pyrazole moiety within analogues 10−
14 appeared to drive target affinity via favorable π-stacking
interactions within the P4 pocket of BCL-XL. The tethered
amines within analogues 13 and 14, in turn, were putatively
An X-ray cocrystal structure of BCL-XL-bound to A-1155463
(2.1 Å resolution, Figure 4) revealed the 2-fluoro substituent to
be proximal to the side chains of Val141 and Phe97 within the
hydrophobic P4 pocket. These close C−F distances (<4.0 Å)
suggested the presence of significant van der Waals contacts.20
A-1155463 showed picomolar binding affinity to BCL-XL,
and >1000-fold weaker binding to BCL-2 (Table 2) and related
proteins BCL-W (Ki = 19 nM) and MCL-1 (Ki > 440 nM).
Figure 5. (a) Kinetics of platelet reduction and rebound following a single IP dose of A-1155463 in SCID-Beige mice. Nontumor bearing female
SCID-Beige mice were treated with a single dose of A-1155463 (5 mg/kg, IP). Blood samples were collected by means of retro-orbital bleeds at
regular time intervals. The number of platelets was plotted as a function of time. Each point reflects the average of 5 biological replicates. Each box
indicates the median SEM. The error bars reflect minimum and maximum values (n = 5 per time point). The colored boxes represent upper and
lower quartiles. The horizontal lines within each colored box represent the mean, and the whiskers are the upper and lower range. (b) Inhibition of
H146 SCLC xenograft growth by A-1155463. Female SCID-Beige mice bearing NCI-H146 subcutaneous xenografts were treated with A-1155463 (5
mg/kg IP, QDx14) or an equal volume of the vehicle (5% DMSO, 10% EtOH, 20% Cremophor ELP, and 65% D5W; n = 5 per time point) and
changes in tumor volume determined as a function of time. Each point reflects the average volume of 5 tumors. Points with significant (Student’s t
test, p < 0.05) difference between treated and untreated groups are indicated by means of asterisks. Tumor volume was calculated by W2·L·0.5 where
W and L are two perpendicular diameters indicating width and length, respectively.
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Moreover, the cellular activity in the BCL-XL-dependent H146
cell line was improved by nearly an order of magnitude relative
to the starting hydrazone WEHI-539. Because the aqueous
solubility of this and related compounds compromised the
generation of quality in vitro ADME data, we elected to directly
investigate the pharmacokinetic properties of A-1155463 in
nontumor bearing SCID-Beige mice following a single
intraperitoneal (IP) dose. As shown in Table 3, a 5 mg/kg
dose afforded target coverage of 12 h relative to the H146 EC50,
which was sufficient for an expectation of in vivo activity.6−8
We next measured the effects of A-1155463 on platelets,
which serve as a convenient biomarker for BCL-XL inhibition in
vivo.8,9,11,12 Following a single 5 mg/kg IP dose of A-1155463
in nontumor bearing SCID-Beige mice, platelet counts fell
dramatically as measured at 6 h postdose and then rebounded
to normal levels within 72 h (Figure 5a). The kinetics of
platelet depletion and recovery were similar to those previously
reported with the dual inhibitor navitoclax.8 To provide
additional evidence that A-1155463 was conferring on-target
in vivo activity, we then administered this compound to SCID-
Beige mice that had been inoculated with BCL-XL-dependent
H146 tumor cells. Daily dosing at 5 mg/kg IP for 14 days
caused a statistically significant inhibition of tumor growth
(maximum tumor growth inhibition = 44%), which was
alleviated upon cessation of dosing (Figure 5b).
In summary, we used iterative structure-based design to
generate a highly potent and selective BCL-XL inhibitor that is
devoid of the labile hydrazone linkage of the earlier tool
compound WEHI-539. The ability of A-1155463 to exert in
vivo on-target activity was demonstrated through a rapid and
reversible reduction in platelets in SCID-Beige mice following a
single IP dose. Additionally, administration of A-1155463 to
tumor bearing SCID-Beige mice afforded modest but statisti-
cally significant tumor growth inhibition. Detailed mechanistic
studies of A-1155463 and combination activity with relevant
chemotherapy across multiple tumor types will be reported in
an accompanying manuscript.
Council of Victoria (fellowship to P.M.C.; grant-in-aid 461239),
and the Australian Cancer Research Foundation. Infrastructure
support from the NHMRC Independent Research Institutes
Infrastructure Support Scheme grant no. 361646 and a
Victorian State Government OIS grant are gratefully acknowl-
edged.
Notes
The authors declare the following competing financial
interest(s): Z.-F.T., L.H., L.W., X.W., A.M.P., C.H.P., E.R.B.,
N.D.C., J.C., S.J., R.A.J., P.J.K., M.J.M., P.N., A.O., D.C.P.,
M.L.S., S.K.T., Y.X., J.X., H.Z., S.H.R., C.T., J.D.L., S.W.E., and
A.J.S. are employees of AbbVie Inc.; K.D., W.J.F., J.A.F., S.G.H.,
M.F.T.K., C.O.N., H.E.P., and K.Z. are employees of
Genentech Inc.; P.M.C., P.E.C., G.L., B.E.S., B.J.S., and
K.G.W. are employees of The Walter and Eliza Hall Institute
of Medical Research. AbbVie, Genentech, and WEHI
participated in the study conduct, design, interpretation of
data, review, and approval of the publication.
ACKNOWLEDGMENTS
■
We thank Drs. Mike Michaelides, Andrew Judd, Kent Stewart,
Michael Wendt, and Gerard Sullivan at AbbVie for critical
reading of the manuscript and Dr. David Huang for helpful
discussion during this work. We thank the beamline staff at the
Australian Synchrotron where diffraction data for the BCL-XL-
compound 10 complex were collected.
ABBREVIATIONS
■
BCL-2, B-cell lymphoma 2; BCL-XL, B-cell lymphoma-extra
large; MCL-1, Myeloid cell leukemia sequence 1
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ASSOCIATED CONTENT
* Supporting Information
■
S
Fragment screening procedures, biological assay protocols,
synthetic description and procedures, and analytic data
including NMR, MS, and crystallographic methods. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(Z.-F.T.) E-mail: zhi-fu.tao@abbvie.com. Tel: (847) 938-
6772.
■
Present Address
#(B.J.S.) La Trobe Institute for Molecular Science, La Trobe
University, VIC 3086, Australia.
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Funding
This work was sponsored by AbbVie, Inc., and Genentech, a
member of the Roche group. Part of this work was supported
by fellowships and grants from the Australian Research Council
(fellowship to P.E.C.), the National Health and Medical
Research Council (NHMRC, fellowships to P.M.C.; develop-
ment grant 305536 and program grants 257502, 461221, and
1016701), the Leukemia and Lymphoma Society (specialized
center of research grant nos. 7015 and 7413), the Cancer
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