Structure-guided rescaffolding of selective antagonists of BCL-X L

Article abstract of DOI:10.1021/ml500030p

Because of the promise of BCL-2 antagonists in combating chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), interest in additional selective antagonists of antiapoptotic proteins has grown. Beginning with a series of selective, potent BC

Full text of DOI:10.1021/ml500030p

Letter
pubs.acs.org/acsmedchemlett
Structure-Guided Rescaffolding of Selective Antagonists of BCL‑XL
Michael F. T. Koehler,*,† Philippe Bergeron,† Edna F. Choo,‡ Kevin Lau,† Chudi Ndubaku,†
Danette Dudley,† Paul Gibbons,† Brad E. Sleebs,∥,⊥ Carl S. Rye,∥,⊥,¶ George Nikolakopoulos,∥,⊥
Chinh Bui,∥,⊥ Sanji Kulasegaram,∥,⊥,∇ Wilhelmus J. A. Kersten,∥,⊥ Brian J. Smith,∥,⊥,◆
Peter E. Czabotar,∥,⊥ Peter M. Colman,∥,⊥ David C. S. Huang,∥,⊥ Jonathan B. Baell,∥,⊥,#
Keith G. Watson,∥,⊥ Lisa Hasvold,○ Zhi-Fu Tao,○ Le Wang,○ Andrew J. Souers,○ Steven W. Elmore,○
John A. Flygare,† Wayne J. Fairbrother,§ and Guillaume Lessene∥,⊥
‡
§
†Departments of Discovery Chemistry, Drug Metabolism and Pharmacokinetics, and Early Discovery Biochemistry, Genentech,
Inc., 1 DNA Way, South San Francisco, California 94080, United States
∥Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia
⊥Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia
○AbbVie, Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States
S
* Supporting Information
ABSTRACT: Because of the promise of BCL-2 antagonists in
combating chronic lymphocytic leukemia (CLL) and non-
Hodgkin’s lymphoma (NHL), interest in additional selective
antagonists of antiapoptotic proteins has grown. Beginning with a
series of selective, potent BCL-XL antagonists containing an
undesirable hydrazone functionality, in silico design and X-ray
crystallography were utilized to develop alternative scaffolds that
retained the selectivity and potency of the starting compounds.
KEYWORDS: BCL-XL, BCL-2, apoptosis, cancer
poptosis, or programmed cell death, is a tightly regulated
biological process that is critical for development, for
which operate immediately downstream of both pathways have
been the target of drug development efforts.8,9
A
maintenance of cellular homeostasis, and for fighting off
infection. The extrinsic1 apoptotic pathway involves the binding
of pro-apoptotic ligands such as Apo2L/TRAIL, TNF, and
FasL to specific cell surface receptors. This begins a cascade of
events that culminates in the activation of caspase-3, the
committed step in apoptosis. The intrinsic2 apoptotic pathway
is initiated when cellular stress stimulates the production of or
increased persistence of the pro-apoptotic BH3-only proteins
BIM, BID, PUMA, NOXA, BAD, HRK, MBF, and BIK. These
proteins interact with the pro-survival proteins BCL-2, BCL-XL,
BCL-w, MCL-1, and A1 and possibly directly with the
apoptotic effector proteins BAX and BAK. Regardless of the
interaction they engage in, the pro-apoptotic proteins
ultimately induce the permeabilization of the mitochondrial
membrane, followed by the activation of caspase-3 and cell
death.
Both the intrinsic and extrinsic apoptotic pathways have been
targeted for cancer therapy. While research into the extrinsic
pathway has focused on Apo2L/TRAIL and its synergies with
existing therapy,3 the focus in the intrinsic pathway has been on
the inhibition of pro-survival proteins overexpressed in tumors,
including BCL-2, BCL-XL, and BCL-w.4 Navitoclax, an orally
available small molecule that potently binds BCL-2, BCL-XL,
and BCL-w, has already shown promise in human clinical
trials.5−7 Additionally, the inhibitor of apoptosis (IAP) proteins,
Broad inhibition of the pro-survival proteins carries with it
the risk of undesired activities in vivo arising from the fact that
these proteins are responsible for maintaining various cell
populations under normal conditions. For example, BCL-2 is
required for B and T lymphocyte survival,10 while BCL-XL is
responsible for platelet survival.11,12 MCL-1 has been shown to
be required for hematopoietic stem cell survival,13 and BCL-w
is essential for developing sperm cells.14 Narrowly targeting the
pro-survival protein responsible for tumor survival or resistance
to chemotherapy could thereby allow for a reduced set of side
effects, as has been demonstrated by the efficacy without
significant thrombocytopenia observed for ABT-199 (GDC-
0199), which is a BCL-2 selective inhibitor, in CLL patients.15
To this end, a series of selective BCL-XL antagonists were
developed16 to target solid tumors without concomitant effects
on BCL-2 dependent hematopoietic cells.5,7
We recently reported compounds 1 and 2 (Figure 1) as
potent and selective BCL-XL inhibitors. While these represent
good lead structures, we were concerned about the presence of
a hydrazone in the molecules, as they are known to hydrolyze,
Received: January 22, 2014
Accepted: March 21, 2014
Published: March 21, 2014
© 2014 American Chemical Society
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There is little space for the addition of functionality in either
the P2 pocket or in the hydrophobic groove containing the
tetrahydronaphthalene core. In our initial designs, we therefore
attempted to preserve both the hydrophobic nature of the
molecules as well as to mimic the overall shape of compounds 1
and 2 as closely as possible.
Our previous structures and structure−activity relationship
(SAR) experience made it clear that we needed to preserve the
multiple contacts made by the polar atoms in the picolinic acid
with Arg139 of BCL-XL. Similarly, it was clear that the
hydrazone nitrogen closest to the benzothiazole served as a
hydrogen bond donor to the backbone carbonyl of Ser106 and
that the benzothiazole nitrogen accepts a hydrogen bond from
the main chain NH of Asp107. We therefore incorporated the
picolinic acid as well as a hydrogen bond donor/acceptor pair
of the 2-amino benzothiazole ring into our designs.
The initial complement of analogues prepared included the
replacement of the hydrazone with an amine, an amide, and a
urea while varying the degree of saturation in the core
naphthalene/tetrahydronapthalene to afford compounds 3−7
(Figure 3). In preparing compounds 5 and 6, we did not
attempt to control the stereochemistry, although docking
experiments indicate that the R enantiomer should be preferred
in both cases. The enantiomers were separated following
synthesis and assayed individually.
Following their synthesis (described in Schemes S1−S4,
Supporting Information), compounds 3−7 were examined for
their ability to inhibit binding of a 26-mer BIMBH3 peptide to
BCL-XL, as described previously.16 The naphthalene amide 4
and tetrahydroquinoline urea 7 were the strongest inhibitors of
the group, but were 36- to 50-fold weaker binders, respectively,
than hydrazone 1. Although the compounds lacking the
hydrazone functionality still retained measurable binding to
BCL-XL, refinement of our designs was clearly necessary in
order to attain biologically relevant levels of inhibition.
Docking experiments with higher affinity ligands 5b and 7
pointed to two new unfavorable interactions introduced in the
carbonyl-containing compounds. One straightforward explan-
ation for the loss of affinity was the desolvation penalty
incurred by the introduction of a hydrophilic carbonyl group
into the highly lipophilic environment of the binding pocket
formed by the side chains from Phe97, Phe105, and Ala142.
More subtly, our docking experiments indicated that 4, 6, and 7
might not be able to align their hydrogen bond donating NH
optimally toward Ser106 as this would force the carbonyl
oxygen into an unfavorable steric interaction with the adjacent
aryl ring.
Figure 1. BCL-XL antagonists 1 and 2 incorporating a hydrazone
linker between the benzothiazole and bicyclic core. Rat PK data
represents an average of three animals dosed at 1 mg/kg IV or 5 mg/
kg PO.
potentially releasing a toxic 2-hydrazinylbenzothiazole upon
exposure to water. Indeed, when compound 1 was dosed
intravenously in rats, the 3,4-dihydronaphthalene-1-one ex-
pected from hydrolysis of the parent hydrazone was observed at
approximately 1% of the level of 1, implying the release of 2-
hydrazinyl benzothiazole.
In addition to our concerns about the presence of a potential
toxicophore, we were concerned about the generally poor
pharmacokinetic (PK) properties of these molecules, as they
suffered from high clearance, low oral bioavailability, or both in
rats. We sought to address these issues through replacement of
the hydrazone with a hydrolytically stable linkage while
preserving the other favorable attributes of the molecules.
The known structure (PDB: 3ZLN) of compound 1 in
complex with BCL-XL (Figure 2) was used as a guide for our
designs. Our first consideration was the observed enlargement
of the hydrophobic P2 pocket upon ligand binding, which
appears to be an induced fit to accommodate the benzothiazole.
As our most potent new analogues all included the carbonyl
oxygen proposed to be producing these unfavorable inter-
actions, we set out to mitigate these effects. We initially
attempted to relieve the steric clash between the carbonyl and
the proton at the 1-position of the naphthalene through
exploration of [5,6]-fused heterocycles. Accordingly, we
prepared analogues 8−10 (Schemes S5−S7, Supporting
Information) and evaluated their ability to bind to BCL-XL as
before.
Unfortunately, none of the compounds showed improved
affinity for the target. Although 8 was predicted to have an
improved ability to engage Ser106, the inclusion of new polar
functionality into the biaryl ring system may have introduced
additional detrimental interactions. A measure of polarity
change can be seen in the increased topological polar surface
area (TPSA) of imidazopyridine 8 (109 Å2) relative to 6 (92
Figure 2. Interactions of compound 1 with BCL-XL observed in a 2.3
Å resolution crystal structure (PDB: 3ZLN). The surface displayed is
one van der Waal’s radius above the analytic Connolly surface of the
protein. Hydrogen bonds are indicated by dashed lines.
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Figure 3. Initially prepared analogues of 1 along with their inhibitory activity toward BCL-XL. Compounds 5 and 6 were assayed as single
enantiomers. Their absolute configuration was not determined.
Å2). Compound 9, whose benzothiophene ring is very similar in
size and geometry to the naphthalene amide 4, binds 5-fold
more weakly for reasons that are not clear. The second
imidazopyridine, 10, may be able to interact more beneficially
in the region of the benzothiazole, but docking experiments
point to the altered spatial relationship of the key polar binding
interactions of the amino benzothiazole and the picolinate as
the reason for its lack of activity. It is also possible that an
intramolecular hydrogen bond between the amide NH and the
imidazopyridine leaves the amide unable to interact with
Ser106.
Having failed to improve upon the amides through altered
ring size, we returned to urea 7, the highest affinity binder in
the first set of ligands prepared. This compound demonstrates
that the inclusion of a saturated ring can preserve the key
binding interactions. Interchanging the position of the saturated
and unsaturated rings was proposed in order to allow the amide
carbonyl oxygen additional flexibility, enabling the amide NH
to optimally engage Ser106. When synthetic considerations
were taken into account, tetrahydroisoquinolines 13 and 14
were selected as our next targets.
The starting point for synthesis of these compounds was the
Boc-protected tetrahydroisoquinoline 11, which was coupled to
2-aminobenzothiazole using standard peptide coupling con-
Figure 4. Tetrahydroisoquinolines with and without extensions into
ditions. Removal of the Boc group gave 12, which could be
the P4 pocket along with AlphaScreen measured IC50 values for BCL-
coupled to t-butyl-6-fluoropicolinate or to methyl 2-chlorothia-
XL. Compounds 19 and 20 were synthesized as the trans isomer.
zole-4-carboxylate to give compounds 13 and 14, respectively,
following hydrolysis of the ester. When evaluated for their
ability to bind BCL-XL, these compounds were found to exhibit
significantly improved affinity (Figure 4). Compound 14 is
particularly potent and exhibits an IC50 value only 7-fold higher
than that of the hydrazone 2, the starting point for our
rescaffolding experiments.
Having minimized the loss of potency resulting from the
rescaffolding effort, we next sought to improve the binding
affinity of our molecules through extension into a known,
adjacent binding region on the protein. Multiple structures,
determined both as NMR solution structures17,18 and as X-ray
cocrystal structures,16,19 have shown that there exists a
lipophilic P4 pocket in the region immediately adjacent to
the picolinic acid region of our molecules. We therefore sought
to extend the tetrahydroisoquinoline analogues from the 3-
position of the picolinic acid 13 and from the 5-position of the
thiazole 14 into this pocket.
Following their synthesis (described in Schemes S9−S16,
Supporting Information), compounds 15−23 provided a clear
picture of the optimal length and flexibility required to take
advantage of the P4 pocket. Beginning from the picolinic acid,
compounds 15−20 showed improvements in affinity as the
number of heavy atoms included before the phenyl was
increased from two to four. Adding a further methylene
reduced the potency greater than 2-fold, indicating that the
entropic cost of organizing the longer carbon chain and/or a
reduced enthalpy of binding due to inappropriate positioning of
the P4 binding element resulted in a reduction in affinity for the
target.
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The potency of these compounds (Scheme 1) was further
improved through the incorporation of a trans double bond
that inhibition of the lone survival factor BCL-XL induces
apoptosis. Treatment of these cells with compounds 1 and 2
produces IC50 values of 0.29 and 0.26 μM, respectively. As
would be expected from a compound with 10-fold reduced
affinity in the Alpha-Screen assay, compound 13 has an IC50
value of 2.4 μM (Table 1). Compound 22 has a 14 nM IC50 in
this assay and is therefore significantly more active than the
compounds from which it was derived.
a
Scheme 1. Synthesis of Tetrahydroisoquinolines 13 and 14
The selectivity of our compounds was assessed through
surface plasmon resonance binding experiments. Curiously,
although our core compounds 13 and 14 are highly selective for
BCL-XL over other pro-survival proteins, compound 22 bound
BCL-w unexpectedly well with a Kd of 62 nM. This still results
in greater than 12-fold selectivity due to its increased affinity for
BCL-XL, but additional SAR studies of the P4 binding group
and its linker will be required to improve selectivity against
BCL-w.
We next characterized the pharmacokinetics of the
rescaffolded molecules in rats. We were gratified to find that
both compounds 13 and 14 exhibited low clearance and long
half-lives when dosed intravenously at 1 mg/kg (Table 1).
Compound 13 was found to have excellent oral bioavailability
(F%) with compound 14 being significantly lower. When the
phenoxy alkyl tail was appended to the scaffold, clearance was
increased more than 10-fold to 7.4 mL min−1 kg−1 and oral
bioavailability was reduced nearly to zero. However, the
enhanced stability of the core in vivo gave us confidence that
these parameters could be improved upon through further
optimization of the portion of the molecule accessing the P4
pocket, initially by reducing its high clogP and multiple
metabolic soft spots.
To further our understanding of the interactions that we had
sought to optimize, the structure of compound 20 was
determined to 2.35 Å resolution in complex with BCL-XL
(Figure 5). This structure confirmed that our molecules were
indeed binding in a fashion very similar to hydrazone 1. The
hydrogen bonds from the 2-aminobenzothiazole to Ser106 and
Leu108 are very similar to those observed in the structure of 1
bound to BCL-XL. The tetrahydroisoquinoline core packs
against Leu130 and Phe105 much as 1 does, and the
interactions of Asn136 and Arg139 with the picolinic acid
carbonyl group are preserved.
a
Reagents and conditions: (a) benzo [d]thiazol-2-amine, EDCI,
DMAP, DCM; (b) 2 N HCl/Et2O, DCM; (c) t-butyl 6-
fluoropyridine-2-carboxylate, Cs2CO3, DMA, sieves, 100 °C, 29%;
(d) HCl, EtOH, H2O, 67%; (e) methyl 2-chlorothiazole-4-carboxylate,
Cs2CO3, DMA, 50 °C, 67%; (f) 2 N NaOH, THF/MeOH, 50 °C.
into the carbon chain, reducing the number of degrees of
freedom available to the spacer and thereby lowering the
entropy of binding. This reduction led to a nearly 7-fold
improvement in the IC50 of the two-atom spacer and to an 8-
fold improvement in the IC50 of the three-atom spacer. Parallel
results were obtained for the thiazole containing compounds 14
and 21−23. Inclusion of the optimal four-atom spacer and a
phenyl ring produced the 1 nM inhibitor 22; increasing the
length of the spacer to five atoms led to a 7-fold increase in the
measured IC50, again suggesting that a four atom linker is
optimal.
Key compounds 13, 14, and 22 were subsequently examined
for their ability to induce apoptosis in an engineered cell line.
All three compounds are highly selective for BCL-XL over the
closely related pro-survival proteins BCL-2, BCL-w, and Mcl-1.
As previously described,16 mouse embryonic fibroblast (MEF)
cells were engineered to be MCL-1 deficient (mcl-1−/−) such
In addition to the expected interactions, we were able to
observe for the first time the interactions of our ligand with the
P4 pocket. The pocket is flanked by aromatic and hydrophobic
side chains including Phe97, Tyr101, Val141, and Tyr195,
which all make van der Waals’ contacts with the phenyl ring.
The only significant residue shift was seen for Tyr101, which
rotates and shifts away from the P4 pocket’s center to allow
insertion of the phenyl ring. We were intrigued to observe the
position of Glu96 along the vectors extending from the 3- or 4-
a
Table 1. Selectivity, Cellular, and Pharmacokinetic Data Obtained in Rat for Compounds 13, 14, and 22
rat PK
surface plasmon resonance Kd (μM)
iv (1 mg/kg)
CLp (mL min−1 kg−1
Vss (L/kg)
po (5 mg/kg)
EC50 Mcl-1−/− MEF, 1% FBS (μM)
BCL-xL
BCL-2
BCL-w
Mcl-1
)
t1/2 (h)
F%
13
14
22
2.4
0.038
0.010
<0.005
>20
9.2
>20
7.8
>20
>20
14.4
0.20
0.47
7.4
0.12
0.16
0.31
8.3
6.0
3.6
60
16
0.2
n/d
0.014
4.4
0.062
a
Selectivity was measured using a surface plasmon resonance competition assay (n = 3). n/d indicates that the compound was not evaluated in a
given assay.
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ACS Medicinal Chemistry Letters
Letter
Present Addresses
∇(S.K.) Monash University, Chemistry Department, Clayton,
VIC 3800, Australia.
#(J.B.B.) Peter MacCallum Cancer Centre, Melbourne, VIC
3006, Australia.
¶(C.S.R.) The Institute of Cancer Research, Belmont, Sutton,
Surrey, SM2 5NG, U.K.
◆(B.J.S.) La Trobe Institute for Molecular Science, La Trobe
University, VIC 3086 Australia.
Funding
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 J.M.A., J.B.B., P.M.C., and D.C.S.H.; 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
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.
Figure 5. Interactions of compound 20 with BCL-XL observed in a
2.35 Å resolution crystal structure. The surface displayed is one van
der Waal’s radius above the analytic Connolly surface of the protein.
Hydrogen bonds are indicated by dashed lines.
Notes
position of the phenyl ring, which suggested additional polar
contacts that might be accessible via the addition of functional
groups to this scaffold.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
In summary, application of a combination of docking studies
and rational design enabled the conversion of an undesirable
hydrazone-based core to amide and urea-based ligands, which
preserve many of the binding interactions and nearly all of the
affinity of the parent compounds. As exemplified by
compounds 13 and 14, these rescaffolded molecules exhibit
significantly better clearance and oral bioavailability than the
parent hydrazones 1 and 2 when dosed in rats.
Building on this more desirable core, both the biochemical
and cellular affinity of the rescaffolded molecules for BCL-XL
was increased through incorporation of aryl rings at the 3-
position of the picolinic acid or the corresponding 5-position of
the thiazole ring. These were demonstrated to be interacting as
designed with the lipophilic P4 pocket and increased the affinity
of our compounds for BCL-XL to single-digit nanomolar levels
in the best case. This improved affinity was also reflected in
activity in our mcl-1−/− MEF cell line, with the most potent
molecule exhibiting an IC50 of 14 nM, a nearly 20-fold
improvement in cellular potency. Having determined the
structure of 20 in complex with BCL-XL, we confirmed our
hypotheses about the binding mode of these molecules and
observed additional possibilities for refinement, which will be
reported in due course.
■
We thank members of the DMPK and purification groups
within Genentech Small Molecule Drug Discovery for analytical
support and Ahmad Wardak and Geoff Thompson for technical
support.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic schemes, procedures, and analytical data describing
the preparation of compounds 3−23 as well as experimental
procedures for determining IC50 and EC50 values and PK. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Enschede, S. H.; Humerickhouse, R. Substantial susceptibility of
chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I
AUTHOR INFORMATION
Corresponding Author
*(M.F.T.K.) Phone: 650-225-8135. Fax: 650-467-8922. E-mail:
mkoehler@gene.com.
■
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