Utilizing PROTAC technology to address the on-target platelet toxicity associated with inhibition of BCL-X

Article abstract of DOI:10.1039/c9cc07217a

BCL-X_L, an anti-apoptotic BCL-2 family protein, plays a key role in cancer cell survival. However, the potential of BCL-X_L as an anti-cancer target has been hampered by the on-target platelet toxicity because platelets depend on BCL-X_L to maintain their viability. Here we report the development of a PROTAC BCL-X_L degrader, XZ424, which has increased selectivity for BCL-X_L-dependent MOLT-4 cells over human platelets compared with conventional BCL-X_L inhibitors. This proof-of-concept study demonstrates the potential of utilizing a PROTAC approach to achieve tissue selectivity.

Full text of DOI:10.1039/c9cc07217a

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Utilizing PROTAC technology to address the
on-target platelet toxicity associated with
inhibition of BCL-XL†
Cite this: Chem. Commun., 2019,
55, 14765
Received 14th September 2019,
Accepted 12th November 2019
Xuan Zhang, ‡a Dinesh Thummuri,‡b Yonghan He,b Xingui Liu,b Peiyi Zhang,a
a
Daohong Zhoub and Guangrong Zheng
*
DOI: 10.1039/c9cc07217a
rsc.li/chemcomm
BCL-XL, an anti-apoptotic BCL-2 family protein, plays a key role in subset of leukemia and lymphoma cells.14 In addition, it has been
cancer cell survival. However, the potential of BCL-XL as an anti- well established that BCL-XL inhibition can sensitize cancer cells
cancer target has been hampered by the on-target platelet toxicity to chemotherapies.15–17 More recently, we and others discovered
because platelets depend on BCL-XL to maintain their viability. Here that BCL-2/BCL-XL dual inhibitors, navitoclax (also known as
we report the development of a PROTAC BCL-XL degrader, XZ424, ABT-263) and ABT-737, and BCL-XL specific inhibitors,
which has increased selectivity for BCL-XL-dependent MOLT-4 cells A-1331852 and A-1155463, are able to selectively kill senescent
over human platelets compared with conventional BCL-XL inhibitors. cells (SnCs).18–20 This is because BCL-XL is a key anti-apoptotic
This proof-of-concept study demonstrates the potential of utilizing a protein in many types of SnCs. Accumulating evidence indicates
PROTAC approach to achieve tissue selectivity.
that cellular senescence plays an important role in many age-
related pathologies.21 Studies on ABT-263 in mouse models have
The B-cell lymphoma 2 (BCL-2) family proteins, consisting of demonstrated that clearance of chemotherapy-induced SnCs
pro- and anti-apoptotic members, play a critical role in deter- reduces several short- and long-term adverse effects of the
mining cell life and death through regulation of the intrinsic therapy, and inhibits cancer relapse and metastasis.22 Thus,
apoptotic pathway.1 The anti-apoptotic BCL-2 family proteins, BCL-XL has also been considered as a promising therapeutic
including BCL-2, BCL-XL, and MCL-1, are upregulated in many target for the treatment of a range of age-related diseases and
human cancers and associated with tumor initiation, progression, cancer therapy-induced adverse effects.
and resistance to chemotherapy and targeted therapies.2,3 These
However, the clinical applications of BCL-XL specific or
proteins inhibit apoptosis by binding the a-helical BCL-2 BCL-2/BCL-XL dual inhibitors currently in development are greatly
homology-3 (BH3) domain of pro-apoptotic proteins Bax and limited by their on-target and dose-limiting thrombocytopenia
Bak, thereby preventing their activation of the mitochondrial toxicity. This is because platelets are solely dependent on BCL-XL
apoptotic pathway.4 Thus, inhibiting the protein–protein inter- for survival.23,24 Thus, traditional structural modifications of
action between anti- and pro-apoptotic BCL-2 proteins, and BCL-XL inhibitors are unlikely to address this on-target toxicity.
thus overcoming the apoptotic resistance of cancer cells, is a Here we report the utilization of proteolysis targeting chimera
highly attractive cancer therapeutic strategy.5–7
Significant progress has been made in developing ‘‘BH3 associated with targeting BCL-XL.
(PROTAC) as an approach to minimize the platelet toxicity
mimetic’’ small-molecule inhibitors of the anti-apoptotic BCL-2
Originally described by Crews and Deshaies in 2001,25 PROTAC
proteins.8–11 Importantly, this therapeutic strategy has been has emerged as a powerful drug discovery technology.26,27
validated by the FDA approval of venetoclax, a BCL-2 specific PROTACs are bivalent small-molecules containing a pharmaco-
inhibitor, for chronic lymphocytic leukemia in 201612 and acute phoric unit that recognizes the target protein linked to a second
myeloid leukemia in 2018.13 BCL-XL is the most common BCL-2 pharmacophoric unit that binds to a specific E3 ubiquitin
family member overexpressed in solid tumors, as well as in a ligase. They can recruit the target protein to an E3 ligase, promote
proximity-induced ubiquitination of the target protein, and lead
a Department of Medicinal Chemistry, College of Pharmacy, University of Florida,
1333 Center Drive, Gainesville, FL, 32610, USA. E-mail: zhengg@cop.ufl.edu
b Department of Pharmacodynamics, College of Pharmacy, University of Florida,
1333 Center Drive, Gainesville, FL, 32610, USA
to its degradation through the ubiquitin proteasome system
(UPS).28 Because PROTACs rely on E3 ligases to induce protein
degradation, it is possible for them to achieve cell/tissue selectivity
even when their target proteins are ubiquitously expressed, if they
target an E3 ligase that is differentially expressed in different
cells or tissues. To our delight, by analysing published human
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9cc07217a
‡ These authors contributed equally.
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Scheme 1 (i) 4 or 5, CuSO4Á5H2O, sodium L-ascorbate, tBuOH, THF,
and H2O, at 55–65 1C. (ii) Chloromethyl methyl ether, Na2CO3, and
DMF. (iii) HCl in 1,4-dioxane, DCM, and MeOH.
Fig. 1 (A) Left panel: representative immunoblot analyses of CRBN
expression in three human cancer cell lines and human platelets from
three individuals indicated by units 1–3; right panel: densitometric analyses
of CRBN expression. (B) X-ray crystal structure of A-1155463 (green)
bound to BCL-XL (PDB code: 4QVX). (C) Structures of A-1155463, com-
pounds 1–3 and XZ424. (D) Binding affinities of various compounds to
BCL-XL; n Z 2.
alkaline hydrolysis conditions. Methoxymethyl (MOM) ester 6,
which was converted from 3 in an 84% yield, was then employed.
The click reaction between 6 and 4, as well as the following
cleavage of the MOM group under mild acidic conditions,
platelet RNA-seq data,29,30 we found that cereblon (CRBN), one underwent smoothly to afford XZ424 (84%, 2 steps).
of the two most popular E3 ligases being recruited by PROTACs As expected, XZ424 had similar BCL-XL binding affinity
to induce targeted protein degradation, is modestly expressed compared with A-1155463 (Fig. 1D). The BCL-XL degradation
in human platelets. This finding was confirmed by Western ability of XZ424 was examined in MOLT-4, a human T-cell acute
blot, which indicates a significantly lower CRBN level in human lymphoblastic leukemia cell line primarily dependent on BCL-XL
platelets compared to a number of cancer cell lines (Fig. 1A).
for survival.33 XZ424 dose-dependently induced BCL-XL degrada-
Through the analysis of the co-crystal structure of A-1155463, a tion in MOLT-4 cells, with a DC50 value (the concentration for 50%
potent and selective BCL-XL inhibitor (Fig. 1C), in complex with protein degradation) of 50 nM under 16 h treatment (Fig. 2A). In
BCL-XL (Fig. 1B),31 we found that the N,N-dimethylamino moiety on contrast, no significant changes in BCL-XL protein levels were
A-1155463 is solvent-exposed, thus representing a potential linker observed in human platelets treated with up to 1.0 mM of XZ424
tethering position. To confirm that this position is amenable to for 16 h (Fig. 2B). In addition, the BCL-XL degradation induced by
linker attachment without a major loss of BCL-XL binding affinity, XZ424 in MOLT-4 was time-dependent, starting within 2 h, and
we synthesized compounds 1 and 2, in which the dimethylamino after drug treatment for 16 h, more than 85% protein was
group was replaced with a piperazine ring, and compound 3, an degraded with 100 nM of XZ424 (Fig. 2C). The effects of XZ424
azide derivative of 2 (Fig. 1C and Scheme S1, ESI†). All three on BCL-XL protein levels in MOLT-4 were long-lasting and also
compounds exhibited BCL-XL binding affinities, measured by a reversible, as indicated in the ‘‘washout’’ assay (Fig. 2D). Further-
bead-based AlphaScreen competition binding assay,32 that are more, pre-incubation of MOLT-4 cells with an excess of the CRBN
comparable to A-1155463 (Fig. 1D). We therefore generated a ligand pomalidomide (POM) or a proteasome inhibitor MG132
focused series of PROTACs by conjugating 3 with the CRBN ligand blocked XZ424-induced BCL-XL degradation (Fig. 2E), indicating
pomalidomide (POM). The most potent PROTAC in inducing that the degradation depends on both CRBN E3 ligase and the
BCL-XL degradation, XZ424 (Fig. 1C), was selected for the proof- UPS. To further confirm that the CRBN E3 ligase is involved in
of-concept studies described below.
XZ424-induced BCL-XL degradation, we synthesized a negative
XZ424 was initially synthesized via Huisgen cycloaddition of control compound XZ424-NC (Scheme 1), in which a methyl
azide 3 with alkyne 4 derived from POM (Scheme 1). The group is installed on the amino group in the POM moiety of
reaction suffered from low yields and the product was difficult XZ424. It has been shown that adding the methyl to thalidomide
to purify, due to the presence of the carboxylic acid group which analogues abolishes their binding to CRBN.28f,34 Not surprisingly,
is attributed to the low solubility of XZ424 in organic solvents. XZ424-NC did not induce BCL-XL degradation in MOLT-4 cells
Azide 3 was then converted to the corresponding benzyl and ethyl (Fig. 2F).
esters. The cycloaddition of both esters with 4 and the subsequent
We next evaluated the effects of XZ424 on the viability
purification of the products were straightforward. However, of MOLT-4 and human platelets, along with A-1155463 and
removal of the benzyl and ethyl groups from the corresponding ABT-263. As expected, A-1155463 and ABT-263 exhibited no selec-
product appeared to be difficult due to the presence of a triple tive cytotoxicity for MOLT-4 over platelets (Fig. 3A), confirming the
bond, which prevents the use of hydrogenolysis for the cleavage on-target platelet toxicity of BCL-XL inhibitors. In contrast, XZ424
of benzyl groups, and an imide moiety that is unstable under showed potent cytotoxicity against MOLT-4 cells with an IC50 value
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Fig. 2 XZ424-induces BCL-XL degradation. (A) Western blot showing the
BCL-XL protein levels in MOLT-4 cells treated with the indicated concen-
tration of XZ424 for 16 h. (B) Western blot analysis of BCL-XL levels after
treatment of human platelets with the indicated concentration of XZ424
for 16 h. (C) Time-dependent experiments in MOLT-4 cells after treatment
with 100 nM XZ424 at the indicated time points. (D) MOLT-4 cells were
incubated with 100 nM of XZ424 for 16 h followed by drug washout,
resuspension and incubation of the cells for an additional time as indicated
in drug-free medium. (E) Pretreatment with 10 mM pomalidomide (POM) or
1 mM MG132 for 2 h blocked the degradation of BCL-XL by XZ424. Data are
presented as representative figures of two independent experiments.
(F) Western blot analysis of BCL-XL in MOLT-4 cells treated with XZ424-NC
at the indicated concentrations for 16 h.
Fig. 4 Characterization of XZ424 mediated apoptosis in MOLT-4 cells. (A)
Western blot analysis of PARP and caspase-3 after XZ424 treatment for
16 h. (B–D) Flow cytometry analysis of apoptosis using Annexin-V and PI
staining. Cells were treated with DMSO and XZ424 (100 nM) for 48 h, and
XZ424 (100 nM) significantly increased the percentage of apoptotic cells
and QVD (10 mM) pre-treatment for 2 h inhibited the apoptosis induced by
XZ424. Data are representative figures of two independent experiments.
pre-incubation of MOLT-4 with POM resulted in 11-fold
reduction of the cytotoxicity of XZ424 (Fig. 3C), suggesting that
the effect of XZ424 on MOLT-4 viability is largely derived from
BCL-XL degradation.
Western blot analysis showed that XZ424 dose-dependently
increased the poly (ADP-ribose) polymerase (PARP) cleavage and
caspase-3 cleavage in MOLT-4 cells (Fig. 4A), suggesting the
apoptotic cell-death mechanism. Furthermore, to determine that
XZ424 induces cell death through caspase mediated apoptosis,
we did flow cytometry analysis of apoptosis using Annexin V and
propidium iodide (PI) staining. We found that 100 nM of XZ424
treatment for 48 h significantly increased the percentage of
Annexin-V-positive cells in MOLT-4 cells compared to the vehicle
group (Fig. 4B and C), whereas pretreatment with 10 mM of the
pan-caspase inhibitor Q-VD-OPh (QVD) for 2 h inhibited the
XZ424 induced apoptosis, which confirms that XZ424 induces
cell death through caspase-dependent apoptosis (Fig. 4D).
Taken together, we demonstrate the development of novel
BCL-XL-PROTACs that can degrade BCL-XL selectively in MOLT-4
cells but not in platelets. Western blot analyses confirmed that the
PROTACs induced BCL-XL protein degradation in a dose- and
Fig. 3 Human platelet toxicity studies. (A) Platelets and MOLT-4 cells
were cultured with BCL-XL inhibitors or XZ424 for 24 h and 72 h,
respectively. The selectivity for MOLT-4 over platelets was calculated.
(B and C) The competitive cytotoxicity assay with 10 mM POM co-treatment
in platelets and MOLT-4 cells. Data are presented as mean Æ SD (n = 3
replicates) of two independent experiments.
of 51 nM and a 22-fold selectivity over platelets (Fig. 3A). The time-dependent manner, and mediated by E3 ligase and UPS.
improved selectivity of XZ424 in comparison to A-1155463 is Compared with conventional BCL-XL inhibitors, XZ424 possesses
likely due to the different BCL-XL degradation efficiency in a unique selectivity for MOLT-4 cells over platelets, suggesting
MOLT-4 and platelets. The cytotoxicity of XZ424 to platelets that an improved therapeutic window can be achieved by the
most likely derived from BCL-XL inhibition rather than degra- conversion of an inhibitor into a PROTAC. This study demon-
dation as pre-incubation of platelets with POM did not affect strated an additional utility of the PROTAC technology. A similar
the cytotoxicity of XZ424 to platelets (Fig. 3B). Since XZ424 and strategy could be used to reduce on-target toxicities of other
A-1155463 had similar binding affinity to BCL-XL, the largely antitumor agents by taking advantage of the tissue-specific
reduced toxicity of XZ424 to platelets is likely due to a decrease expression of E3 ligases. In addition, because XZ424 is a potent
in cell permeability compared to A-1155463. On the other hand, and selective BCL-XL degrader, it might be a useful toolkit to
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This work was supported in part by NIH grants R01CA211963,
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Conflicts of interest
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M. J. Roy, C. Whitworth, E. Diers, N. Trainor, D. Zollman, S. Steurer,
J. Karolyi-Oezguer, C. Riedmueller, T. Gmaschitz, J. Wachter, C. Dank,
M. Galant, B. Sharps, K. Rumpel, E. Traxler, T. Gerstberger, R. Schnitzer,
O. Petermann, P. Greb, H. Weinstabl, G. Bader, A. Zoephel, A. Weiss-
X. Z., D. T., X. L., P. Z., D. Z. and G. Z. are inventors of pending patent
application(s) for the use of BCL-XL PROTACs as anticancer agents.
D. Z. and G. Z. are co-founders of and have equity of Dialectic
Therapeutics, which develops BCL-XL PROTACs to treat cancer.
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