Induction of apoptosis in MDA-MB-231 breast cancer cells by a PARP1-targeting PROTAC small molecule

Article abstract of DOI:10.1039/c8cc07813k

Poly (ADP-ribose) polymerase-1 (PARP1) is a major member of the PARP superfamily that is involved in DNA damage signalling and other important cellular processes. Here we report the development of a small molecule targeting PARP1 based on the PROTAC strategy. In the MDA-MB-231 cell line, the representative compound 3 can induce significant PARP1 cleavage and programmed cell death.

Full text of DOI:10.1039/c8cc07813k

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Induction of apoptosis in MDA-MB-231 breast
cancer cells by a PARP1-targeting PROTAC small
molecule†
Cite this:DOI: 10.1039/c8cc07813k
Received 30th September 2018,
Accepted 5th December 2018
b
a
Qiuye Zhao, ‡a Tianlong Lan, ‡a Shang Su
and Yu Rao
*
DOI: 10.1039/c8cc07813k
rsc.li/chemcomm
Poly (ADP-ribose) polymerase-1 (PARP1) is a major member of the of breast cancers especially triple negative breast cancer (TNBC),
PARP superfamily that is involved in DNA damage signalling and in consideration of its poor prognosis, high resistance to stan-
other important cellular processes. Here we report the development dard treatment and complicated heterogeneity, other thera-
of a small molecule targeting PARP1 based on the PROTAC strategy. peutics with novel mechanisms are still highly needed.22
In the MDA-MB-231 cell line, the representative compound 3 can
induce significant PARP1 cleavage and programmed cell death.
Proteolysis-targeting chimeras (PROTACs) have emerged
as a novel and valuable tool for the chemical knockdown of
a protein of interest. The concept can be dated back to 2001
Poly (ADP-ribose) polymerases (PARPs) are DNA-dependent when Crews reported the first example of seeking to artificially
nuclear enzymes that transfer negatively charged ADP-ribose target a protein to an E3 complex for ubiquitination and
moieties from cellular nicotinamide-adenine-dinucleotide (NAD+) degradation.23 PROTAC molecules (PROTACs) are heterobi-
to a variety of protein substrates.1–3 Poly (ADP-ribose) polymerase-1 functional small molecules consisting of a target protein binding
(PARP1) is a fundamental member of the enzyme superfamily.4 ligand, an E3 recruiting ligand and an interval linker (Fig. 1A).
Following DNA damage, PARP1 can rapidly sense and bind to These molecules are capable of bringing the target protein close
single-strand breaks (SSBs). Then, SSBs are repaired by the base to an E3 ligase of interest, causing consequent degradation of
excision repair (BER) pathway. Inactivation of SSB repair by PARP1 the target protein.24 In 2015, the Bradner25 and Crews26 groups
inhibition can cause the formation of DNA double-strand breaks independently demonstrated the efficient degradation of BRD4 by
after stalling and collapse of progressing DNA replication forks.5–7
Due to its pivotal role in DNA damage response, PARP1 is
considered as a suitable therapeutic target for the potential
treatment of cancers.8 The rationale for PARP-targeting thera-
peutics in cancer treatment is the selective induction of synthetic
lethality in some BRCA1/2 mutant cancers (especially, ovarian
cancer and breast cancer).9–11 Currently, a number of PARP1
inhibitors, such as olaparib,12–14 niraparib15,16 and iniparib17,18
,
are under development in different stages of clinical trials.19,20
Among these inhibitors, olaparib has been approved for the
treatment of germline BRCA mutated (gBRCAm) advanced
ovarian cancer and gBRCAm metastatic breast cancer. Although
the synthetic lethal strategy is very promising, BRCA1 or BRCA2
mutant cancers only account for 3–5% of all breast cancers and a
greater proportion of ovarian cancers.21 For the remaining part
a MOE Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences,
MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology,
Tsinghua University, Beijing 100084, P. R. China. E-mail: yrao@tsinghua.edu.cn
b MOE Key Laboratory of Protein Sciences, School of Life Sciences,
Tsinghua University, Beijing 100084, P. R. China
Fig. 1 Design of PROTACs for PARP1. (A) Schematic representation
of protein degradation mediated by proteolysis targeting chimeras
(PROTACs). (B) Crystal structure of niraparib bound to the PARP1 catalytic
domain (PDB code: 4R6E). (C) A ‘‘click chemistry’’ strategy was utilized to
construct the PROTAC candidates targeting PARP1.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc07813k
‡ These authors contributed equally.
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PROTACs employing pomalidomide as the CRBN E3 ligase assisted click reaction and the yielded 1,2,3-triazol group may to
ligand, which has encouraged more to develop PROTACs some extent mimic the binding mode of the piperidine ring of
targeting other proteins, such as ALK,27,28 BCR-ABL,29,30 CDK9,31,32 niraparib to PARP1 (Fig. 1C). As for the E3 ligase binding part, 11,
and TRIM24.33 In our previous study, we developed efficient a racemic mixture of nutlin-3 (enantiomer a IC50 = 13.6 mM,
PROTACs targeting BTK, which are also effective to degrade enantiomer b IC50 = 0.09 mM), was synthesized using the MDM2
mutant C481S BTK and may be used as a novel therapy for recruiting moiety based on a literature reported method.35
ibrutinib-resistant B-cell malignancies induced by mutation.34 Afterwards, the condensation of 11 with amine 12 yielded the
Considering the promising therapeutic utility of degrading azido intermediate 13. Finally, through a ‘‘click chemistry’’
PARP1, the successful examples of PROTACs reported so far method, an enantiomeric mixture of compound 3 was prepared
have encouraged us to develop potential PARP1 degraders.
as a potential PARP1 degrader. In addition, an alkyne analogue
To develop PARP1 targeting PROTACs, a PARP1 ligand should of olaparib was synthesized as another PARP1 binding ligand.
be tethered to an E3 ubiquitination ligase ligand through a Other E3 ligase binding ligands targeting CRBN or VHL were
flexible linker. By simultaneously binding to PARP1 and E3 also employed in this study. Subsequently, through the ‘‘click
ligase, the PROTACs should bridge the unnatural interaction chemistry’’ strategy, PARP1 targeting PROTAC candidates 1, 2, 4
between PARP1 and the E3 ligase to enable the ubiquitination and 5 were synthesized as well (for details, please see the ESI†).
and subsequent proteasome degradation of PARP1. To initiate
All PROTACs were then tested for PARP1 degradation in
the study, niraparib was selected as the PARP1 binding moiety. TNBC cell lines. Among the different tested TNBC cell lines
The analysis of the crystal structure of niraparib in the complex (Fig. S1, ESI†), it was found that compound 3 could selectively
with PARP1 (Fig. 1B) suggests that the piperidine ring on induce significant PARP1 cleavage in the MDA-MB-231 cell line
niraparib experiences the opening of the ligand binding (Fig. 3A). Control assays clearly demonstrated that niraparib,
pocket and thus may represent a suitable site for modification nutlin-3 or their combination at equimolar or higher concentra-
without losing too much binding affinity. As a consequence, tions could not induce the cleavage of PARP1 in MDA-MB-231
intermediate 6, an alkyne analogue of niraparib was designed as cells (Fig. 3A).
a synthetic precursor to PARP1 PROTACs. The synthesis of 10 is
To explore whether the cleavage of PARP1 is dependent on
depicted in Fig. 2. The radical benzylic bromination of 6 followed binding to both PARP1 and MDM2, we pre-treated MDA-MB-231
by the oxidation of benzyl bromide yielded 7. Then, substitution cells with niraparib at 50 mM or nutlin-3 at 20 mM. It was found
of the nitro group with the azido group gave 8. Heating 1c with that niraparib abolished compound 3 induced PARP1 cleavage
commercially available 4-ethynylaniline at 100 1C in DMF under successfully while nutlin-3 only partially blocked PARP1 cleavage
acidic conditions generated intermediate 9. Subsequent ammono- (Fig. S2A and B, ESI†). Higher concentrations of nutlin-3 might be
lysis of 9 produced intermediate 10. The advantage of employing more effective in blocking the PARP1 cleavage, however it is toxic
intermediate 10 as a precursor is that the desired PROTACs can be to the cells. As MDM2 is a RING-type E3 ubiquitin ligase,36
readily obtained from intermediate 10 through a versatile copper we used MLN4924, an inhibitor of the NEDD8-activating enzyme
Fig. 2 The synthesis of compound 3 and schematic representation of compounds (i) NBS, AIBN, MeCN, reflux, and 3 h. (ii) 4-Methylmorpholine N-oxide,
MeCN, and 3 h. (iii) NaN3, DMF, 80 1C, and 1 h. (iv) 4-Ethynylaniline, HOAc, DMF, 100 1C, and 12 h. (v) Ammonium hydroxide, EtOH, 70 1C, and 11 h.
(vi) (a) HATU, Et3N, DMF/DCM, and 5 min; (b) 14-azido-3,6,9,12-tetraoxatetradecan-1-amine, and 5 h. (vii) CuSO4, sodium ascorbate, DMF/H2O, and 6 h.
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Fig. 4 Effect of compound 3 and PARP1 inhibitors on the cell viability.
(A and B) CCK-8 assay was performed after incubation of MDA-MB-231
cells (5 Â 103 cells per well) with serially diluted compound 3, niraparib,
nutlin-3 or in combination for 24 h and 48 h in 96-well plates in triplicate.
(C) CCK-8 assay was performed after incubation of MDA-MB-231 cells
(5 Â 103 cells per well) with serially diluted compound 3, niraparib, olaparib
and veliparib for 48 h in 96-well plates in triplicate. (D) Viability of BEAS-2B,
MCF10A, HeLa and HepG2 cells that were cultured with serially diluted
compound 3, niraparib, nutlin-3 or in combination for 48 h in 96-well
plates was measured by a CCK-8 assay. Data are mean Æ SD of 3 inde-
pendent experiments.
Fig. 3 Characterization of compound 3 mediated PARP1 degradation and
apoptosis in MDA-MB-231 cells. (A) Immunoblot of PARP1 and actin
following 24 h incubation with DMSO or indicated small molecules.
(B) Immunoblot of PARP1 and actin following 24 h incubation with DMSO
or the indicated concentrations of compound 3 in MDA-MB-231 cells.
(C and D) MDA-MB-231 cells were treated with DMSO and compound 3
(10 mM) for 24 h, and apoptosis was assayed by flow cytometry after
annexin-V/PI co-staining. The annexin V/PI intensity dot plots show a
significantly increased dot intensity in the Q2 region after treatment with
compound 3 (10 mM) for 24 h. (E) Immunoblot for PARP1, cleaved caspase
3 and actin after treatment with DMSO, compound 3 (10 mM), nutlin-3
(10 mM), niraparib (10 mM) or a combination of nutlin-3 (10 mM) and
niraparib (10 mM) for 24 h.
25.19 Æ 0.62 mM and 28.64 Æ 0.89 mM, respectively) (Fig. 4B).
In addition, we also compared compound 3 with other PARP1
(NAE), to pre-treat MDA-MB-231 cells at 1 mM for 4 h, which inhibitors, including olaparib and veliparib, both of which
effectively blocked compound 3-induced PARP1 cleavage exhibited only 10% inhibition at 10 mM after 48 h treatment
(Fig. S2C, ESI†). These results verified that PARP1 cleavage (IC50 = 6.55 Æ 0.16 mM, 28.6 Æ 0.89 mM, 50.1 Æ 4.8 mM and
induced by compound 3 was dependent on both specific 90.88 Æ 10.29 mM, respectively) (Fig. 4C). Furthermore, we used
ligand-binding and proteasome.
MCF10A, a non-tumorigenic breast epithelial cell line, BEAS-2B,
Given that PARP1 cleavage is a well-known marker for cell a normal bronchial epithelium cell line, HeLa cells and HepG2
apoptosis,37–39 we conjectured that PARP1 cleavage induced cells to evaluate the cytotoxicity of compound 3. Surprisingly,
by compound 3 in MDA-MB-231 cells is a characterization of compound 3 showed 40% inhibition or no toxic effect on
cell apoptosis. To verify this hypothesis, we performed flow the cell viability even at 100 mM after incubation for 48 h.
cytometric analysis to evaluate the occurrence of apoptosis. Meanwhile, there was no significant change of the PAPR1
After incubation of 10 mM compound 3 for 24 h and then protein in theses cell lines detected by immunoblot (Fig. S4,
staining with annexin V-FITC/PI, we could significantly detect ESI†), which indicated that compound 3 exhibited very high
enhanced PS externalization in MDA-MB-231 cells (Fig. 3C and D). selectivity (Fig. 4D).
Furthermore, the appearance of cleaved caspase-3 also indicated
In conclusion, we have reported the development of a PROTAC-
that compound 3 could induce an enhanced apoptotic response type small molecule based on a niraparib derivative and nutlin-3
in MDA-MB-231 cells (Fig. 3E). Under the same conditions, derivative. The small molecule specifically induces PARP1 cleavage
niraparib and nutlin-3 alone or in combination failed to and cell apoptosis in the MDA-MB-231 cell line, the mechanism
externalize PS (Fig. S3, ESI†) and induce any detectable of which should be due to the PARP1 degradation or some
caspase-3 cleavage (Fig. 3E).
other complicated reasons. We propose that some genes may
Next, we examined the effect of compound 3 on the cell be mutated and defective in MDA-MB-231 cells, which leads to
viability in the MDA-MB-231 cell line. Treatment with compound synthetic lethal effects to make cells undergo suicide in the presence
3 at 10 mM inhibited the cell growth by about 70% at 24 h, of compound 3. According to our observations, compound 3 is
whereas, only marginal or no inhibitory effects were observed 5-fold more potent than niraparib, olaparib and veliparib in
upon treatment with niraparib, nutlin-3 alone or in combination MDA-MB-231 cells. Besides, compound 3 exhibited no cytotoxi-
(IC50 = 8.45 Æ 0.54 mM, 33.22 Æ 1.7 mM, 28.47 Æ 0.75 mM and city to the normal breast epithelial cells. Considering the com-
60.91 Æ 4.5 mM, respectively) (Fig. 4A). Upon prolonging the plicated heterogeneity of triple negative breast cancer (TNBC),
treatment time to 48 h, treatment with compound 3 at 10 mM these PARP 1-targeting PROTAC-type compounds are of great
produced 80–90% inhibition, whereas, niraparib, nutlin-3 or their potential application value for the therapy of the MDA-MB-231
combination produced about 30%, 30% and 40% inhibition, cells-like subtype of TNBCs. The detailed mechanism study is
respectively (IC50 = 6.12 Æ 0.204 mM, 18.2 Æ 0.35 mM, underway in our laboratory and will be reported in due course.
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This work was supported by the National Natural Science
Foundation of China (#81573277, 81622042, 81773567).
We thank Prof. Wei Wu of Tsinghua University, Prof. Jiadong
Wang of Peking University and Prof. Ming Ji of Institute of
Materia Medica, Peking Union Medical College, and the Chinese
Academy of Medical Sciences for helpful discussion.
Conflicts of interest
There are no conflicts to declare.
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