PARP inhibitor cyanine dye conjugate with enhanced cytotoxic and antiproliferative activity in patient derived glioblastoma cell lines

Article abstract of DOI:10.1016/j.bmcl.2020.127252

We describe the synthesis and in vitro activity of drug-dye conjugate 1, which is a combination of the PARP inhibitor rucaparib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed strong cytotoxic activity with nanomolar potency (EC₅₀: 128 nM), which was a 780 fold improvement over rucaparib itself. We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard drug for treatment for glioblastoma even though these cell lines were resistant to TMZ treatment. We envisage such conjugates to be worth exploring for their utility in the treatment of various brain cancers.

Full text of DOI:10.1016/j.bmcl.2020.127252

Journal Pre-proofs
PARP inhibitor cyanine dye conjugate with enhanced cytotoxic and antiproli‐
ferative activity in patient derived Glioblastoma cell lines
Peter J. Choi, Elizabeth Cooper, Patrick Schweder, Edward Mee, Clinton
Turner, Richard Faull, William A. Denny, Mike Dragunow, Thomas I-H.
Park, Jiney Jose
PII:
S0960-894X(20)30357-7
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127252
BMCL 127252
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
31 March 2020
3 May 2020
6 May 2020
Please cite this article as: Choi, P.J., Cooper, E., Schweder, P., Mee, E., Turner, C., Faull, R., Denny, W.A.,
Dragunow, M., Park, T.I-H., Jose, J., PARP inhibitor cyanine dye conjugate with enhanced cytotoxic and
antiproliferative activity in patient derived Glioblastoma cell lines, Bioorganic & Medicinal Chemistry Letters
(2020), doi: https://doi.org/10.1016/j.bmcl.2020.127252
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PARP inhibitor cyanine dye conjugate with enhanced cytotoxic and antiproliferative
activity in patient derived Glioblastoma cell lines.
Peter J. Choia*, Elizabeth Coopera,b,c*, Patrick Schwederc,d, Edward Meec,d, Clinton
Turnere, Richard Faullc, William A. Dennya, Mike Dragunow,b,c†, Thomas I-H. Parkb,c†
Jiney Josea†
,
aAuckland Cancer Society Research Centre, School of Medical Sciences, University
of Auckland, Private Bag 92019, Auckland 1142, New Zealand
bDepartment of Pharmacology & The Centre for Brain Research, University of
Auckland, Private Bag 92019, Auckland 1142, New Zealand
cNeurosurgical Research Unit, The Centre for Brain Research, University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand
dDepartment of Neurosurgery, Auckland City Hospital, Private Bag 92024, Auckland
1142, New Zealand
eDepartment of Anatomical Pathology, LabPlus, Auckland City Hospital, 2 Park Road,
Auckland, New Zealand
* Denotes equal contribution to this work and are both regarded as primary
authors.
†
Corresponding authors.

Abstract:
We describe the synthesis and in vitro activity of drug-dye conjugate 1, which is
a combination of the PARP inhibitor rucaparib and heptamethine cyanine dye IR-
786. The drug-dye conjugate 1 was evaluated in three different patient-derived
glioblastoma cell lines and showed strong cytotoxic activity with nanomolar
potency (EC50:128 nM), which was a 780 fold improvement over rucaparib itself.
We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard
drug for treatment for glioblastoma even though these cell lines were resistant
to TMZ treatment. We envisage such conjugates to be worth exploring for their
utility in the treatment of various brain cancers.
N
H
F
N
N
N
O
N
H
I
N
1
EC50: 128 nM
potent antiproliferative activity in three different
patient derived glioblastoma cell lines
Key words:
Glioblastoma, temozolomide, PARP inhibitors, rucaparib, heptamethine cyanine
dyes.
Cancer treatment has evolved since the discovery of nitrogen mustards in the 1940s to the
current targeted therapies. The understanding of the biology of tumour initiation and
progression has helped us to design drugs with improved therapeutic indexes. The
challenge we face today in drug discovery is to translate the mechanistic revelation of cancer
progression into meaningful drug design and delivery. This is particularly true for brain
cancers where chemotherapy has not been able to have a significant impact.1-2 The
multifaceted challenges in designing central nervous system (CNS) drugs and the huge
attrition rate has made it difficult to make any lasting progress in this area.3 Among brain
tumours, glioblastoma (GBM) is the most lethal, and it poses a challenge to known
therapeutic intervention.4 The median survival period of 15 to 20 months for patients
suffering from GBM has not improved in the past decade. The DNA alkylating agent,
temozolomide (TMZ) the mainstay in treating primary and recurrent GBM is ineffective in half
of the patient population. This is because approximately 50–60% of GBM tumours have
unmethylated O-6-methylguanine-DNA methyltransferase (MGMT), an enzyme which
repairs the DNA damage caused by TMZ, thereby protecting cancer cells from cytotoxic
alkylating agents.5 Expression of the gene coding for this enzyme when methylated

(epigenetic silencing) is suppressed resulting in reduced levels and hence activity of MGMT,
which promotes the cytotoxic actions of TMZ. The recent advances in understanding the
embryonic origin of brain tumours have shed light on the fact that mechanisms that regulate
stem cell behaviour in the embryo could be sabotaged in a tumour microenvironment leading
to increased proliferation and loss of cellular distinction.6-7 The subtle difference in normal
and malignant brain tissues makes it difficult for meaningful therapeutic intervention without
invoking damage to surrounding normal brain tissues. In this scenario, a selective approach
such as targeted drug delivery would be highly desirable. Although this has brought many
benefits in treating peripheral cancers,8-9 in the field of brain cancers, there is much to be
achieved.
Among recently emerging targeted therapies, the poly ADP ribose polymerase (PARP)
inhibitors have shown promise in treating cancer such as ovarian10-11 and breast cancer12
with many undergoing clinical trials for various other cancers.13-16 These inhibitors work by
inhibiting the DNA damage repair function of PARP enzymes, which can ultimately result in
cell death. These inhibitors are particularly effective in cancer patients with tumours having
mutations in homologous recombination repair enzymes BRCA1, BRCA2, and PALB2,17
which increases the accumulation of unrepaired DNA double-strand breaks that lead to
apoptosis.18-19 There are marketed therapies currently which take advantage of genomic
instability in cancer to achieve selectivity in targeting cancer cells over normal cells.20 This
has brought about an increased efficacy in treatment but off-target toxicity still persists.
PARP inhibitors (PARPi) can radiosensitize glioblastoma cancer stem cells (GSCs) which
makes them a useful combination to use with alkylating agents such as temozolomide.21-22
Although this is very encouraging, their ability to cross the blood-brain barrier (BBB) to
achieve effective therapeutic doses is very limited.23-26 Literature reports certain PARP
inhibitors being efflux pump substrates which are partly responsible for development of drug
resistance over the period of treatment.27-29 Besides, it is reported that the PARP inhibitor
Veliparib radiosensitizes both normoxic and hypoxic cell lines which could invoke systemic
toxicity.30-31 Therefore improving the tumour specificity of PARPi will expand their utility
beyond the treatment of breast and ovarian cancer in oncology.
A class of near infrared-emitting dyes called heptamethine cyanine dyes (HMCDs) is reported
to possess BBB crossing properties and selectivity towards tumour compared to normal
tissue.32 These dyes evade efflux pumps33-34 and utilize tumour hypoxia and organic anion-
transporting polypeptides (OATPs) overexpressed in cancer cells to achieve their specificity.35
There are literature examples of cytotoxic drug-dye conjugates showing efficacy in treating
brain cancers in mouse models.36-37 The drug-dye conjugate incorporating crizotinib, an
anaplastic lymphoma kinase (ALK) inhibitor showed 100-fold enhanced cytotoxicity over the
parent kinase inhibitor.38 This improvement was even more pronounced (492-fold) when the
above conjugate was combined with TMZ, the standard drug for treatment for GBM.
Encouraged by these results, we have applied this strategy to improve the efficacy of PARP
inhibitors in the treatment of GBM. Herein, we conjugated HMCD, IR-786 7 with modified
PARPi 6, an analogue of rucaparib. Rucaparib is a PARPi which is reported to be an efflux
pump substrate and therefore not amenable to cross the BBB.24 To the best of our knowledge,
there are no reported studies exploring HMCD-modified rucaparib conjugate for the treatment
of GBM. Such conjugates may provide novel ways of treating brain cancers.
The synthesis of conjugate 1 is described in scheme 1. Lithium halide exchange with bromide
2 afforded boronic acid 3 which subsequently underwent Suzuki-Miyaura cross coupling
reaction with commercially available bromide 4 to obtain intermediate 5. Removal of the tert-
butyloxycarbonyl protecting group using trifluoroacetic acid afforded rucaparib analogue 6. 6
was then conjugated to HMCD IR-786 by facile displacement of chloro group to obtain

conjugate 1. This modification was affected after careful consideration of published crystal
structure of rucaparib with PARP and Tankyrase protein so as not to perturb the interaction of
rucaparib with the protein hot spots.39-40 Both rucaparib and analogue 6 showed similar modest
activity in patient-derived glioblastoma cell line (Figure 1D).
n-BuLi, triisopropylborate,
O
O
OH
OH
O
O
THF, -70 °C, 30 min
N
N
B
N
N
Br
O
74%
2
3
H
N
H
Br
N
N
O
F
H
4
O
Pd(dppf)Cl2.DCM, Na2CO3
1,4-Dioxane, 90 °C, 1h
N
N
N
O
F
H
76%
5
H
N
O
TFA, DCM, 4h
HN
N
N
H
85%
F
6
F
O
NH
HN
Cl
N
N
I
IR-786 iodide
7
DMF, 80 oC, 6 h
N
N
31 %
N
N
I
1
Scheme 1. Synthesis of drug-dye conjugate 1.
The drug-dye conjugate 1, IR-786 7 and rucaparib were screened against three patient-
derived cell lines obtained from Auckland City Hospital (Sup. Table S1). As shown in Table 1,

and Figure 1, the drug-dye conjugate 1 was more potent at reducing cell numbers when
compared to rucaparib (EC50 of 128 nM vs >100,000 nM, respectively after 48 h). Treatment
with rucaparib was unable to result in enough cell death to determine an EC50 within the range
of concentrations used.
Table 1. EC50 of the viability of the GBM cells as indicated by total cell count which is
normalised to the vehicle DMSO after 48. Data represents mean ± SEM (n=3).
Compound
(EC50 nM)
Compound with TMZ
(EC50 nM)
IR-786 (7)
1
1735 ± 249
128 ± 30
390 ± 45
56 ± 6
Rucaparib
>100,000
>100,000
The reported mode of uptake for HMCDs are through the activated hypoxia-inducible factor
1α-organic anion transporting polypeptide (OATP) axis. Therefore, we investigated whether 1
maintained the same mode of cellular uptake. We utilized a known OATP pump blocker
sodium taurocholic acid (250 µM) and the HIF1 α-OATP axis activator, 3,4-DHB (1 mM).
Figure 3 clearly illustrates the increase in uptake of 1 with a 24 h pre-incubation of 3,4-DHB,
and the dramatic decrease in uptake with a 30 min pre-incubation with the blocker, sodium
taurocholic acid. These results suggest the conjugate 1 has a high reliance on the OATPs for
their uptake into the tumour cells.
Additionally, Figure 1 highlights that the three GBM patient cell lines were largely unresponsive
to TMZ. However, co-treatment of 1 with 100 µM of TMZ increased the cytotoxic potency of 1
to 56 nM, which was two-fold higher than treatment with 1 alone (Figure 1). This response is
suggestive of synergism of conjugate 1 with TMZ. Additionally, the co-administration of TMZ
with 7 had an appreciable increase in potency in relation to treatment with 7 alone. In contrast,
co-administration of rucaparib with TMZ did not produce a change in potency for either
compound.
Despite the lack of cytotoxic activity exhibited by rucaparib on GBM cells, there was an
apparent antiproliferative effect (IC50 of 53 M). However, by conjugating rucaparib to our dye
(drug-dye conjugate 1), it significantly increased the cytotoxic activity on the GBM cells
(EC50:128 nM). Furthermore, conjugate 1 significantly increased the antiproliferative activity
on the GBM cells when compared to rucaparib alone (IC50: 21 nM vs 53 µM; Table 2, p <
0.001). No cytotoxic activity was observed at this concentration. In terms of proliferation,
despite treatment with up to 100 µM, TMZ failed to exert a 50% reduction in proliferating cells
across all three primary GBM patient-derived cell lines. Additionally, co-treatment of rucaparib
with 100 µM of TMZ resulted in a 24-fold increase in the potency of rucaparib in
antiproliferative activity (2262 nM) (Figure 3). However, drug-dye conjugate 1 did not show
any evidence of synergism with TMZ in terms of the proliferation of GBM cells (IC50 of 21nM
to 20 nM with TMZ; Table 2).
The ability of tumour specific accumulation of a class of near infra-red emitting dyes, HMCD
and their application in conjugating various drug-like molecules to enhance potency and
reduce off-target toxicity as reported in the literature36-37, 41-42 prompted us to try a similar
strategy to conjugate a PARPi to HMCD IR-786 7. This effort resulted in drug-dye conjugate
1 with nanomolar potency in three different patient-derived GBM cell lines. The observed
potency is at least 780 fold higher than rucaparib or the modified rucaparib 6. The potency of
compound 1 was positively influenced by the addition of TMZ (two-fold improvement
compared to 1 alone), although TMZ itself did not have any effect in reducing cell number, due

to the inherited resistance of these cell lines to TMZ treatment. IR-786 7 itself was 13.5 fold
and 7 fold less potent than 1 alone or in combination with TMZ. Such dramatic improvements
in potency exhibited by 1 could be due to altered transport mechanism of such compounds
since rucaparib itself is reported to be an efflux pump substrate43 contributing to its diminished
activity. Conjugate 1 appears to rescue this activity by targeting an alternate pathway of
transport, mainly the OATPs, which is activated by hypoxic tumour conditions. Our
experiments demonstrate that this mode of transport was indeed present, and is consistent
with similar compounds reported in the literature.44-46 Although other mechanisms cannot be
ruled out, the activity of conjugate 1 gives a good rationale for exploring OATPs in oncology
to overcome drug resistance, which is a formidable barrier to overcome in CNS tumours.
The prognosis of GBM patients remains dismal despite the aggressive treatment regimen.
The PARP family of enzymes has a pleiotropic role in DNA repair, and therefore, has attracted
attention for the development of inhibitors as monotherapies and a chemo-radiation sensitizer.
However, the use of PARP inhibitors like rucaparib in the treatment of GBM has been
challenged by poor brain penetration and tumor heterogeneity which is a hallmark of most
brain cancers.25, 47 Although this might be the case, these classes of drugs are extremely
valuable for the treatment of CNS tumors due to the pleiotropic role of PARP enzymes in DNA
repair. With increasing evidence of unsuitability in targeting cancer stem cells in brain cancer
due to the transient nature of the biomarkers expressed by them, and compounded by tumour
plasticity48-49, a shift in therapeutic approach is warranted. The drug-dye conjugate 1 serves
as an example of the beneficial effects of combining PARP inhibitors like rucaparib with
HMCDs to afford a significantly more potent compound with good efficacy in human
glioblastoma cell lines. Despite rucaparib being relatively inactive across the human
glioblastoma cell lines, conjugation to IR-786 7, afforded a cytotoxic compound with nanomolar
potency. The co-incubation of 1 with TMZ provided evidence for synergism by reducing the
EC50 value by two-fold. This highlights the potential of 1 as both a monotherapy and a
chemotherapy sensitizer. The radiation-sensitisation effects of these compounds and their
mechanisms of action are currently being investigated. Future work is directed towards
improving physiochemical properties of similar conjugates to explore their utility in animal
models of glioblastoma and in human patients.

Figure 1. Viability and proliferative activity of GBM cells as indicated by total cell count after
48 h (A-B) incubation with conjugate (1), rucaparib, IR-786 (7) and TMZ, and co-incubation
with 100 µM TMZ (C). The EC50 was determined using Hoechst counts to represent the total
number of cells (B -C). The activity of rucaparib compared to 6 is seen in D. A non-linear
curve was fitted using Graphpad Prism using the concentration of each compound versus
the total number of Hoechst-positive cells normalised to DMSO (three parameters). Data
represents mean ± SEM ns = P > 0.05 to rucaparib (one-way ANOVA with Dunnett’s post-
test). Data displayed is from three independent GBM cases (T115, T84 and T141)

Table 2. IC50 of the proliferation of the GBM cells as indicated by the proportion of EdU-
positive cells which is normalised to the vehicle DMSO after 48 h. Data represents mean ±
SEM. * = P < 0.05, *** = P > 0.005 relative to rucaparib.
Compound
(IC50 nM)
Compound with TMZ
(IC50 nM)
IR-786 (7)
1
357 ± 39 ***
21 ± 4 ***
120 ± 30 *
20 ± 7 *
Rucaparib
53443 ± 473
2262 ± 488
Figure 2. Proliferative activity of GBM cells as indicated by an EdU proliferation assay after
48 h (A) incubation with conjugate (1), rucaparib, IR-786 (7) and TMZ, and co-incubation
with 100 µM TMZ (B). The IC50 was determined using the percentage of EdU-positive cells
as a measure of proliferation. A non-linear curve was fitted using Graphpad Prism using the
concentration of each compound versus the percentage of EdU-positive cells normalised to
DMSO (three parameters). Data represents mean ± SEM. Data displayed is from three
independent GBM cases.
Figure 3. Fluorescence microscope images illustrating the uptake of the drug-dye conjugate
1 (red) into patient-derived GBM cells. 1 (1 µM) was added to the cells for 1 h in all
conditions before imaging. Pre-incubation with 1 mM of 3,4-DHB greatly increased 1’s
uptakes as shown by the increased far-red fluorescence signal. Conversely, blocking the
OATP pump with pre-incubation with sodium taurocholic acid greatly attenuated this
fluorescence signal. The bar-graph is a quantification of the average cell integrated intensity
of the far-red signal . Data represents mean ± SEM (n=3).

Acknowledgment:
We thank Dr Adrian Blaser for the proton and carbon NMR, Sisira Kumara for the
analytical HPLC, Sree Sreebhavan for high resolution mass spectra, and Miranda
Aalderink, Sheryl Feng and Yewon Jung for cell biology technical support. We thank
the donors and their families for their generous gift of brain tissue for research and the
Neurosurgical team at Auckland hospital for their continued support of this research
programme. We also thank the Neurological Foundation of New Zealand, the
Auckland Cancer Society Research Centre, the Health Research Council (Programme
grant to MD and RLMF), the School of Medicine Foundation (project grant to MD, co-
Director of the Neurosurgical Research Unit) for funding the Douglas Neurosurgical
Research Fellow (TP) and the Hugh Green Foundation, for their generous funding (to
MD, Director of the Hugh Green Biobank) to establish and run the Centre for Brain
Research Hugh Green Biobank - for financial and technical support.
Supplementary material:
Detailed synthetic procedures, characterization of compounds, biological assays can
be found in the online version at
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