Enhancing the activity of platinum-based drugs by improved inhibitors of ERCC1–XPF-mediated DNA repair

Article abstract of DOI:10.1007/s00280-020-04213-x

Purpose: The ERCC1–XPF 5′–3′ DNA endonuclease complex is involved in the nucleotide excision repair pathway and in the DNA inter-strand crosslink repair pathway, two key mechanisms modulating the activity of chemotherapeutic alkylating agents in cancer ce

Full text of DOI:10.1007/s00280-020-04213-x

Cancer Chemotherapy and Pharmacology (2021) 87:259–267
https://doi.org/10.1007/s00280-020-04213-x
ORIGINAL ARTICLE
Enhancing the activity of platinum‑based drugs by improved
inhibitors of ERCC1–XPF‑mediated DNA repair
Gloria Ciniero · Ahmed H. Elmenoufy · Francesco Gentile4,9 · Michael Weinfeld5,6 · Marco A. Deriu ·
1
2,3
1
Frederick G. West2 · Jack A. Tuszynski
,6
1,4,5
· Charles Dumontet · Emeline Cros‑Perrial · Lars Petter Jordheim
7,8
7
7
Received: 8 June 2020 / Accepted: 10 December 2020 / Published online: 5 January 2021
The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021
©
Abstract
Purpose The ERCC1–XPF 5′–3′ DNA endonuclease complex is involved in the nucleotide excision repair pathway and in
the DNA inter-strand crosslink repair pathway, two key mechanisms modulating the activity of chemotherapeutic alkylating
agents in cancer cells. Inhibitors of the interaction between ERCC1 and XPF can be used to sensitize cancer cells to such
drugs.
Methods We tested recently synthesized new generation inhibitors of this interaction and evaluated their capacity to sensi-
tize cancer cells to the genotoxic activity of agents in synergy studies, as well as their capacity to inhibit the protein–protein
interaction in cancer cells using proximity ligation assay.
Results Compound B9 showed the best activity being synergistic with cisplatin and mitomycin C in both colon and lung
cancer cells. Also, B9 abolished the interaction between ERCC1 and XPF in cancer cells as shown by proximity ligation
assay. Results of diꢀerent compounds correlated with values from our previously obtained in silico predictions.
Conclusion Our results conꢁrm the feasibility of the approach of targeting the protein–protein interaction between ERCC1
and XPF to sensitize cancer cells to alkylating agents, thanks to the improved binding aꢂnity of the newly synthesized
compounds.
Keywords DNA repair · Protein–protein interaction · Chemical synthesis · Cancer
Introduction
cells, they can be self-defeating in cancer therapy, as they
can interfere with the DNA damage inꢃicted by therapies in
tumour cells. The heterodimer ERCC1–XPF is a 5′–3′ endo-
nuclease formed by ERCC1, which is involved in DNA–pro-
tein and protein–protein interactions, and XPF, which retains
the endonuclease active site. This enzyme belongs to
A well-functioning DNA repair apparatus naturally allows
cells to be protected from endogenous and exogenous
damages, preserving health status and integrity of tissues.
Although the DNA repair mechanisms are physiological in
6
*
Lars Petter Jordheim
Cancer Research Institute of Northern Alberta, University
lars-petter.jordheim@univ-lyon1.fr
of Alberta, Edmonton, AB T6G 2E1, Canada
7
Univ Lyon, Université Claude Bernard Lyon 1, Centre de
1
2
3
Department of Mechanical and Aerospace Engineering,
Politecnico di Torino, 10129 Turin, Italy
Recherche en Cancérologie de Lyon, INSERM U1052,
CNRS UMR 5286, Centre Léon Bérard, 8 Avenue
Rockefeller, 69008 Lyon, France
Department of Chemistry, University of Alberta, Edmonton,
AB T6G 2G2, Canada
8
Laboratoire de Biochimie-Toxicologie, Centre Hospitalier
Department of Pharmaceutical Chemistry, College
of Pharmacy, Misr University for Science and Technology,
P.O. Box: 77, 6th of October City 12568, Egypt
Lyon-Sud, Hospices Civils de Lyon, 69495 Pierre Bénite,
France
9
Present Address: Vancouver Prostate Centre, University
4
5
Department of Physics, University of Alberta, Edmonton,
AB T6G 2E1, Canada
of British Columbia, Vancouver, BC V6H 3Z6, Canada
Department of Oncology, Cross Cancer Institute, University
of Alberta, Edmonton, AB T6G 1Z2, Canada
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Cancer Chemotherapy and Pharmacology (2021) 87:259–267
structure-speciꢁc endonucleases, since its mechanism of
action involves the cleavage of hanging single strand por-
tion of DNA from double stranded ꢁlaments. ERCC1–XPF
is part of the nucleotide excision repair (NER) pathway,
responsible for repairing lesions such as bulky helix distor-
tions like cyclobutane pyrimidine dimers induced by UV
irradiation. Other DNA repair mechanisms in which this
enzyme is involved are inter-strand crosslinks repair (ICL)
and double-strand breaks repair (DSB) [1–3]. NER and ICL
are the mechanisms primarily involved in development of
resistance to DNA damaging agents such as cisplatin, mito-
mycin C and cyclophosphamide; therefore, the inhibition
of these mechanisms may overcome cancer resistance and
increase eꢀects of chemotherapy on tumours [4, 5]. Down-
regulation of ERCC1 and XPF by siRNA has been shown to
decrease DNA repair and enhance the sensitivity of several
cancer cell lines to cisplatin [6–8].
drugs has been investigated in the present study through
synergy studies between the DNA crosslinking drugs and
the ERCC1–XPF inhibitors.
Materials and methods
Overview of in silico design strategy for ERCC1–XPF
inhibitors
Since the design and screening of F06 analogues were per-
formed as described previously [10, 15], we only provide a
very brief outline. The chemical structures were obtained by
modifying diꢀerent F06 sites using an in-house collection of
molecular fragments [10] and Molecular Operating Environ-
ment (MOE) MedChem transformations (Chemical Com-
puting Group Inc, 2015, Molecular Operating Environment,
MOE, 2015). Molecules were docked on the XPF surface
to a pocket involved in key interactions with ERCC1 using
Pharmacophore docking in MOE Dock, and scored using
generalized Born Volume Integral/Weighted Surface Area
(GBVI/WSA) function [17]. Rescoring was performed using
2 ns of molecular dynamics simulations of the ligand–recep-
tor complexes, and Molecular Mechanics/Generalized Born
Surface Area (MM/GBSA).
One approach to inhibiting ERCC1–XPF is through the
use of small molecules that target ERCC1–XPF interac-
tion [9, 10]. Properly executing this strategy allows one to
employ combination cancer therapy using genotoxic chemo-
therapeutic agents with DNA repair inhibitors. Indeed, the
co-administration of the two agents to patients may improve
the eꢂcacy of widely used DNA crosslinking drugs such as
the platinum based agents [11, 12]. Cancer chemotherapy
outcomes for patients treated with DNA crosslinking drugs
depend on the tumoral stage and type, but above all on the
cancer cell biology. Indeed, the outcome of platinum-based
chemotherapy is inꢃuenced by diꢀerent cellular processes
including those upstream of DNA damage/repair mecha-
nisms such as cellular uptake and eꢄux and detoxiꢁcation
by cytoplasmic proteins, and in cell death-triggering pro-
cesses such as DNA damage detection and apoptosis [13,
Synthesis and characterization of ERCC1–XPF
inhibitors
Synthesis of F06, A2 and A4 has been previously described
[15]. The synthesis of B9 (4-((6-chloro-2-methoxyacridin-
9-yl)amino)-2-((4-(2-(dimethylamino)ethyl) piperazin-1-yl)
methyl) phenol) was previously reported as B5 [10]. General
synthetic route of D7 was performed through nucleophilic
aromatic substitution reaction by mixing 6,9-dichloro-
hydroxyacridine and 2-amino-4,5-dimethoxybenzonitrile.
1
4].
In previous studies we designed and synthesized vari-
ous ERCC1–XPF inhibitors aiming to improve eꢀects of
widespread DNA crosslinking drugs like cisplatin and mito-
mycin C [9, 10]. Preliminary in vitro assays conꢁrmed that
F06 shows promising inhibitory eꢀect against ERCC1–XPF
endonuclease activity and acts synergistically with cisplatin
and mitomycin C [9]. However, the activity of F06 is sub-
optimal in terms of clinical properties, including its potency
and pharmacokinetic proꢁle, and a derivatization strategy
was adapted to optimize the action of the compound [10, 15,
MTT cytotoxicity and synergy assays
Cytotoxicity assays were performed as described before
[10], using human lung cancer (A549) and human colon
cancer (HCT-116) cell lines purchased from American Type
Culture Collection (Manassas, VA). Cells (3000 per well)
were seeded in 96-well plates in 100 µL media and allowed
to adhere before diꢀerent concentrations of compounds were
added. After 72 h in culture, MTT (1 µg per well) was added
16]. After a successful synthesis of the top in silico screened
compounds, they were subjected to several cell-free and cell-
based assays. The results yielded two potent compounds, A4,
previously named as compound 4, and B9, that showed a sig-
niꢁcant sensitization of colorectal cancer cells to cyclophos-
phamide and UV radiation [10, 15, 16]. Here, we continue
our eꢀort to evaluate these improved molecules intended
for combination cancer therapy. The inꢃuence of the new
compounds on the action of traditional DNA crosslinking
and replaced by 100 µL isopropanol/H O/HCl 90/9/1 v/v/v
2
after 2 h incubation. Finally, absorbance was determined
at 570 and 690 nm with a Multiskan EX bench-top micro-
plate reader (ThermoFisher, Waltham, USA). For synergy
assays, ERCC1/XPF inhibitors and alkylating agents were
added in ꢁxed ratios (close to ratios of the IC ). Values
5
0
for IC and combination index 95 (CI95) were calculated
5
0
1
3

Cancer Chemotherapy and Pharmacology (2021) 87:259–267
261
using CompuSyn software 1.0 (ComboSyn, Inc., Paramus,
NJ, USA). Eꢀect of associations was indicated as synergy
if CI95<0.9, additive if 0.9<CI95<1.1, and antagonistic
if CI95>1.1.
(data not shown). Synthesis of compounds F06, A2 and A4
was achieved through a one-pot sequential addition reac-
tion in three steps as reported before [15]. In summary, this
Mannich-type reaction of p-acetamidophenol with formal-
dehyde and the appropriate secondary amine in 2-propanol
was carried out under reꢃux for 12 h. The solvent and the
excess of unreacted formaldehyde from the resulting mix-
ture were removed under vacuum, and without isolating the
compound, the resulting viscous residue was treated with
6 M HCl to deacetylate the acetamido group and furnish
the primary amine. Afterwards, an equimolar amount of
6,9-dichloro-2-methoxyacridine was added, aꢀording, after
heating, compounds F06, A2 and A4 in moderate to good
yields after isolation. The synthesis is general, easy, and
reproducible. All synthesized compounds were character-
Apoptosis assay
A549 and HCT-116 cells were seeded in 24-well plates at
a density of 50,000 cells per well. Cells were incubated in
complete media and left overnight to adhere before add-
ing compounds at indicated concentrations. Upon a further
incubation of 45 h, cells were washed with PBS and stained
with AnnexinV-Fluos Staining kit (Roche) as indicated by
the manufacturer. Cells were analysed by FACS (Fortessa,
BD Biosciences, Franklin Lakes, NJ, USA) and the percent-
age of living cells (AnnexinV negative and propidium iodide
negative), was used to measure the activity of drugs and
combinations.
1
13
ized by H NMR, C NMR, HRMS, IR, and the purity of
compounds A4 and B9 was determined by HPLC (≥95%
purity) as reported before [15]. The synthesis of B9 was
accomplished through the same synthetic route as A4 except
the last step, where 1 eq of the unprotected aniline inter-
mediate reacts with 1 eq 6,9-dichloro-2-hydroxyacridine.
Nucleophilic aromatic substitution reaction was carried out
to synthesize D7 by reacting 6,9-dichloro-hydroxyacridine
and 2-amino-4,5-dimethoxybenzonitrile. Figure 1 shows the
chemical structures of these studied compounds.
Proximity ligation assay
A549 cells were seeded in an 8-well Nunc Lab-Tek Chamber
Slide system at a density of 30,000 cells per well. The cells
were left to adhere for 24 h before adding the compounds
alone or in combinations as indicated in the Results section.
The plate was incubated for 24 h and then processed for
protein proximity analysis using the Duolink assay (Olink
Bioscience, Uppsala, Sweden) with an ERCC1 antibody
Synergy analysis for the inhibitors with cisplatin
and mitomycin C
(FL-297, 1/100; Santa Cruz Biotechnology, Santa Cruz, CA,
USA) and an XPF antibody (LS-C173159, 1/100; LifeSpan
BioSciences, Seattle, WA). The samples were then ꢁxed
and stained with DAPI. Cells were observed using a ZEISS
Axio Scan.Z1 slide scanner (ZEISS, Oberkochen, Ger-
many). Images of the red dots representing the interaction of
ERCC1 and XPF were analysed using the ImageJ software
The classic MTT assay was chosen to perform synergy stud-
ies with the conventional chemotherapy drugs cisplatin and
mitomycin C in association with the new inhibitors of the
ERCC1–XPF interaction. Studies were performed on A549
and HCT-116 cell lines, which were chosen as models for
lung and colon cancer, respectively, as these pathologies are
often treated using DNA crosslinking agents and that they
have already been shown to be sensitive to such an approach
[9].
(
LOCI, University of Wisconsin, USA). Both cell number
and dots quantity were assessed by automatic counting in
each microscopic ꢁeld. A total of eight diꢀerent microscopic
ꢁ
elds and more than 1400 cells were analysed per condition.
The intrinsic activity (IC50 for antiproliferative activ-
ity), as determined by MTT assay, was similar for the new
compounds, although a little bit lower for compound D7
Results are expressed as mean values from two experiments
conducted independently.
(
p<0.01 for comparisons with all other compounds on both
cell lines), and in the low micromolar range (Table 1).
Furthermore, we assessed the synergistic activity between
ERCC1/XPF inhibitors and the DNA damaging agents cis-
platin and mitomycin C. CI95 values obtained from the syn-
ergy experiments performed on A549 and HCT-116 (Fig. 2),
indicate that the optimized compounds A4 and B9 exhibit
a synergistic behaviour with cisplatin, comparable with the
one displayed by F06. Synergy of the inhibitors with mito-
mycin C is less pronounced compared with the same results
for cisplatin, even if a slight diꢀerence is evident compar-
ing the synergistic compounds and the negative controls.
Results
Synthesis of F06‑based analogues
A2, A4 and B9 were top-ranked analogues, with MM/GBSA
scores of -11.60, -13.12, and −12.44 kcal/mol, respectively,
and thus were selected for synthesis and testing. For com-
parison, the corresponding value for F06 was −17.78 kcal/
mol. D7 was manually designed as a non-active compound,
screened in the in vitro ERCC1–XPF endonuclease assay
1
3

262
Cancer Chemotherapy and Pharmacology (2021) 87:259–267
Fig. 1 Structures of ERCC1/
XPF inhibitors studied. Struc-
tural diꢀerences with F06 are
highlighted in red
N
N
N
N
N
N
N
HO
Cl
HO
Cl
HO
Cl
NH
NH
NH
O
O
O
N
N
N
F06
A2
A4
N
N
N
HO
O
O
CN
NH
NH
OH
OH
Cl
N
Cl
N
B9
D7
Table 1 Intrinsic antiproliferative activity of new compounds
Synergies were either moderate (0.7<CI95<0.85) or ꢁrm
0.3<CI95<0.7). In contrast, strong antagonism between
(
Compound
A2
D7
A4
B9
F06
D7 and cisplatin was observed in both A549 and HCT-116
cells. Compound A2 was found to have a slight synergis-
tic or additive behaviour, and this was evident mostly in
the A549 cells if used together with mitomycin C (CI95
A549 (IC50
4.3± 1.1 7.9± 0.8 2.0± 0.3 4.2± 0.7 3.2± 0.5
(
µM))
HCT-116 (IC50 4.6± 0.9 12.0± 1.4 1.0± 0.3 1.3± 0.2 1.3± 0.3
(
µM))
0
.46–0.61). A2 displays an additive eꢀect also in the A549
Data are mean IC50± SEM (µM) of seven independent experiments
cells, together with cisplatin, with an estimation of CI95 in
the interval of 0.50–1.5.
Fig. 2 CI95 values detected
in A549 and HCT-116 cells
exposed to cisplatin or mito-
mycin C in association with
ERCC1–XPF inhibitors. Results
are means and error bars are
SEM from at least ꢁve diꢀer-
ent experiments. Dotted lines
indicate values of CI95=0.9
and 1.1
4
2
1
0
.8
0
.6
0.4
0.2
A2 D7 A4 B9 F06 A2 D7 A4 B9 F06 A2 D7 A4 B9 F06 A2 D7 A4 B9 F06
Cisplaꢀn
Mitomycin C
Cisplaꢀn
Mitomycin C
A549
HCT116
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Cancer Chemotherapy and Pharmacology (2021) 87:259–267
263
B9 potentiates apoptosis induced by alkylating
agents
B9, 33.0%± 3.8% vs 70.2%± 14.2% live cells for 1 µM mito-
mycin C with and without B9), as compared to the eꢀects of
each compound alone (Fig. 3). A4 did not show any potenti-
ating eꢀect in these experiments.
To conꢁrm the results obtained from the synergy experi-
ments with the MTT assay, we determined the cell survival
after exposure to A4 or B9 together with cisplatin and mito-
mycin C using AnnexinV and propidium iodide staining.
Data clearly shows that B9 in combination with both cis-
platin and mitomycin C strongly enhances the cytotoxic
action in both A549 (4.8% ± 4.7% vs 71.0% ± 2.0% live
cells for 10 µM cisplatin with and without B9, 4.5%± 2.3%
vs 69.1% ± 2.4% live cells for 1 µM mitomycin C with
and without B9) and HCT-116 cells (38.6% ± 11.6% vs
Interaction between ERCC1 and XPF is disrupted
by ERCC1–XPF inhibitors in cells
Our hypothesis is based on the inhibition of the pro-
tein–protein interaction between ERCC1 and XPF, result-
ing in decreased NER activity and subsequently in a better
activity of alkylating agents. To conꢁrm that our compounds
are able to disrupt the interaction between these proteins in
cells, we performed a proximity ligation assay using A549
7
8.7%± 4.4% live cells for 5 µM cisplatin with and without
Fig. 3 Survival of A549 and
HCT-116 cells exposed to cispl-
atin or mitomycin C (µM) alone
or in combination with 1 µM A4
or B9. Graphs show mean val-
ues of cell survival from three
independent experiments per-
formed in duplicate, and error
bars are standard deviation. Sta-
tistical analysis was performed
using one-way ANOVA tests.
A549
1
00
8
0
***
***
*** ***
6
0
4
0
*
*p<0.01 and ***p<0.001 as
compared either to cells without
cisplatin or mitomycin C or to
cells without B9
***
*
**
2
0
***
*** ***
***
0
-
B9 A4
B9 B9 B9 A4 A4 A4
B9 B9 B9 A4 A4 A4
10 10
10
20 50 10 20 50 10 20 50
Cisplatin
1
2
10
1
2
1
2
Mitomycin C
HCT-116
100
80
***
**
60
***
40
***
***
***
***
***
20
0
-
B9 A4
B9 B9 B9 A4 A4 A4
10 20 10 20
Cisplatin
B9 B9 B9 A4 A4 A4
5
10 20
5
5
1
2
10
1
2
10
1
2
10
Mitomycin C
1
3

264
Cancer Chemotherapy and Pharmacology (2021) 87:259–267
cells exposed to compounds alone or in combination. Upon
addition of cisplatin, an increase of ERCC1 and XPF interac-
tion as shown by the foci is observed, going from 15.6 foci
per cell in the unexposed cells to 56.6 foci per cell (Fig. 4
and Table 2). This enhanced interaction was compromised
by F06 as we only observed 18.2 foci per cell when cisplatin
was combined with F06. Even stronger results were obtained
with A4 and B9 for which we observed 13.8 and 2.2 foci
per cell after exposure together with cisplatin, respectively.
The important decrease of interaction between ERCC1 and
XPF by B9 was also observed in the absence of cisplatin.
Altogether, these results show that both A4 and in particular
B9, possess improved eꢂciency in inhibiting the formation
of the ERCC1–XPF complex in cells as compared to the
ꢁrst-generation compound F06. This is in line with their
better biological activity and strengthens our hypothesis for
the mechanism of action of the association between the new
compounds and the alkylating agents.
Conclusions and discussion
Cisplatin and mitomycin C are widely used cancer chemo-
therapy agents and constitute the elective therapy for many
tumour types [10, 17]. However, discontinuation of plati-
num-based therapies is common among patients because of
the development of drug resistance and the high toxicity and
side eꢀects brought about by the therapy [18, 19]. Therefore,
Fig. 4 Representative PLA images on A549 cells exposed to F06
lower-right zones, zoomed images show the ERCC1–XPF interaction
complexes, visible as red dots, and cellular nuclei in blue, by DAPI
staining
(
1 µM), A4 (1 µM), B9 (1 µM) and cisplatin (20 µM) alone or in
combination for 24 h. Images were obtained at 40X magniꢁcation,
analysed ꢁelds are shoved on the background of each square. On the
Table 2 ERCC1/XPF interaction in A549 cells exposed to cisplatin or ERCC1–XPF inhibitors alone or in combination at indicated concentra-
tions for 24 h
Condition
NT
Cisplatin 20 µM F06 1 µM Cisplatin
A4 1 µM Cisplatin
20 µM+A4
1 µM
B9 1 µM Cisplatin
20 µM+B9
1 µM
2
1
0 µM+F06
µM
Mean number of foci per cell 15.6± 7.2 56.6± 19.8**
Total number of cells counted 2097 1455
13.7± 5.0 18.2± 4.7**
2268 1831
13.5± 3.5 13.8± 3.6** 5.1± 3.9* 2.2± 2.0**
1949 1701 2122 1506
Data represent mean number of foci per cell± SD counted in eight diꢀerent microscopic ꢁeld, belonging to two independent experiments
p<0.05 and **p<0.0001 as compared to condition without ERCC1/XPF inhibitor (or as compared to NT for cisplatin) using one-way
ANOVA test
*
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Cancer Chemotherapy and Pharmacology (2021) 87:259–267
265
ꢁ
nding compounds able to signiꢁcantly increase sensitivity
and possibly reducing toxic eꢀects. Recently, Thomas et al.
[23] designed and optimized a ꢃuorescence-based technique
able to assess enzyme activity. Their aim was to generate a
robust assay for high-throughput screening, based on the
ꢃorescence signal generated by the enzymatic cleavage of
specially tagged oligonucleotide substrates upon binding
with the full-length enzyme ERCC1–XPF.
to cisplatin in cisplatin-resistant tumours has a great poten-
tial in therapy and could lead to clinical advantages such
as increasing the eꢂcacy of chemotherapy at lower dos-
ages, which would be better tolerated by the patient. Cispl-
atin and mitomycin C are DNA intrastrand and interstrand
crosslinking agents, and the lesions they induce in DNA
can be repaired by the NER or ICL DNA repair pathways,
respectively. The use of cisplatin and mitomycin C has
allowed us to test the inhibition of the ERCC1–XPF com-
plex acting on these two diꢀerent DNA repair pathways [9].
In this paper we report on our investigations of the activities
of earlier published improved ERCC1–XPF inhibitors and
their capacity to potentiate the cytotoxic activity of cisplatin
and mitomycin C.
In our work, compounds tested were designed through
docking-based virtual screening, then optimized in silico by
further functionalization. This has resulted in improved bind-
ing aꢂnity, speciꢁcity and activity of the new compounds
A4 and B9 [15, 16], even if their pharmacological properties
can be compared to the previously discovered compounds
[21, 24] due to their chemical similarity based on similar
scaꢀolds. However, because the design of A4 and B9 was
based on precisely targeting the structure of the dimerization
HhH2 domain of XPF, it most likely renders these com-
pounds more speciꢁc for the ERCC1–XPF complex, and
less likely to interfere with other proteins. However, we do
not have any experimental proof of speciꢁc targeting of the
ERCC1–XPF interaction in the cells. This could eventually
be obtained using ERCC1- and/or XPF-deꢁcient cells for
antiproliferation assay or synergy experiments.
ERCC1–XPF interaction and activity are mainly depend-
ent on the dimerization of the two C-terminal regions of the
dimer as well as the endonuclease activity of XPF [5]. The
formation of the heterodimer is principally due to the inter-
action of double helix–harpin–helix motifs, HhH2, which are
present at the C-terminus dimerization interface of the two
monomers. This domain is a promising target for inhibitors
[5, 9] able to disrupt the interaction and, therefore, the activ-
ity of the enzyme. Despite the importance of this mechanism
as a potential therapeutic route, few suitable inhibitors have
been identiꢁed so far [9, 20–23]. McNeil et al. [22] investi-
gated the dimerization HhH2 domain of XPF as a pharmaco-
logical target. By employing in silico screening techniques,
they identiꢁed diꢀerent inhibitors of the dimer, discovering
a small NER inhibiting compound, able to improve eꢂcacy
of cisplatin in melanoma cells, even though the IC and
In the past, we have extensively demonstrated how F06
interacts with XPF, using diꢀerent techniques such as ꢃuo-
rescence quenching, immunoprecipitation and surface plas-
mon resonance assays. Moreover, proof of synergy between
F06, cisplatin and mitomycin C has been provided, together
with the ability of F06 to interact with ERCC1–XPF in vitro,
impair DNA repair, and disrupt the protein–protein interac-
tion in cells [9]. Previous characterization and synthesis of
F06 has allowed us to use this compound as benchmark in
the study of the new generation of improved molecules able
to target and suppress ERCC1 and XPF interaction.
5
0
K values of this molecule were suboptimal [22]. They also
d
identiꢁed inhibitors of the active site that were able to sen-
sitize cancer cells to cisplatin when used at low micromolar
concentrations. In addition, Arora et al. [21] and Chapman
et al. [24] were able to identify and optimise several inhibi-
tors of NER activity with IC values within the nanomolar
In this work, we further investigated whether our top-
ranked compounds, A4 and B9, display a synergistic eꢀect
with cisplatin and mitomycin C in diꢀerent cancer cell lines.
Our MTT assay indicates that A4 and B9, like F06, show
synergy with both cisplatin and mitomycin C. The synergy
appears to be slightly stronger with cisplatin than with mito-
mycin C. This may be because mitomycin C DNA monoad-
ducts, which may not be repaired by NER or ICL pathways,
can contribute to mitomycin C cytotoxicity [25]. We further
examined the synergistic interactions to see if they induced
an apoptotic response. Intriguingly, we observed a strong
apoptotic response for B9 with both cisplatin and mitomycin
C compared with the crosslinking agents when used alone,
but a substantially weaker response for A4 and the crosslink-
ing agents. This will require further exploration.
5
0
range that also improved the cytotoxicity of platinum-based
chemotherapeutic agents in cancer cells. Compounds from
Arora’s paper did not alter the DNA binding of ERCC1/
XPF and are, therefore, supposed to target the endonucle-
ase activity. For compounds in Chapman’s paper, the tar-
get is not described, although a clear inhibition of NER is
observed. Although in these previously described works the
speciꢁcity of inhibitors towards ERCC1–XPF endonucle-
ase was assessed, a detailed knowledge of the molecular
structure of the inhibitor-XPF complexes was not provided.
Indeed, rationally designed inhibitors are speciꢁcally stud-
ied to adapt to the enzymatic binding pocket, and this was
the approach we used for targeting the ERCC1/XPF interac-
tion site. They are usually more speciꢁc, may exhibit lower
oꢀ-target interactions due to similarities among binding
pockets of other endonucleases, helping to increase eꢂcacy
We also showed, using the proximity ligation assay,
that the compounds markedly reduce interaction between
ERCC1 and XPF in the assayed cell lines. The new genera-
tion inhibitors were found to be strikingly more eꢀective in
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3

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Cancer Chemotherapy and Pharmacology (2021) 87:259–267
disrupting the ERCC1–XPF interaction compared to F06.
B9, in particular, reduced the level of interaction, as meas-
ured by the assay, to almost zero in cells treated or untreated
with cisplatin. This is, to our knowledge, the most eꢂcient
compounds reported thus far.
human cancer cells. Nutrients 10:1644. https://doi.org/10.3390/
nu10111644
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McNeil EM, Melton DW (2012) DNA repair endonuclease
ERCC1–XPF as a novel therapeutic target to overcome chemore-
sistance in cancer therapy. Nucleic Acids Res 40:9990–10004.
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bcp.2006.04.025
The most important ꢁnding of this work is that the newly
developed compounds exhibit synergistic properties and
appear to improve the cytotoxicity of both mitomycin C
and cisplatin in the cancer cell lines tested. The reported
data also conꢁrm improved inhibitory activity towards the
complex ERCC1–XPF, as shown before [15]. Therefore,
we believe the new inhibitors, and in particular B9, show
excellent promise to impede NER and ICL repair processes
in cancer cells and thereby address drug resistance issues
associated with well-known chemotherapeutic agents such
as cisplatin and mitomycin C.
7
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In conclusion, the new inhibitors have been shown to
exhibit the predicted mode of action and can be adopted as
a new route to target cancerous pathologies. They address
drug resistance issues, paving the way for the develop-
ment of innovative combination treatments, which may be
clinically applied in combination with existing well-known
chemotherapy agents such as cisplatin and mitomycin C. The
next step in the development of this strategy will involve the
in vivo validation of our culture-based experiments.
Jordheim LP, Barakat KH, Heinrich-Balard L, Matera EL,
Cros-Perrial E, Bouledrak K, El SR, Perez-Pineiro R, Wishart
DS, Cohen R, Tuszynski J, Dumontet C (2013) Small molecule
inhibitors of ERCC1–XPF protein–protein interaction synergize
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Acknowledgements This research was partly supported by funds from
the Alberta Cancer Foundation. FG was supported by an Alberta Inno-
vates scholarship and a Novartis Pharmaceuticals Canada Inc. scholar-
ship. LPJ received funding from Olav Raagholt og Gerd Meidel Raa-
gholts stiftelse for forskning. The authors are grateful to Bruno Chapuis
and Denis Ressnikoꢀ at CIQLE, Lyon for valuable assistance in image
acquisition and analysis.
1
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sztein DC, Rudel T, Ryan KM, Sayan E, Scorrano L, Shao F,
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Compliance with ethical standards
Conflict of interest The authors have no conꢃict of interest to declare.
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