Design, Biological Evaluation, and Molecular Modeling of Tetrahydroisoquinoline Derivatives: Discovery of A Potent P-Glycoprotein Ligand Overcoming Multidrug Resistance in Cancer Stem Cells

Article abstract of DOI:10.1021/acs.jmedchem.8b01655

P-Glycoprotein is a well-known membrane transporter responsible for the efflux of an ample spectrum of anticancer drugs. Its relevance in the management of cancer chemotherapy is increased in view of its high expression in cancer stem cells, a population of cancer cells with strong tumor-promoting ability. In the present study, a series of compounds were synthesized through structure modulation of [4′-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-ylmethyl)biphenyl-4-ol] (MC70), modifying the phenolic group of the lead compound. Among them, compound 5b emerged for its activity against the transporter (EC₅₀ = 15 nM) and was capable of restoring doxorubicin antiproliferative activity at nontoxic concentration. Its behavior was rationalized through a molecular modeling study consisting of a well-tempered metadynamics simulation, which allowed one to identify the most favorable binding pose, and of a subsequent molecular dynamics run, which indicated a peculiar effect of the compound on the motion pattern of the transporter.

Full text of DOI:10.1021/acs.jmedchem.8b01655

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Article
Design, Biological Evaluation and Molecular Modelling of
Tetrahydroisoquinoline Derivatives: Discovery of A Potent P-glycoprotein
Ligand Overcoming Multi-Drug Resistance in Cancer Stem Cells.
Chiara Riganti, Marialessandra Contino, Stefano Guglielmo, Maria Grazia Perrone,
Iris Chiara Salaroglio, Vladan Milosevic, Roberta Giampietro, Francesco Leonetti,
Barbara Rolando, Loretta Lazzarato, Nicola Antonio Colabufo, and Roberta Fruttero
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01655 • Publication Date (Web): 25 Dec 2018
Downloaded from http://pubs.acs.org on December 25, 2018
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Design, Biological Evaluation and Molecular
Modelling of Tetrahydroisoquinoline Derivatives:
Discovery of A Potent P-glycoprotein Ligand
Overcoming Multi-Drug Resistance in Cancer Stem
Cells.
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Chiara Riganti,⸹ Marialessandra Contino,*,§ Stefano Guglielmo,*,‖ Maria G. Perrone,§
Iris C. Salaroglio,⸹ Vladan Milosevic,⸹ Roberta Giampietro,§ Francesco Leonetti, §
Barbara Rolando,‖ Loretta Lazzarato,‖ Nicola A. Colabufo,§,# and Roberta Fruttero.‖
⸹Dipartimento di Oncologia, Università degli Studi di Torino, via Santena 5/bis, 10126,
Torino, Italy
§Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO
MORO, via Orabona 4, 70125, Bari Italy
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‖Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via
P. Giuria 9, 10125 Torino, Italy
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#Biofordrug s.r.l., Spin-off dell’Università degli Studi di Bari ALDO MORO, Via Orabona
4, 70125 Bari, Italy
KEYWORDS Multi-drug resistance, P-glycoprotein, cancer stem cells, drug design,
molecular modelling.
ABSTRACT P-glycoprotein (P-gp) is a well-known membrane transporter responsible
for the efflux of an ample spectrum of anticancer drugs. Its relevance in the
management of cancer chemotherapy is increased in view of its high expression in
cancer stem cells (CSCs), a population of cancer cells with strong tumour-promoting
ability. In the present study, a series of compounds were synthesized through structure
modulation of MC70 ([4’-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-ylmethyl)biphenyl-
4-ol]), modifying the phenolic group of the lead compound. Among them compound 5b
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emerged for its activity against the transporter (EC50 = 15 nM) and was capable of
restoring Doxorubicin anti-proliferative activity at non-toxic concentration. Its behaviour
was rationalized through a molecular modelling study consisting of a well-tempered
metadynamics simulation, that allowed to identify the most favourable binding pose, and
of a subsequent molecular dynamics run which indicated a peculiar effect of the
compound on the motion pattern of the transporter.
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INTRODUCTION
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Resistance to chemotherapy is to date the main obstacle in cancer treatment.
Although the genetic and biochemical mechanisms involved in drug resistance have
been widely investigated, there is an increasing awareness that the cause of therapy
failure and disease relapse is in part due to tumour heterogeneity. Tumours can be
defined as a “complex ecosystem” consisting of heterogeneous cells types able to
create an hypoxic environment with a hierarchic organization.1,2 Cancer stem cells
(CSCs) are at the apex of this hierarchy. CSCs are a small population with great
tumourigenicity, self-renewal ability and intrinsic resistance to conventional and targeted
cancer therapy. Thus, CSCs intrinsic chemoresistance may be the cause of disease
progression and relapse in different types of cancer.3,4 The current antitumour
strategies, either chemotherapy- or radiotherapy-based, inhibiting bulk replicating
tumour cells are not efficacious vs CSCs owing to their multiple intrinsic mechanisms of
resistance.
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One of the main mechanisms of CSCs chemoresistance is the abundance of ATP
Binding Cassette (ABC) transporters,4–6 a family of transporters actively involved in the
efflux of chemotherapuetic agents and targeted-therapies.
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ABC transporters are structurally constituted by two nucleotide-binding domains
(NBD) and two transmembrane domains (TMD).1 The substrate binding site is placed at
TMD level. It is widely accepted that ABC transporters may play a pivotal role in
regulating stem cell renewal, survival, differentiation and chemoresistance.7 The
tumour-promoting activity is linked to the involvement of these transporters in the
regulation of: (i) cellular redox status; (ii) release of signalling molecules and hormones;
(iii) membrane lipid composition; (iv) cellular metabolism and release of nutrients and
metabolites; (v) changes in tumour microenvironment.1 Overall, ABC proteins transport
cell-signalling molecules that could maintain the tumour and promote tumourigenesis.
ABGC2/BCRP (Breast Cancer Resistance Protein) is one of the hallmark of
stemness,8 but it has a limited spectrum of substrates compared to other transporters
such as ABCB1/P-glycoportein (P-gp).9 Recently, we and others highlighted the
overexpression of P-gp in SC and CSCs, where it exerts protective functions from toxic
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catabolites, xenobiotics and chemotherapeutic drugs.6,10–13. In the tumour context,
blocking the activity of P-gp in CSCs will represent a promising approach to improve the
chemosensitivity to different chemotherapeutic drugs in the most chemorefractory
tumour component.
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With this in mind, we developed a new class of P-gp ligands, starting from our
previously studied P-gp inhibitor, MC70 ([4’-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-
2-ylmethyl)biphenyl-4-ol]).14,15 MC70 is a potent P-gp inhibitor (EC50 = 0.69 M), with
moderate activity towards the Multidrug Resistance Related Protein MRP1 (EC50 = 9.30
M) and breast cancer resistance protein BCRP (EC50 = 73 M).
We have previously performed SAR studies on MC70 focusing on two structural
aspects: i) the functionalization of phenolic group with alkyl and oxyalkyl chains,16 and
with more complex moieties containing variously substituted furazan (1,2,5-oxadiazole)
ring17 and ii) the “decoration” of the biphenyl core.18 The present work is a further
development of the structural modulation of MC70 and was inspired by the well-known
application of bioisosteres in drug design.19 In particular, the study was focused on the
phenolic group of MC70: the isostere substitution was fulfilled using an NH group made
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“acidic” through the introduction of an electron-withdrawing moiety or through the
inclusion in a heteroarene. The synthesized compounds display a variety of structural
features, which determine properties of key impact on molecular recognition: besides
the possibility of preventing phenol metabolic transformations, the isosteric substitutions
aimed at the improvement of activity against P-gp through wide modulation of shape,
lipophilicity, steric and electronic properties. This structural tolerance has been already
experienced by the authors in previous works, in which even subtle changes made on
MC70 scaffold gave rise to series of derivatives characterized by a large range of
activity, and can be partially explained through the well-known polyspecifity of the
target.20
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RESULTS AND DISCUSSION
Chemistry. The synthetic routes followed in the present study are depicted in the
schemes reported below. Target compounds 3a-g (Scheme 1) and 9a-c (Scheme 2)
were synthesized according to the same path: the key step is represented by the
synthesis of the diphenyl intermediates obtained through the Suzuki coupling of an
appropriately built bromoarene with 4-(hydroxymethyl)benzeneboronic acid. The
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reaction conditions were adapted from a reported procedure:21 the coupling reaction
was conducted in aqueous media in presence of an excess of diisopropylamine and
using a ligand-free catalyst (palladium (II) acetate). The products of the coupling
reaction were then converted to the corresponding benzylic chlorides which were readily
reacted with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline in mild conditions.
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In the case of compounds 5a and 6 the coupling reaction between the functionalized
bromoarene and the boronic acid, carried out in several conditions, afforded only traces
of the desired compounds; the two products were thus obtained through the
functionalization
of
the
aniline
group
after
the
introduction
of
dimethoxytetrahydroisoquinoline moiety (Scheme 1). As for the synthesis of 6, the
reaction of aniline intermediate with phenylsulfonyl chloride gave the (N-
phenylsulfonyl)phenylsulfonamido- derivative 5b, which was subsequently converted to
6 through basic hydrolysis. (Scheme 1).
Scheme 1. Synthesis of compounds 3a - g, 5a, 5b and 6a
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Ders.
R
NHR
HO
Br
CONHMe
CONHEt
CONHnPr
CONHnBu
CONHPh
COMe
a
b
c
d
e
f
a
b,c
O
NHR
N
NHR
O
1a-g
2a-g
3a-g
SO2Me
g
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NR'R"
HO
b,c,d
O
O
N
NH2
4
5a: R' = COPh, R" = H
5b: R' = R" = SO2Ph
NHSO2Ph
e
O
5b
N
O
6
aReagnets and Conditions: (a) 4-(hydroxymethyl)benzeneboronic acid, iPr2NH,
Pd(OAc)2, H2O, 80 °C; (b) 37% HCl, 90 °C; (c) 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride, Et3N, CH3CN, 60 °C; (d) Et3N, PhCOCl (for 5a) or
PhSO2Cl (for 5b), CH2Cl2, room temperature; (e) LiOH, DMF, 120 °C.
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Scheme 2. Synthesis of compounds 9a - c, 11, 12, 15, 17, 18 and 20a
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Ders.
R
NH
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R
R
O
O
b,c
O
O
a
R
NH
N
HO
b
Br
7a-d
8a-d
9a-c
O
THP
N
N
7c,8c
NH
N
9c
NH
7d,8d
SO2Ph
N
Cl
HO
c
d
O
O
N
N
N
H
10
8d
11
SO2Ph
H
N
O
O
e
N
12
H
N
HO
Br
Cl
O
H
N
b,f
c
O
NH2
NH2
N
H
O
N
N
H
O
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13
15
N
a,b,c
O
N
H
N
THP
N
N
O
17
16
H
N
Cl
Br
O
O
O
i
b,g
O
8a
N
N
N
H
18
19
O
O
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aReagents and Conditions: (a) 4-(hydroxymethyl)benzeneboronic acid, iPr2NH,
Pd(OAc)2, H2O, 80 °C; (b) 37% HCl, 90 °C; (c) 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride, Et3N, CH3CN, 60 °C; (d) PhSO2Cl, NaOH,
catalytic nBu4NHSO4, 1,2-dichloroethane, 70 °C; (e) NaOH, tetrahydrofuran/CH3OH 2/1,
reflux; (f) triphosgene, Et3N, CH2Cl2, room temperature; (g) bromotrimethylsilane,
DMSO, tetrahydrofuran, 0 °C to room temperature; (i) 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline hydrochloride, KHCO3, CH3CN, 0 - 10 °C.
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In case of indole derivative 12, the Suzuki coupling product was reacted with
phenysulfonyl chloride, affording the N-phenylsulfonyl benzylic chloride 10 as result of
the nucleophilic substitution of the benzylic sulfonate by chloride ions formed in situ
(Scheme 2).
Compound 15 was synthesized starting from intermediate 13:22 also in this case
coupling reaction carried out on the bromoarene with imidazolone moiety could not
afford the desired product in useful yields; for this reason, intermediate 13 was
converted to the benzylic chloride and readily reacted with triphosgene in mild
conditions (Scheme 2).
Benzimidazole compound 17 was obtained starting from the mixture of isomers 16:23
the isomers coming from coupling reaction were not separated and were reacted with
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concentrated hydrochloric acid, giving concomitant deprotection of the tetrahydropiranyl
group and conversion to benzylic chloride, which was then substituted with 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline (Scheme 2).
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Compound 19 was obtained through a double nucleophilic substitution with 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline in very mild conditions on intermediate 18.
The latter was synthesized using an oxidative bromination procedure, reported in Kajita
et al.,24 in presence of borotrimethylsilane and dimethylsulfoxide (Scheme 2).
Biology
P-gp interacting profile and ABC transporters selectivity
All the compounds were tested for their P-gp interacting profile by three combined
assays: i) inhibition of the transport of a pro-fluorescent probe (Calcein-AM) in MDCK-
MDR1 cells; ii) Apparent Permeability (Papp) determination (BA/AB) in Caco-2 cell
monolayer; iii) ATP cell depletion in MDCK-MDR1 cells. As previously reported, the first
assay measures the potency of the interaction of each ligand towards P-gp. The second
assay measures the ratio between two fluxes: 1) BA, representative of passive diffusion,
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from the basolateral to apical compartments, and (2) AB, representative of active
transport, from the apical to basolateral compartment.
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If BA/AB is < 2, the compound can be considered as an inhibitor, if BA/AB > 2, the
compound is classified as a substrate. This last information is completed by the third
assay (ATP cell depletion), that measures the total ATP cell level in the MDCK cells
overexpressing only MDR1: in this setting the observed effect can be mainly ascribed to
the overexpressed transporter.
Only a P-gp unambiguous substrate (category I), as transported by the pump, induces
an ATP consumption, while a P-gp inhibitor does not induce ATP consumption.
Compounds displaying a BA/AB > 2 but not inducing an ATP cell depletion are
classified as class IIB3 substrates.25
Moreover, to assess the selectivity of all compounds, their activity towards the other
two MDR sister proteins, MRP1 and BCRP, has been detected by measuring the
inhibition of the efflux of Calcein-AM (MRP1 substrate) in cells overexpressing MRP1
(MDCK-MRP1 cells) and of the fluorescent probe Hoechst 33342 (BCRP substrate) in
cells overexpressing BCRP (MDCK-BCRP cells).
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As shown in Table 1, all compounds were active towards P-gp with EC50 values
ranging from 0.015 to 29.5 M, except for derivative 5a that was completely inactive
towards all the MDR proteins.
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With respect to the reference compound MC70 (EC50 = 0.69 M), all the developed
compounds were found less active (3a-c, 3f, 3g, 9a, 9b, 11, 12, 15, 17, 19) or displaying
comparable activity (9c, 6, 3e). Only compound 5b, bearing
a
N-
(benzenesulfonyl)benzenesulfonamide moiety, was potent and selective towards P-gp
(EC50 = 15 nM).
All compounds were selective towards P-gp and showed a category IIB3 substrate
profile since the Apparent Permeability Papp (BA/AB) values were > 2 for all compounds
and they were all unable to induce ATP cell depletion.
The only exception was compound 12, which displayed a weak P-gp inhibitor profile
having a Papp < 2, not inducing an ATP consumption and exerting a low P-gp inhibiting
effect (EC50 = 21.2 M).
Table 1. Biological Activity Profile of Target Compounds.
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O
O
R
N
EC50 ± SEM (M)a
MRP1 BCRP
8
9
Compoun
d
b
R
ATP Papp
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MDR1
NH NH
O
3a
3b
3c
3d
3e
3.1 ± 0.60
1.7 ± 0.32
1.1 ± 0.20
0.32 ± 0.06
0.98 ± 0.20
NA
NA
NA
NA
NO 12.8
NO 9.3
NO 17.6
NH NH
O
NA
NA
NA
NA
NH NH
O
NH NH
O
46 ± 9.20 NO 12.8
NH NH
O
NA
NA
NO > 20
NO 13.6
NH
O
3f
1.52 ± 0.30
5.2 ± 1.00
NA
NA
O
O
S
NH
3g
NA
NA
YES 5
NH
O
5a
5b
NA
NA
NA
NO 19.9
O
S
N
O
O
S
0.0153
0.003
±
O
NA
NO > 20
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Journal of Medicinal Chemistry
Page 16 of 61
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O
O
S
6
0.87 ± 0.20
NA
NA
NO 15
NH
8
9
H
N
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13
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19
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60
9a
9b
9c
1.47 ± 0.30
21.4 ± 4.20
0.90 ± 0.10
NA
NA
NA
NA
NA
NA
NO 3.6
NO 8.1
NO 12
O
H
N
O
O
H
N
N
O
S
O
11
19.2 ± 3.80
NA
NA
NO 4.2
N
H
N
12
15
21.2 ± 4.20
29.5 ± 5.78
NA
NA
NA
NA
NO 1.9
NO 15
H
N
O
N
H
H
N
17
6.93 ± 1.30
NA
NA
NO 9.2
N
H
N
O
1.8
±
O
O
N
19
1.2 ± 0.24
0.69
NA
NO 8.5
NO 1.3
0.30
OH
MC7015
9.30
73
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Page 17 of 61
Journal of Medicinal Chemistry
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a
Values are the means ± SEM of three independent experiments carried out in
triplicate.
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b Apparent permeability ratio (the value is from two independent experiments).
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Co-administration assay
Compound 5b, displaying the best P-gp activity profile (EC50 = 15nM), has been
tested in a co-administration assay in order to study its ability to revert MDR, restoring
Doxorubicin cytotoxic effect in MDCK-MDR1 cells.
Doxorubicin alone did not exert cytotoxicity being a P-gp substrate, while in presence
of 1 M of compound 5b, it was able to induce cell death. Compound 5b was tested at
different doses in a range near its EC50 value and showed the best reverting activity at 1
M as depicted in Figure 1. To exclude any effect of 1% v/v of DMSO, the solvent used
to dissolve 5b, we preliminarily tested that cells treated with Doxorubicin with or without
1% v/V of DMSO did not differ in their drug content (data not shown).
Figure 1. In vitro cell growth experiments performed on MDCK-MDR1 cells in presence
of 10 M Doxorubicin, Doxo, alone (white bar), compound 5b at different doses, alone
and in co-administration of Doxo. Each bar represents the mean ± SEM of two
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Journal of Medicinal Chemistry
Page 18 of 61
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experiments performed in triplicate. One-way ANOVA analysis: ***p < 0.0005, ****p <
0.0001 vs control.
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60
100
***
80
60
****
40
20
0
M
oxo
oxo
Doxo
10nM
10nM+D
100n
100n
M+Doxo
1microM
1microM+D
Efficacy of compound 5b against P-gp-expressing cancer stem cells
Given the central role of CSCs in mediating drug resistance in tumours, and the
emerging role of ABC transporters in the drug resistance of CSCs, we tested the
efficacy of compound 5b against primary CSCs, in order to assay its activity in a model
closer to the biology of patients’ tumours. We focused on glioblastoma (GB) and
malignant pleural mesothelioma (MPM), two tumours that differ for pathogenesis,
genetic background and histological origin, but share a high chemoresistance due to
CSCs component.3,26 From three patients affected by GB and MPM we separated the
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Page 19 of 61
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differentiated cells (GDC or MDC, respectively) from the SCs of the same tumour (GSC
or MSC, respectively), as detailed in the Supporting Information. The phenotypic
characterization of DC and SC are reported in the Supplementary Tables S1 and S2. By
screening the expression levels of ABC transporters in SC versus DC, several ABC
transporters involved in drug resistance were found significantly more expressed in SC
compared to DC. ABCB1/P-gp/Mdr1, ABCB2/Mdr2, ABCC1/MRP1 and ABCG2/BCRP
were significantly more expressed in 3 out of 3 SC patients in both GB and MPM
(Figure 2A). Given the high specificity of compound 5b for inhibiting P-gp, we focused
on this transporter. The high expression of P-gp at protein levels in GSC and MSC
compared to GDC and MDC was validated by immunoblotting (Figure 2B).
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Figure 2. Expression of ABC transporters in human cancer stem cells of glioblastoma
and mesothelioma. Differentiated cells from primary glioblastoma (GDC1-3) and
mesothelioma (MDC1-3) from 3 patients and stem cells from the same patients (GSC1-
3 and MSC1-3) were isolated and put in culture. A. Hitmap of ABC transporters,
represented in a colorimetric logarithmic scale. The expression of the each gene in the
corresponding DC was considered 1 (not shown in the figure). B. Immunoblotting
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Journal of Medicinal Chemistry
Page 20 of 61
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analysis of P-gp expression in DC and SC of each patient. Actin was used as control of
equal protein loading.
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Page 21 of 61
Journal of Medicinal Chemistry
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In line with this pattern, GDC and MDC accumulated more Doxorubicin, a substrate of
P-gp, than GSC and MSC (Figure 3A-D). The third-line generation P-gp inhibitor
Elacridar significantly increased Doxorubicin accumulation at low micromolar
concentration, in line with its Ki14 (Figure 3B and Figure 3C) in SC. This effect was
stronger in SC than in DC of both tumours (Figure 3A-D), consistently with the higher
expression of P-gp in SC. Notably, compound 5b produced an analogue increase of
Doxorubicin at nanomolar concentration (Figure 3A-D) in SC of both tumours.
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Figure 3. Intracellular Doxorubicin accumulation in human cancer stem cells of
glioblastoma and mesothelioma. Differentiated cells from primary glioblastoma (GDC,
panel A) and mesothelioma (MDC, panel C) from 3 patients and stem cells from the
same patients (GSC, panel B, and MSC, panel D) were incubated for 3 h with medium
containing 5 μM Doxorubicin (Doxo), alone or with the indicated concentrations of
Elacridar or compound 5b. DMSO 1% v/v was the concentration of solvent present in
cells treated with compound 5b. The intracellular accumulation of Doxorubicin was
measured spectrofluorimetrically in duplicates (n=3). Data are means ± SEM of each
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cell categories. One-way ANOVA analysis: *p < 0.05 Elacridar or compound 5b + Doxo
vs Doxo; **p < 0.005 Elacridar or compound 5b + Doxo vs Doxo; ***p < 0.0001
compound 5b+Doxo vs Doxo.
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Page 23 of 61
Journal of Medicinal Chemistry
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5
A
B
6
7
2.0
1.5
8
9
***
1.5
1.0
0.5
0.0
1.0
0.5
0.0
10
11
12
13
14
15
16
17
18
19
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44
45
46
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48
49
50
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52
53
54
55
56
57
58
59
60
***
***
***
***
M
M
l
M
M
M
M
M
M
%
M
M
l
M
M
M
M
5
M
M
%
n
n
n
n
1




n
n
n
n
+
D
1




0
0
0
0
o
1
0
1
0
O
+
0
0
1
0
0
o
1
0
0
1
0
1
0
1
1
O
a
b
1
0
1
0
1
S
o
1
1
1
a
b
S
a
l
b
5
a
l
5
b
1
1
a
l
b
M
E
a
b
a
b
5
l
E
l
M
E
+
a
5
b
E
5
D
5
l
+
E
E
D
+
E
+
D
5
+
+
+
+
E
+
+
o
+
+
o
x
+
o
x
+
+
o
x
x
o
o
o
+
x
o
o
x
o
x
o
o
x
x
o
x
o
x
o
x
o
o
x
o
o
o
x
o
o
x
D
o
x
o
o
x
o
o
D
o
D
o
x
D
o
D
o
D
o
D
D
o
D
D
D
D
D
D
D
D
C
D
1.5
1.0
0.5
0.0
1.0
*
***
0.8
0.6
0.4
0.2
0.0
***
***
***
**
*
M
M
l
M
M
M
M
M
M
%
M
M
l
M
M
M
M
5
M
M
%
n
n
n
n
1




n
n
n
n
+
D
1




0
0
0
0
o
1
0
1
0
0
0
0
0
o
O
+
1
0
1
0
O
+
1
0
1
0
1
1
1
0
1
0
1
1
a
b
S
a
b
1
1
S
1
1
a
l
b
a
l
5
b
5
a
l
b
a
b
M
E
l
a
5
b
M
E
l
a
5
b
5
E
+
E
D
l
D
5
E
E
D
+
D
5
+
E
+
+
+
E
+
+
+
+
+
o
x
+
+
x
o
+
o
x
o
x
o
x
+
o
o
x
o
x
o
o
x
o
o
o
x
o
o
x
o
x
o
x
o
x
x
o
o
x
o
o
x
o
x
D
x
o
o
o
o
o
D
D
o
o
o
D
D
o
o
D
D
o
D
D
D
D
D
D
D
D
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Journal of Medicinal Chemistry
Page 24 of 61
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In line, with this trend, compound 5b enhanced the acute cytotoxicity, measured as
release of lactate dehydrogenase (LDH) (Supporting Figure 1A-B), and reduced cell
viability (Figure 4A-B) induced by Doxorubicin in SC, where Doxorubicin alone was
ineffective, as a consequence of its low intracellular accumulation. Again, the effect of
compound 5b on these parameters was achieved at 10 nM, while Elacridar produced
the same effects at 1 µM. Overall, these results pointed out that compound 5b was
more effective than the most potent P-gp inhibitor presently used in preclinical models
and in clinical trials. According to the in vitro results, the compound was not toxic when
used alone. To exclude any effect of DMSO 1% v/v, the solvent used to dissolve 5b, we
checked that at this concentration DMSO did not change the doxorubicin accumulation
(Figure 3A-D), nor the cell damage (Supporting Figure S1A-B) or the cell viability
(Figure 4A-B) also in primary cells.
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Figure 4. Viability of cancer stem cells of glioblastoma and mesothelioma in the
presence of compound 5b and Doxorubicin. Differentiated cells from primary
glioblastoma (GDC) and stem cells from the same patients (GSC, panel A),
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differentiated cells from primary mesothelioma (MDC) and stem cells from the same
patients (MSC, panel B) were incubated for 72 h with fresh medium (ctrl) or medium
containing 5 μM Doxorubicin (doxo), alone or 10 nM compound 5b or 1 μM Elacridar.
DMSO 1% v/v was the concentration of solvent present in cells treated with compound
5b. Cell viability was measured by a chemiluminescence-based assay in quadruplicates
(n=3). Data are means ± SEM of each cell categories. One-way ANOVA analysis: ***p <
0.0001 Doxo or Elacridar or compound 5b vs ctrl; °° < 0.0002 Elacridar or compound 5b
vs Doxo alone; °°°p < 0.0001 Elacridar or compound 5b vs Doxo alone.
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Journal of Medicinal Chemistry
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A
150
7
8
9
GDC
°°°
GSC
°°°
100
50
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°°°
***
°°°
***
***
***
***
0
l
l
r
o
b
o
o
a
+
a
r
t
o
x
b
o
x
a
+
a
l
O
l
l
O
l
t
x
x
5
5
c
c
S
E
E
l
S
E
E
o
o
o
o
M
M
D
D
D
D
D
D
o
o
x
+
+
x
%
%
b
b
o
1
1
5
5
D
D
+
l
+
r
r
t
t
c
c
B
150
100
50
MDC
°°
MSC
°°
°°°
***
***
***
°°°
***
***
0
l
l
r
o
x
b
o
x
a
+
a
+
r
t
o
b
o
o
a
a
l
O
l
l
O
l
t
x
x
5
5
c
c
S
E
E
S
E
E
o
o
o
o
M
M
+
D
D
D
D
D
D
o
o
x
+
+
x
%
%
b
b
o
1
1
5
5
D
D
+
l
l
r
r
t
t
c
c
Although the absence of toxicity must be proved in in vivo experiments, that are
ongoing in our laboratory, this data suggests that compound 5b achieves good efficacy
at low concentrations and thus can be less affected by the side-effects characterizing all
the P-gp inhibitors tested until now, including Elacridar.27
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Compound 5b did not increase the toxicity of Doxorubicin in DC, in keeping with the
absence or very low level of P-gp in these cells. This data indirectly confirms that the
compound is a specific modulator of P-gp.
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To further prove this point, since SC express different ABC transporters, we stably
knocked-down P-gp in GSC and MSC derived from one patient, using a CRISR-Cas
vector. Knocked-out (KO) SCs had undetectable P-gp, in line with DC of the
corresponding patient (Figure 5A).
Figure 5. Effects of compound 5b on cancer stem cells of glioblastoma and
mesothelioma knocked-out for P-gp. Stem cells from glioblastoma patients #2 (GSC)
and from mesothelioma patient #1 (MSC) were not untreated (nt) or transfected with a
scrambled CRISPR-Cas vector (scr) or with a CRISPR-Cas vector knocking-out P-gp
(KO). Glioblastoma and mesothelioma differentiated cells (GD and MD) of the same
patient were included as control of P-gp-lowly expressing cells. A. Immunoblotting
analysis of P-gp expression in DC and SC of each patient. Actin was used as control of
equal protein loading. B-C. Cells were incubated for 3 h with medium containing 5 μM
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Doxorubicin (Doxo), alone or with 10 nM compound 5b. The intracellular accumulation
of Doxorubicin was measured spectrofluorimetrically in duplicates (n=3). Data are
means ± SEM of each cell categories. One-way ANOVA analysis: *p< 0.05 compound
5b + Doxo vs Doxo GSC/MSC nt; **p< 0.005 compound 5b + Doxo vs Doxo GSC/MSC
nt;°°° p< 0.0008 compound 5b-treated KO cells vs compound 5b-treated nt or scr-cells,
°° p< 0.007 compound 5b-treated KO cells vs compound 5b-treated nt or scr-cells.
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Page 29 of 61
Journal of Medicinal Chemistry
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B
1.5
GSC KO
GDC
8
9
°°
1.0
0.5
0.0
10
11
12
13
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GSC scr
GSC ctrl
*
**
o
o
o
o
o
o
o
o
x
x
x
x
x
x
x
x
o
o
o
o
o
o
o
o
D
D
D
D
D
D
D
D
+
+
+
+
b
b
b
b
5
5
5
5
C
1.0
MSC KO
MDC
##
°°°
0.8
0.6
0.4
0.2
0.0
MSC scr
MSC ctrl
*
**
o
o
o
o
o
o
o
o
x
x
x
x
x
x
x
x
o
o
o
o
o
o
o
o
D
D
D
D
D
D
D
D
+
+
+
+
b
b
b
b
5
5
5
5
Compared to untreated SC or SC treated with a not-targeting (scrambled) CRISR-Cas
vector, used as internal control of specificity, KO SC of both GB and MPM accumulated
significantly more doxorubicin (Figure 5B-C) and the amount of doxorubicin was similar
to GD or MD, respectively. Compound 5b, that increased the cytotoxic effects of
ACS Paragon Plus Environment
29