Discovery of Novel Flavonoid Dimers to Reverse Multidrug Resistance Protein 1 (MRP1, ABCC1) Mediated Drug Resistance in Cancers Using a High Throughput Platform with "click Chemistry"

Article abstract of DOI:10.1021/acs.jmedchem.8b00834

A 300-member flavonoid dimer library of multidrug resistance-associated protein 1 (MRP1, ABCC1) modulators was rapidly assembled using "click chemistry". Subsequent high-throughput screening has led to the discovery of highly potent (EC₅₀ ranging from 53 to 298 nM) and safe (selective indexes ranging from >190 to >1887) MRP1 modulators. Some dimers have potency about 6.5- to 36-fold and 64- to 358-fold higher than the well-known MRP1 inhibitors, verapamil, and MK571, respectively. They inhibited DOX efflux and restored intracellular DOX concentration. The most potent modulator, Ac3Az11, was predicted to bind to the bipartite substrate-binding site of MRP1 in a competitive manner. Moreover, it provided sufficient concentration to maintain its plasma level above its in vitro EC₅₀ (53 nM for DOX) for about 90 min. Overall, we demonstrate that "click chemistry" coupled with high throughput screening is a rapid, reliable, and efficient tool in the discovery of compounds having potent MRP1-modualting activity.

Full text of DOI:10.1021/acs.jmedchem.8b00834

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Article
Discovery of novel flavonoid dimers to reverse multidrug
resistance protein 1 (MRP1; ABCC1)-mediated drug resistance in
cancers using a high throughput platform with “click chemistry”
Iris L. K. Wong, Xuezhen Zhu, Kin Fai Chan, Man Chun Law, Aya
M. Y. Lo, Xuesen Hu, Larry M C CHOW, and Tak-Hang Chan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00834 • Publication Date (Web): 23 Oct 2018
Downloaded from http://pubs.acs.org on October 23, 2018
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Discovery of novel flavonoid dimers to reverse multidrug resistance protein 1 (MRP1;
ABCC1)-mediated drug resistance in cancers using a high throughput platform with “click
chemistry”
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#
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Iris L.K. Wong , Xuezhen Zhu , Kin-Fai Chan , Man Chun Law , Aya M.Y. Lo , Xuesen Hu ,
,1
,1,2
Larry M.C. Chow* and Tak Hang Chan*
1Department of Applied Biology and Chemical Technology and State Key Laboratory of
Chemical Biology and Drug Discovery, Hong Kong Polytechnic University, Hong Kong SAR,
China
2Department of Chemistry, McGill University, Montreal, Quebec, H3A 2K6 Canada
#These three authors contributed equally to this work.
*Co-corresponding authors: Tak Hang Chan and Larry M. C. Chow
For L. M. C. C., Phone: (852)-34008662: Fax: (852)-23649932; E-mail: larry.chow@polyu.edu.hk
For T. H. C., Phone: (852)-34008670; Fax: (852)-23649932; E-mail: tak-hang.chan@polyu.edu.hk
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ABSTRACT
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A 300-member flavonoid dimer library of multidrug resistance-associated protein 1 (MRP1;
ABCC1) modulators was rapidly assembled using “click chemistry”. Subsequent high-throughput
screening has led to the discovery of highly potent (EC50 ranging from 53 to 298 nM) and safe
(
selective indexes ranging from >190 to >1887) MRP1 modulators. Some dimers have potency
about 6.5- to 36-fold and 64- to 358-fold higher than the well-known MRP1 inhibitors, verapamil
and MK571, respectively. They inhibited DOX efflux and restored intracellular DOX
concentration. The most potent modulator, Ac3Az11, was predicted to bind to the bipartite
substrate-binding site of MRP1 in a competitive manner. Moreover, it provided sufficient
concentration to maintain its plasma level above its in vitro EC50 (53 nM for DOX) for about 90
minutes. Overall, we demonstrate that “click chemistry” coupled with high throughput screening
is a rapid, reliable and efficient tool in the discovery of compounds having potent MRP1-
modualting activity.
Keywords: Click chemistry, CuAAC reaction, multidrug resistance, multidrug resistance-
associated protein 1, MRP1 modulators, flavonoids
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1
. INTRODUCTION
Multidrug resistance (MDR) is a major impediment to successful treatment of many forms of
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malignant cancers. MDR is often associated with overexpression of an ATP-binding cassette (ABC)
transporter which can extrude a wide variety of structurally-unrelated anticancer drugs and
decrease intracellular drug accumulation below toxic levels. P-gp/ABCB1, MRP1/ABCC1 and
BCRP/ABCG2 are the three major ABC members that confer cancer MDR. Multidrug resistance–
associated protein 1 (MRP1/ABCC1) was first identified in 1992 in a drug-selected human small
lung cancer cell line H69AR.1, 2 It pumps out organic anions, glutathione-, glucuronate- or sulfate-
conjugated drugs, or unconjugated drugs in concert with free glutathione, including the
chemotherapeutic agents, vincristine, doxorubicin and etoposide.3-8 Its elevated protein and
mRNA levels have been reported in many tumors and is correlated with poor patient outcome
including non-small-cell lung cancer, gastrointestinal carcinoma, melanoma, neuroblastoma and
cancers of the breast, ovary and prostate.9-12 Similar to P-gp/ABCB1, MRP1 contains an internally
duplicated structure of two transmembrane domains (TMD1 and TMD2) and two cytosolic
nucleotide binding domains (NBD). However, MRP1 has an extra N-terminal transmembrane
domain containing five membrane-spanning helices (TMD0). The functional role of this third
TMD0 is currently unclear. 3, 4, 7, 8
Considerable efforts have been placed to overcome MDR by designing modulators which can
inhibit the function of ABC transporter and restore intracellular accumulation of drugs. A large
number of modulators have been identified for P-gp/ABCB1 (verapamil, cyclosporine A, PSC-
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33, biricodar, zosuquidar, tariquidar, elacridar and ontogen)13-15 and BCRP/ABCG2 (elacridar,
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ko143, pantoprazole, tariquidar and biricodar), 16-20 some with high potencies having low nM range
of EC50 (effective concentration that can lower the IC50 of a drug to resistant cancer cells by 50%)
in vitro. In contrast, there are fewer MRP1 inhibitors including probenecid and MK-571.21, 22 These
MRP1 inhibitors are far from satisfactory because of their relatively high EC50 which may cause
toxicity and side effects in clinical trials. Therefore, development of modulators which are potent
and safe against MRP1 is highly desirable.
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We have previously synthesized some potent and safe P-gp and MRP1 modulators using
flavonoid as the structural motif.23-28 Many natural flavonoids are known to be modest modulators
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of these ABC transporters. Flavonoids are commonly found in fruits, vegetables, and plant-
derived products of human diet and generally considered as safe compounds. Because of the
pseudo-dimeric structure of many ABC transporters, we reasoned that a bivalent approach by
combining two flavonoid moieties linked with different polyethylene glycol linkers would yield
selective and potent modulators. Indeed, we discovered that some flavonoid dimers showed
effectiveness to inhibit P-gp and MRP1 transporters with nanomolar EC50 values (<170 nM).23-28
The flavonoid dimer FD-4e, with PEG linker length n=5, displayed a low EC50 value of 73 nM in
reversing DOX resistance in a MRP1 overexpressing ovarian (2008/MRP1) cell line (Figure 1).27
However, its relatively low aqueous solubility rendered it a poor candidate for further in vivo
animal study.
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Figure 1. Chemical structures of FD-4e, MK571 and verapamil used in this study.
Triazoles are amphoteric in nature, acting as both acids and bases. Such properties make them
usually soluble in aqueous medium. In order to rapidly generate a large number of diverse and
dimeric MRP1 modulators which may have better physiochemical properties as potential drug
candidates, we employed the copper (I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction
between azides and alkynes to yield 1,4-disubstituted 1,2,3 triazoles (commonly known as CuAAC
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reaction). The reaction belongs to a class of reactions known as “click chemistry”. The features
of these reactions include high efficiency (~100% reaction yield), chemoselectivity, and
modularity. They have been found to be useful as a rapid method to assemble compound libraries
and allow direct in vitro screening of the clicked products without the need of purification.30, 31
Click-chemistry based high throughput screening platform has been developed in cases where the
32
33-36
target enzyme is available as reported for the Ras palmitoylation and various other enzymes.
Click-chemistry has also been employed to synthesize bivalent ligands in the study of P-gp-
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mediated cellular efflux.37-39 Because whole cells, instead of a pure enzyme, have to be used for
the bioassay of ABC transporter-mediated cellular efflux, no high throughput screening platform
was developed and only a small library of pure click products was prepared and studied. Herein,
we report a 300-member flavonoid dimer library synthesized using the “click chemistry” approach
and coupled that with a high throughput screening platform to discover novel MRP1/ABCC1
modulators with favorable physiochemical properties.
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2. RESULTS
2
.1 Library Generation by Click Chemistry
Previously, we synthesized a series of bivalent ligands by coupling two flavonoid moieties with
PEG linkers. FD-4e was demonstrated to be a potent MPR1 inhibitor for reversing DOX resistance
with EC50 of 73 nM.27 In terms of chemical structures, our previous dimers contain mainly
symmetrical structures with two identical flavonoid moieties. Based on the previous synthetic
routes, it was difficult to generate a large number of unsymmetrical dimers with two different
flavonoids.
In this work, we take advantage of the appealing ease and chemo-selectivity of the Cu (I)-
catalyzed azide-alkyne cycloaddition as the key dimerizing approach for the construction of a
library of triazole bridged flavonoid bearing molecules. At first, we designed twenty-five terminal
alkynes comprising of mono-acetylenes (Ac1-13, Ac16, Ac19, Ac27, Ac33, Ac35, Ac42),
diacetylenes (Ac15, Ac22, Ac23, Ac29, Ac31) and the triacetylene Ac17 (Figure 2) and twelve
flavonoids bearing azido groups (Az1-5, Az7, Az10-13, Az17 and Az18) (Figure 3).
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Figure 2. Structures of various alkynes.
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Figure 3. Structures of various azides.
2.1.1 Synthesis of alkynes
The flavonoids bearing alkynes Ac1-5, Ac12, Ac35 and Ac42 were prepared by treating 4’ or
7-hydroxyflavones 1a-g with various haloalkynes in high yield (Scheme 1). Ac33 was easily
prepared by treating p-hydroxybenzaldehyde 4d with 6-chloro-1-hexyne (Scheme 4). Base-
catalyzed aldol condensation of aldehyde Ac33 with various 2-hydroxyl acetophenones 3a-e
afforded the chalcones Ac6-10 (Scheme 2). 2-Phenylquinazolin-4(3H)-one derivative Ac11 was
prepared by treatment of 2-aminobenzamide with aldehyde 2a in the presence of catalytic amount
of iodine (Scheme 2). Acetylene Ac13 was simply obtained in two steps: (1) alkylation of hydroxyl
flavone 1e with bromoethanol; (2) alkylation of the hydroxyl group with propargyl bromide in the
presence of sodium hydride (Scheme 3). Acetylenes bearing flavonoid Ac16 were easily prepared
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through ligation of flavone 1e with 2-(benzyl(prop-2-yn-1-yl)amino)ethanol under Mitsunobu
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condition (Scheme 3). Treatment of aniline (4a), piperazine (4b) and N , N -dimethylethane-1,2-
diamine (4c) with propargyl bromide easily furnished mono-acetylene Ac27, diacetylene Ac29
and Ac31 (Scheme 4). Acetylene Ac15, triacetylene Ac17, mono-acetylene Ac19, di-acetylene
Ac22 and Ac23 are commercially available.
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Scheme 1. Synthesis of acetylenes Ac1-5, Ac12 and Ac42.a
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Reagents and condition: (i) K
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, 6-chloro-1-hexyne or 5-chloro-1-pentyne, DMF, reflux;
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Scheme 2. Synthesis of acetylenes Ac6-Ac11.a
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Reagents and condition: (i) KOH, EtOH, rt; (ii) I , DMSO, 150 C;
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Scheme 3. Synthesis of acetylenes Ac13 and Ac16.a
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, 2-bromoethanol, DMF, reflux; (b) NaH, propargyl
, DIAD, THF;
bromide solution, THF; (ii) 2-(benzyl(prop-2-yn-1-yl)amino)ethanol, PPh
3
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Scheme 4. Synthesis of acetylenes Ac27, Ac29, Ac31 and Ac33.a
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, propargyl bromide, DMF, 10 C; (iv) K
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, 6-chloro-1-hexyne, KI,
o
DMF, 80 C;
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.1.2 Synthesis of azides
The synthesis of the required azide bearing flavonoids is shown in Scheme 5. The azides Az1-
, Az10-13, Az17 and Az18 were conveniently prepared from 4’ or 7-hydroxyflavones (1a, d, f,
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g, h, i) with high yield by the following three steps: (1) treatment of 4’ or 7-hydroxyflavones with
various hydroxyl halides such as bromoethanol, 2-(2-chloroethoxy)ethanol and 2-(2-(2-
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chloroethoxy)ethoxy)ethanol in the presence of sodium carbonate; (2) conversion of hydroxyl to
methanesulfonate group by methanesulfonyl chloride; and (3) treatment of the mesylated flavones
with excess sodium azide.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5. Synthesis of azides Az1-5, Az7-13, Az17 and Az18.a
1
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a
Reagents and condition: (i) K
2
CO , 2-bromoethanol (n = 0) or 2-(2-chloroethoxy)ethanol (n = 1)
3
or 2-(2-(2-chloroethoxy)ethoxy)ethanol (n = 2), DMF, reflux; (ii) (a) methanesulfonyl chloride,
o
NEt
3
, DCM, 0 C; (b) NaN
3
, ACN;
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2.1.3 The click reaction
Using the CuAAC reactions, a 300-member “clicked” flavonoid dimers library was constructed
in microtiter plates and in millimolar scale. In general, one millimolar of each acetylene (AcN)
was mixed with each of 1 mM azides (AzM) in 100 PL of THF solvent, followed by addition of
catalytic amount of bromotris(triphenylphosphine)copper(I) to yield about 1 mM of triazole
bridged products (AcN-AzM) without purification. For diacetylenes Ac15, Ac22, Ac23, Ac29, Ac
3
1 and triacetylene Ac17, two millimolar and three millimolar of azides (AzM) were used
o
respectively. The reactions were carried out overnight in microplate heater (70 C). We generated
3
00 triazole compounds in such identical reaction condition. After overnight reaction, all THF
solvent in each well was removed by evaporation and the 300 triazole compounds were then
dissolved in DMSO. Without any further purification of the assembled products, they were directly
screened for MRP1 modulating activity in 96-well plates containing 2008/MRP1 cells and
anticancer drug, doxorubicin.
2
.2 Biological assay results
2
.2.1 Primary and secondary screening of MRP1-modulating activity of clicked flavonoid
dimers
Each of the un-purified 300-member of the clicked homo- and hetero-flavonoid dimer library
contained at least four components: (1) the alkyne AcN, (2) the azide AzM (3) the catalyst
bromotris(triphenylphosphine)copper(I) and (4) the product AcN-AzM. This library was used for
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
primary screening (Table 1) at 2 μM of the product AcN-AzM by assuming a nearly 100% yield
of the click reaction. A hundred nanomolar of DOX was employed for the assay, a concentration
at which it showed no toxicity towards 2008/MRP1 cells (100% of survival, data not shown). The
relative potency of MRP1-modulating activity of the clicked dimers was determined by MTS
proliferation assay and presented as % of survival normalized to those with DOX but without un-
purified clicked dimer. The active clicked dimers would result in a relatively lower % of cell
survival. Controlled experiments with the pure alkyne, or with the pure azide, or with the catalyst
were also performed (Table 2). At 2 μM, all pure alkynes, azides and catalyst showed no or low
MRP1-modulating activity with % of survival ranged from 57% to 103% (Table 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In the primary screening, the relative MRP1-modulating activity of each clicked dimer was
measured and presented as in Table 1. Out of the 300-member library, 53 members were
considered as “hit” compounds with % of survival ranging from 20% to 49% (Table 1, bolded and
underlined). Among these 53 flavonoid dimer “hits”, 18 of them exhibited cytotoxicity towards
2008/MRP1 cells at 2 PM with % of survival less than 70% (Table 3). Interestingly, 7 out of the
12 dimers in the Ac19 sub-library showed cytotoxic effect, suggesting that Ac19 is a poor building
block for making MRP1 modulators (Table 3). After excluding these 18 cytotoxic flavonoid
dimers, the remaining 35 “hit” compounds were further differentiated by testing them at a lower
concentration of 1 PM. A total of 21 compounds consistently maintained promising activity with
<
51% of survival (Table 4).
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Table 1. Primary screening of clicked flavonoid dimers to reverse DOX resistance in 2008/MRP1
cells.a
> 90 % survival
69 – 50% survival
< 50% survival
9
0 – 70% survival
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Az1
20
21
23
26
29
30
31
37
39
39
40
40
42
49
57
58
60
75
79
81
82
87
90
93
Az7 Az12 Az11 Az4
Az3
38
45
55
53
51
51
54
81
61
53
46
63
63
52
61
72
67
72
77
74
71
76
86
82
103
Az2
39
44
54
45
56
57
59
28
65
66
58
60
55
29
55
76
70
70
85
83
77
71
88
79
100
Az18 Az5 Az10 Az13 Az17
Ac5
Ac3
30
55
55
36
39
52
54
39
72
63
65
80
70
70
71
76
82
78
74
77
76
73
81
74
86
33
52
57
51
42
61
60
41
72
76
78
79
67
74
72
60
84
81
74
88
77
88
90
40
89
34
41
45
46
39
53
60
51
58
54
56
68
64
76
62
49
73
82
76
79
83
99
88
111
99
34
42
48
44
46
47
53
28
56
58
60
67
53
54
51
51
72
74
72
83
77
79
90
76
85
55
45
65
73
54
62
77
62
83
74
75
85
68
87
79
67
78
82
89
82
91
83
92
79
84
59
37
63
65
45
63
45
34
61
89
33
77
68
87
71
68
76
75
80
86
81
84
87
75
96
73
60
51
49
50
79
46
57
72
88
68
69
69
62
66
64
68
59
87
82
77
76
91
97
58
75
77
56
80
69
60
68
37
85
81
51
86
73
96
75
67
77
83
90
87
87
90
85
89
93
78
61
78
79
68
86
47
75
74
79
57
76
75
80
78
67
75
76
82
88
86
64
89
86
85
Ac2
Ac16
Ac1
Ac12
Ac13
Ac19
Ac6
Ac4
Ac35
Ac7
Ac10
Ac15
Ac9
Ac11
Ac8
Ac33
Ac22
Ac23
Ac17
Ac31
Ac27
Ac42
Ac29 116
a
A total of 300 clicked dimers was used to screen for the MRP1-modulating activity. 2008/MRP1
cells were incubated with 100 nM DOX and 2 μM of crude clicked dimers together. The % of
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
survival was determined using MTS assay after 72 h incubation and normalized to the control
containing no DOX and any clicked dimer. Each clicked dimer was tested in triplicate in each
experiment. N = 3 independent experiment. The % of survivors was presented as mean here.
Percentage survival less than 50% was bolded and underlined. These dimers displaying potential
MRP1-modulating activity were picked up for further cytotoxicity test.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Table 2. MRP1-modulating activity of pure alkyne, pure azide or Cu(I) catalyst in 2008/MRP1
cells. a
Compounds % Survival
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ac1
Ac2
Ac3
Ac4
Ac5
Ac6
Ac7
Ac8
74 ± 3
85 ± 1
79 ± 3
67 ± 5
88 ± 8
102 ± 2
103 ± 4
100 ± 1
100 ± 2
99 ± 0
86 ± 3
99 ± 2
98 ± 4
81 ± 2
86 ± 2
84 ± 3
83 ± 2
84 ± 3
86 ± 2
87 ± 2
81 ± 3
87 ± 1
86 ± 1
71 ± 3
81 ± 3
78 ± 2
85 ± 7
84 ± 8
85 ± 6
74 ± 8
90 ± 5
72 ± 9
91 ± 7
90 ± 8
79 ± 4
57 ± 4
86 ± 2
Ac9
Ac10
Ac11
Ac12
Ac13
Ac15
Ac16
Ac17
Ac19
Ac22
Ac23
Ac27
Ac29
Ac31
Ac33
Ac35
Ac42
Az1
Az2
Az3
Az4
Az5
Az7
Az10
Az11
Az12
Az13
Az17
Az18
Cu (I) catalyst 103 ± 3
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
A total of 25 pure alkynes, 12 pure azides and Cu(I) catalyst were tested for their MRP1-
modulating activity. 2008/MRP1 cells were incubated with 100 nM DOX and 2 μM of each
monomer or catalyst together. Percentage survival was determined using MTS assay after 5-day
incubation and normalized to the control containing no DOX or monomers. Each monomer was
tested in triplicate in each experiment. N = 3 independent experiment. Percentage survival was
presented as mean ± SEM here. Percentage survival below 50% indicates that the compound
showed promising MRP1-modulating activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Table 3. Cytotoxicity of 53 clicked dimers alone towards 2008/MRP1 cells. a
% of survival
2 μM dimers + 100 nM DOX
2008/MRP1
20 ± 1
% of survival
2 μM dimers alone
Compounds
Ac
5
Az
1
2008/MRP1
85
91
92
97
4
3
1
21 ± 2
23 ± 3
26 ± 3
28 ± 19
28 ± 18
29 ± 0
29 ± 3
30 ± 2
30 ± 6
31 ± 1
33 ± 12
33 ± 1
34 ± 13
34 ± 1
34 ± 2
36 ± 17
37 ± 3
37 ± 15
37 ± 20
38 ± 2
39 ± 2
39 ± 2
39 ± 13
39 ± 4
39 ± 3
39 ± 3
40 ± 2
40 ± 13
40 ± 9
41 ± 3
41 ± 21
42 ± 3
42 ± 2
42 ± 2
44 ± 3
44 ± 7
45 ± 5
45 ± 5
45 ± 2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
1
1
1
6
9
9
1
2
4
10
81
49
99
95
97
53
89
16
100
93
87
97
18
11
100
43
39
14
95
83
68
95
63
105
93
23
49
95
90
93
72
39
98
95
99
74
95
44
80
50
85
88
92
88
65
82
92
1
15
1
2
5
7
1
1
3
2
3
5
1
1
5
5
12
5
1
9
5
4
5
16
3
11
7
5
1
9
1
1
5
9
13
3
1
7
1
11
7
1
9
4
6
5
7
1
1
2
1
3
5
1
4
1
2
3
9
12
11
12
12
4
1
3
10
3
1
2
16
1
4
5
2
3
11
3
3
18
2
45 ± 3
45 ± 5
45 ± 6
46 ± 3
46 ± 6
46 ± 13
46 ± 5
47 ± 8
47 ± 3
48 ± 2
1
6
1
3
5
1
4
16
35
11
3
1
1
1
3
3
2
10
17
4
2
4
1
1
1
1
6
5
11
10
1
49 ± 5
49 ± 2
49 ± 1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
After primary screening, a total of 53 promising clicked dimers were tested for their cytotoxicity
towards 2008/MRP1 cells. The clicked dimers were ranked from highest to the lowest according
to their MRP1-modulating activity from the primary screening. Each clicked dimer was tested in
triplicates and performed in 3 independent experiments. In cytotoxicity towards 2008/MRP1 cells,
the % of survival was presented as mean. The % of survival below 70% was highlighted in grey
color, indicating that these dimers alone showed cytotoxicity towards 2008/MRP1 cells and were
excluded from the secondary screening.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
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Table 4. Secondary screening of 35 promising clicked dimers in reversing DOX resistance in
2
008/MRP1 cells. a
%
of survival
Compounds
1 μM dimers + 100 nM DOX
2008/MRP1
25 ± 0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ac
5
Az
1
3
1
1
29 ± 2
29 ± 1
1
6
2
5
5
1
7
4
1
30 ± 3
38 ± 3
41 ± 1
42 ± 4
1
2
5
11
1
7
4
2
46 ± 1
46 ± 4
46 ± 14
48 ± 3
48 ± 2
1
1
3
6
3
16
1
2
3
5
4
48 ± 2
49 ± 2
2
3
3
16
5
3
11
11
3
4
3
49 ± 1
49 ± 2
50 ± 2
50 ± 2
50 ± 3
51 ± 2
2
1
3
1
5
51 ± 3
53 ± 8
53 ± 3
54 ± 14
54 ± 2
55 ± 6
56 ± 2
56 ± 3
59 ± 1
62 ± 7
1
6
10
12
11
18
1
10
4
5
1
16
3
6
1
1
3
2
1
16
4
2
1
63 ± 3
64 ± 7
5
7
12
1
65 ± 4
66 ± 4
1
11
13
0
1
66 ± 3
70 ± 4
71 ± 5
79 ± 3
11
17
1
1
5
2
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2
2
2
2
2
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3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
a
A total of 35 dimers showing low cytotoxicity towards 2008/MRP1 cells was selected for
secondary screening of MRP1-modulating activity. The cells were incubated with 100 nM DOX
and 1 μM of clicked dimers together. These dimers were ranked from the highest to the lowest
according to the MRP1-modulating activity as determined by % of survival. Each dimer was tested
in triplicate and performed in 3 independent experiments. The % of survival was presented as mean
here. In the presence of 100 nM DOX, the clicked dimers showing 25% to 51% of survival were
selected for determining EC50 and cytotoxicity towards L929 cells.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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0
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2
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8
9
0
2
.2.2 Effective concentration (EC50) and therapeutic indexes of potent clicked flavonoid dimers.
After the secondary screening, the top 21 “hit” compounds (% of survival < 51% at 1 μM) were
re-synthesized, purified and tested for their MRP1-modulating activity. EC50 values were found to
be in nanomolar range from 53 nM to 298 nM and did not show cytotoxicity towards L929 with
IC50 > 50 PM (Table 5). Among them, Ac3Az11 has the lowest EC50 in reversing DOX resistance
in 2008/MRP1 cells (Figure 4). In contrast, under identical protocol, the EC50 of well-established
MRP1 inhibitors verapamil (EC50 = 1925 nM) and MK571 (EC50 = 19000 nM) were in micromolar
range (Table 5). The potencies of these clicked dimers were about 6.5- to 36-fold and 64- to 358-
fold higher than verapamil and MK571, respectively. Moreover, our compounds are relatively
non-toxic towards L929 cells. Their selective indexes ranged from >190 to >1887. Selective index
was calculated by dividing the IC50 of modulators towards L929 cells by the EC50 of modulators
in reversing DOX resistance. EC50 values of pure dimers are in general consistent with the data
from the primary and secondary screening using un-purified clicked dimers, indicating that the
CuAAC reaction coupled with the high throughput screening is a reliable platform for rapid
discovery of MRP1 modulators.
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Table 5. Effective concentrations (EC50) of 21 potent pure clicked dimers and their selective
indexes. a
Pure
L929
EC50 (nM) for reversing
Selective
indexes
>1887
>1282
>1149
>1053
>1010
>553
>971
531
Linker conjugation No. of carbon
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
clicked dimers (IC , μM) Dox resistance in 2008/MRP1
site
A
A
A
A
A
B
B
B
B
B
B
A
A
A
A
A
B
B
B
A
C
B
/
atom in linker
50
Ac3Az11
Ac16Az1
Ac12Az1
Ac2Az4
Ac16Az2
Ac5Az1
Ac3Az4
Ac1Az1
Ac3Az3
Ac5Az3
Ac3Az1
Ac2Az1
Ac16Az7
Ac16Az4
Ac5Az11
Ac13Az5
Ac3Az2
Ac5Az4
Ac5Az7
Ac13Az1
Ac2Az11
>100
>100
>100
>100
>100
>57
53 ± 2
9
9
78 ± 17
87 ± 17
10
11
11
10
11
9
95 ± 5
99 ± 18
103 ± 15
103 ± 6
>100
60±16
>100
>50
113 ± 49
121 ± 13
137 ± 12
140 ± 40
151 ± 14
155 ± 33
156 ± 27
175 ± 18
193 ± 13
208 ± 48
217 ± 25
250 ± 31
263 ± 7
>826
>365
>714
>662
>645
>641
>571
>518
>481
>461
>400
>190
>336
>1370
46
11
12
9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
9
9
11
10
11
11
12
10
9
>100
>100
88±7
>100
298 ± 44
9
FD-4e
Verapamil
MK571
73 ± 13
1925 ± 677
19000 ± 1000
10
/
>5
/
/
a
A total of 21 pure clicked dimers was re-synthesized and determined their EC50 values. EC50
values were presented as mean r standard error of mean. N = 3-7 independent experiments.
Selective index = (IC50 of modulators towards L929)/ (EC50 of modulators for reversing DOX
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resistance). L929 is normal mouse fibroblast cell line. These pure clicked dimers can be divided
into three classes according to the linker conjugation site: (A) linker conjugated at C7 of A-ring
and C4’ of B-ring of flavones, (B) linker conjugated at C4’ of both B-rings of flavones and (C)
linker conjugated at C7 of both A-rings of flavones. “/” means verapamil and MK571 do not
contain linker. The chemical structures of these potent pure clicked dimers are shown in supporting
information (Figure S7 – S27).
0
1
2
3
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5
6
7
8
9
0
1
2
3
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0
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0
1
2
3
4
5
6
7
8
9
0
125
100
75
50
25
EC = 53 nM
5
0
0
-3
-2
-1
0
1
2
3
4
Log conc. of Ac3Az11 (nM)
Figure 4. EC50 of Ac3Az11 for reversing DOX resistance in 2008/MRP1 cells
2.2.3 Dimeric clicked alkyne-azide modulator is much more potent than monomeric alkyne or
azide in reversing DOX resistance in 2008/MRP1 cells
To illustrate the effect of dimerization in improving the MRP1-modulating activity, we
determine the potency of the dimers and monomers. One μM of clicked dimers Ac3Az11 and
Ac12Az1 showed promising MRP1-modulating activity with RF = 13.5 and 12.2, respectively
(Table 6). In contrary, their constituent monomers Ac3, Az11, Ac12 or Az1 were not active with
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RF values below 1.8 even when they were used at double the concentration (Table 6). A
combination of Ac3 and Az11 or Ac12 and Az1 also displayed no activity with RF = 1.3 and 1.5,
respectively (Table 6). This observation of MRP1 being strongly inhibited by dimeric AcN-AzM
modulator, but not by the constituent monomeric AcN or AzM highlights the importance of
dimerization of constituent flavonoid monomers in modulating MRP1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Comparing MRP1-modulating activity of pure Ac3Az11, Ac12Az1 and their respective
monomers in 2008/MRP1 cells.a
Compounds
μM Ac3Az11
μM Ac3
2 μM Az11
μM Ac3 + 1 μM Az11
μM Ac12Az1
2 μM Ac12
μM Az1
μM Ac12 + 1 μM Az1
0.1% DMSO
.1% DMSO
Mean IC50 of DOX (nM) in 2008/MRP1 cells
RF
13.5
1.3
1.1
1.3
12.2
1.0
1.8
1.5
1.0
9.5
1
2
31 ± 10
541 ± 189
487 ± 108
444 ± 101
51 ± 16
566 ± 132
338 ± 87
400 ± 105
597 ± 165
63 ± 5*
1
1
2
1
0
a
2
008/MRP1 cells were incubated with either 1 μM of clicked dimer or 2 μM of alkyne or azide
monomers or a mixture of 1 μM each of alkyne and azide monomers in the presence of DOX for
5 days. After incubation, percentage survival was determined by MTS proliferation assay. Relative
fold (RF) was determined by dividing the IC50 value without modulators / IC50 value with
modulators. *2008 wild type ovarian cancer cells were used. 2008/MRP1 is MRP1 overexpressing
ovarian cancer cell line. Each compound was tested in triplicate and performed in 3 independent
experiments. 0.1% DMSO was used as a solvent control.
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2
.2.4 Effect on intracellular DOX accumulation in 2008/MRP1 cells
The above results showed that these triazole linked flavonoid dimers are effective MRP1
modulators. DOX is a fluorescent MRP1substrate that can be used to monitor intracellular drug
accumulation. We determined whether the modulation of MRP1-mediated drug resistance is
associated with a concomitant increase in drug accumulation. Since Ac3Az11, Ac12Az1 and
Ac16Az1 are effective MRP1 modulators according to their EC50 (Table 5), we selected these
compounds for further characterization. As shown in Figure 5, treatment of 2008/MRP1 cells with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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9
0
1
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0
1
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0
1
2
3
4
5
6
7
8
9
0
2
PM of compounds Ac3Az11, Ac12Az1 and Ac16Az1 resulted in 2.0-fold increase in
intracellular DOX accumulation. Verapamil, being a less potent MRP1 modulator, only increased
the DOX accumulation by about 1.6-fold (Figure 5). MK571, an even less potent modulator, did
not increase DOX accumulation in 2008/MRP1 cells at 2 μM (Figure 5). The result suggests that
these flavonoid dimers inhibit transport activity of MRP1 and restore the intracellular DOX
concentration to a level similar to that of parental 2008/P cells.
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.0
.5
*
*
*
2008/P
008/MRP1
*
2
2.0
1
1
1
1
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1
1
1
1
2
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0
1
2
3
4
5
6
7
8
9
0
1
1
.5
.0
N=3 independent
expts
0.5
.0
0
Figure 5. Effect of pure clicked flavonoid dimers on intracellular DOX accumulation in
o
2
008/MRP1 cells. 2008/MRP1 cells were incubated with 5 PM DOX for 120 minutes at 37 C
with or without 2 PM of Ac3Az11, Ac12Az1, Ac16Az1, FD-4e, verapamil or MK571. 0.2% of
DMSO was used as negative control. After incubation, cells were washed and intracellular
accumulation of DOX was measured by flow cytometry. Experiments were performed in duplicate
and repeated thrice. The florescence level of each sample was normalized to the 0.2% DMSO
negative control and presented as a fold-difference. The results were presented as mean ± standard
error of mean. Student paired t test was conducted relative to 2008/MRP1 cells incubated with
0
.2% DMSO. * P <0.05.
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.2.5 Effect on MRP1 protein expression level
We characterized the effect of Ac3Az11 on MRP1 protein expression by flow cytometry
Figure 6A and 6B). 2008/MRP1 cells has 7.8-fold (P<0.001) higher levels of MRP1 than the
(
0
1
2
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0
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0
parental 2008/P cells (Figure 6B). After incubating with 1, 2 or 5 μM of Ac3Az11 for 3 days, the
high expression level of MRP1 in 2008/MRP1 cells remained unchanged, indicating that Ac3Az11
does not affect the protein expression. After co-incubating with Ac3Az11, the increased DOX
accumulation level observed in 2008/MRP1 cells (Figure 5) might be due to the loss of
functionality of MRP1.
A
2008/P
2008/MRP1
Cell
Count
Isotype
control
Isotype
control
MRP1-
FITC
MRP1-
FITC
Total MRP1-FITC level
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B
2
008/P
1
0000
8000
000
000
000
7
**
.8X
2008/MRP1
1
1
1
1
1
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1
1
1
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9
0
1
2
3
4
5
6
7
8
9
0
N=3 independent expts
6
4
2
0
Figure 6. Effect of Ac3Az11 on MRP1 protein expression. The 2008/MRP1 cells were incubated
with 1, 2 or 5 μM of Ac3Az11 for 3 days. Total MRP1 level was measured by flow cytometer at
FL1 channel. (A) Flow cytometry result of total MRP1 protein expression in 2008/P or
2008/MRP1 cells after incubating with 0.25% DMSO. (B) Total MRP1 protein level of
2
008/MRP1 cells after incubating with 1, 2 or 5 μM of Ac3Az11 for 3 days. N = 3 independent
experiments. The fluorescence units were presented as mean ± standard error of mean. 0.25%
DMSO was used as a solvent control. Student paired t test was conducted between 2008/P and
2008/MRP1 cells after incubating with 0.25% of DMSO for 3 days. ** P<0.001.
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