Download Chalcogenopyrylium Dyes as Modulators of Multidrug Resistance Protein (MRP) 1,

Chalcogenopyrylium Dyes as Modulators of Multidrug Resistance Protein (MRP) 1,
MRP2 and MRP4 Transport Activities
By:
Robert Leonard Myette
A thesis submitted to the Graduate Program in Pathology and Molecular Medicine in conformity
with the requirements for the Degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
Final Submission: November, 2011
Copyright © Robert Leonard Myette, 2011
Abstract
MRPs mediate the ATP-dependent efflux of a structurally diverse array of compounds.
Certain MRPs, including MRP1, MRP2 and MRP4, are involved in multidrug resistance in
tumour cells, while in non-malignant cells these MRPs can influence the distribution and/or
elimination of many clinically important drugs. In addition, these MRPs mediate the efflux of
physiological metabolites, many of which are conjugated organic anions. Modulation of the drug
transporting activity of MRP1 (and to a lesser extent MRP2 and MRP4) has been a long sought
after therapeutic objective. In this study, the modulatory abilities of five structurally distinct
classes (I-V) of chalcogenopyrylium dyes (CGPs) were examined utilizing an in vitro assay
which measures inhibition of radiolabeled estradiol glucuronide ([3H]E217βG) (a prototypical
MRP substrate) uptake into inside-out membrane vesicles prepared from MRP-transfected
human embryonic kidney (HEK) cells. Additionally, some CGPs were tested in a calcein efflux
assay using intact MRP1-transfected HEK cells. Sixteen of 34 CGPs initially tested at a single
concentration (≤30 µM) inhibited MRP1-mediated uptake by >50%, with IC50’s ranging from
0.7-7.6 µM. Of the 9 CGPs with IC50’s ≤2 µM, five belonged to Class I, two to Class II, and two
to Class IV. When tested in the calcein efflux assay, only 4 of 16 CGPs inhibited MRP1mediated cellular efflux by >50% (I-3, -4, -6, IV-1) while a fifth (II-5) inhibited efflux by 23%.
These five CGPs were then tested as modulators of [3H]E217βG uptake by MRP2 and MRP4.
Their effects on MRP2 transport activity were differential with two (I-4, I-6) inhibiting transport
(IC50’s 2.0, 7.1 µM), two (I-3, IV-1) stimulating transport (>2-fold), while II-5 had no effect. On
the other hand, all five CGPs inhibited [3H]E217βG uptake by MRP4, but less effectively than by
MRP1. Finally, five analogs of CGP IV-1 were tested for their effects on MRP1, MRP2 and
MRP4 [3H]E217βG uptake, but none were more efficacious than CGP IV-1. The CGPs tested
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here represent novel MRP1, MRP2 and MRP4 modulators with variable effects on transport
activities. These CGPs may represent a new avenue for the development of clinically applicable
modulators of MRP proteins involved in multidrug resistance.
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Statement of Originality

All experiments described in this thesis were performed by Robert L. Myette.

The synthesis of the CGPs was done in the laboratory of Dr. Michael Detty by Sean P.
Ebert and Bryan Wetzel.

This thesis was written by Robert L. Myette.

Significant editing of the thesis and guidance was provided by my supervisor and mentor
Dr. Susan Cole. Helpful discussions were also provided by my fellow lab members.

The calcein efflux assay was optimized by Dr. Gwenäelle Conseil and Mrs. Kathy
Sparks. Preliminary calcein experiments were performed by Mrs. Kathy Sparks;
however, none of her data are included here.

The stable cell line used throughout this thesis was derived by Dr. Gwenäelle Conseil.
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Acknowledgments
I would like to thank my friends and family for being there for me. Your conversation
was helpful and thoughtful, and your words always encouraging and inspiring. I would also like
to thank my fellow lab members for their constant encouragement and input with respect to my
project and beyond. Thank you to Dr. Susan Cole for taking me on as a M. Sc. student and
instructing and mentoring me over the last two years, I am grateful to you for allowing me to
work on such an interesting, and challenging project. Also, I want to thank Dr. Michael Detty
and his students Sean and Bryan for providing me with the compounds investigated in this study.
Furthermore, thank you to Drs. Greer and LeBrun for helpful advice throughout my MSc. And a
final thank you in advance to my defense committee for agreeing to be here. Lastly, A very big
thank you to Fern McSorley for being there for me when I needed you, throughout it all.
Individually, I would need 300 pages to thank everyone who has helped me along the
way, whether it was going for a pint(s) and a conversation, or proofreading a document and then
a pint(s), although your names are not here, I am definitely indebted to you all.
Lastly, I would like to say, “Although sometimes I can’t see, I know I won, and it is ok,
because it is just him”. Basically, I think most of you will know what that means. Test your
might, for you never know the next time you will be playing MK. The city is destroyed. Je me
souviens. Hennessey.
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Table of Contents
ABSTRACT………………………………………………………………………..……….…….ii
STATEMENT OF ORIGINALITY…...………………………………………………..…..........iv
ACKNOWLEDGMENTS………………………………………………………………..……....v
TABLE OF CONTENTS..............................................................................................................vi
LIST OF TABLES..……………………………………………………………………….…......ix
LIST OF FIGURES…………..…………………………………………………………….….....x
LIST OF ABBREVIATIONS……………………………………………………………........…xi
CHAPTER I: INTRODUCTION AND BACKGROUND
1.1 ATP-Binding Cassette (ABC) Transporters..................................................................1
1.2 An Overview of P-glycoprotein and MRP1..................................................................4
1.2.1 P-glycoprotein (Pgp / gene symbol ABCB1)………......................................4
1.2.2 Multidrug Resistance Proteins (MRP / gene symbol ABCC)…....................5
1.2.3 Multidrug Resistance Protein 1 (MRP1 / gene symbol ABCC1).……..........5
1.2.4 Multidrug Resistance in Cancer Mediated by MRP1.....................................7
1.3 MRPs in Drug Disposition and Drug-Drug Interactions
1.3.1 MRP1……………………...........................................................................11
1.3.2 MRP2 (gene symbol ABCC2)......................................................................14
1.3.3 MRP4 (gene symbol ABCC4)…………………………………..…..……..18
1.4 Reversal of Drug Resistance Mediated by Pgp and MRP1........................................20
1.4.1 Modulators of Pgp........................................................................................20
1.4.2 Modulators of MRP1....................................................................................27
1.5 Rationale, Hypothesis and Objectives……………………………………….............34
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CHAPTER II: MATERIALS AND METHODS
2.1 Materials
2.1.1 Synthesis and Characterization of Chalcogenopyrylium Dyes (CGPs).......36
2.1.2 Other Reagents..............................................................................................36
2.2 Cell Culture and Transfection......................................................................................39
2.3 Preparation of Membrane Vesicles..............................................................................40
2.4 Immunoblotting of MRP1, MRP2 and MRP4 Proteins...............................................41
2.5 MRP-mediated Uptake of [3H]E217βG by Membrane Vesicles..................................42
2.6 MRP1-mediated Calcein efflux from HEK-MRP1 Cells............................................44
CHAPTER III: RESULTS
3.1 Modulation of MRP1 Transport Activity by the CGPs………………………..........45
3.1.i
Initial Screen of CGPs on MRP1-mediated Vesicular Uptake of
[3H]E217βG…………………………………………………………......45
3.1.ii Relative inhibitory Potencies of CGPs on MRP1-mediated [3H]E217βG
Uptake…………………………………………………………………..53
3.1.iii Modulation of MRP1-mediated Calcein Efflux from Intact HEK-MRP1
Cells by CGPs………………………………………………………......56
3.1.iv Modulation of MRP1-mediated [3H]E217βG Uptake by Class IV CGP
analogs.....................................................................................................56
3.2 Modulation of MRP2-mediated [3H]E217βG Uptake by CGPs I-3, I-4, I-6, II-5
and IV-1……………………………………………………………………………...59
3.3 Modulation of MRP4-mediated uptake of [3H]E217βG ………….………….…......65
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CHAPTER IV: DISCUSSION……………………………………………………….…………68
CHAPTER V: CONCLUSIONS AND FUTURE DIRECTIONS…………………………..….78
REFERENCES……………………………………………………………….………….…..…..81
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List of Tables
I.1 A brief list of substrates of MRP1, MRP2 and MRP4.............................................................12
I.2 MRP1 modulators and their respective effects on transport activity........................................33
I.3 The Evolution of the CGPs.......................................................................................................35
III-1 Inhibition of MRP1-mediated uptake of [3H]E217βG by Class I CGPs................................48
III-2 Inhibition of MRP1-mediated uptake of [3H]E217βG by Class II CGP................................49
III-3 Inhibition of MRP1-mediated uptake of [3H]E217βG by Class III CGPs.............................50
III-4 Inhibition of MRP1-mediated uptake of [3H]E217βG by Class IV CGPs…………..….......51
III-5 Inhibition of MRP1-mediated uptake of [3H]E217βG by Class V CGPs..............................52
III-6 Summary of IC50 Data...........................................................................................................54
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List of Figures
I.1 Topological structures of some mammalian ABC transporters………………………............2
I.2 Chemical structures of known stimulators of MRP2-mediated transport activity..................17
I.3 Chemical structures of the rigid chalcogenorhodamines and the flexible
chalcogenopyryliums (CGPs).................................................................................................26
II.1 Basic chemical structures of the five Classes of CGPs examined in this study.....................37
II.2 Chemical structures of CGP IV-1 and its structural analogs..................................................38
III.1 Expression of MRP1, MRP2 and MRP4 proteins in membrane vesicles..............................46
III.2 Concentration dependence of CGP-mediated inhibition of [3H]E217βG uptake by
MRP1.....................................................................................................................................55
III.3 Effect of 5 CGPs on cellular efflux of calcein from HEK-MRP1 cells.................................57
III.4 Chemical structures of I-3, I-4, I-6, II-5 and IV-1 that inhibit MRP1-mediated calcein
efflux from intact HEK-MRP1 cells by >25%......................................................................58
III.5 Effect of Class IV-1 analogs on MRP1-mediated [3H]E217βG uptake activity....................60
III.6 Effect of selected CGPs on MRP2-mediated uptake of [3H]E217βG into inside-out
membrane vesicles................................................................................................................61
III.7 Concentration dependent effects of CGPs on MRP2-mediated uptake of [3H]E217βG
into inside-out membrane vesicles.........................................................................................63
III.8 Effect of Class IV-1 CGP analogs on MRP2-mediated uptake of [3H]E217βG....................64
III.9 Effect of selected CGPs on MRP4-mediated uptake of [3H]E217βG into inside-out
membrane vesicles................................................................................................................66
III.10 Effect of Class IV-1 analogs on MRP4-mediated uptake of [3H]E217βG into insideout membrane vesicles.........................................................................................................67
x
List of Abbreviations
ABC
ATP-binding cassette
ALL
Acute lymphoblastic leukemia
AML
Acute myelogenous leukemia
BBB
Blood brain barrier
BSA
Bovine serum albumin
CAM
Calcein acetoxymethyl ester
CFTR
Cystic fibrosis transmembrane conductance regulator
CGP
Chalcogenopyrylium dye
cMOAT
Canalicular multi-specific organic anion transporter
DJS
Dubin-Johnson Syndrome
DMEM
Dulbeccos modified eagle medium
E217βG
17β-estradiol 17-(β-D-glucuronide)
ECL
Enhanced chemiluminescence
FBS
Fetal bovine serum
GSH
Glutathione
GSSG
Oxidized glutathione (glutathione disulfide)
KO
Knock out
LTC4
Leukotriene C4
MAb
Monoclonal antibody
MDCK
Madine-Darby canine kidney cells
MDR
Multidrug resistance
miRNA
Micro-RNA
MRP
Multidrug resistance protein
MSD
Membrane spanning domain
NBD
Nucleotide binding domain
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NHERF
Na+/H+ exchanger regulatory factor
NMRI
Navy Medical Research Institute
NSCLC
Non-small cell lung cancer
PDZ
PSD-95/Disc Large/ZO-1 Scaffold
PGE2
Prostaglandin E2
Pgp
P-glycoprotein
QSAR
Quantitative structure-activity relationship
SCLC
Small cell lung cancer
siRNA
Small interfering RNA
TM
transmembrane helix
TSB
Tris-sucrose buffer
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CHAPTER I. INTRODUCTION AND BACKGROUND
1.1 ATP-Binding Cassette (ABC) Transporters
The ABC superfamily is a functionally diverse group of polytopic membrane proteins,
whose members transport a vast array of structurally dissimilar compounds at the expense of
ATP binding and hydrolysis (1, 2). The core functional unit of an ABC transporter consists of
two polytopic membrane spanning domains (MSD) and two nucleotide binding domains (NBD)
where ATP binding and hydrolysis takes place (3). All ABC transporter NBDs have three
defining motifs, the Walker A and Walker B motifs (4) and the ABC ‘C’ signature motif (1).
Most MSDs contain 6 transmembrane (TM) helices, but the number can range from 5-10 (2, 5).
Generally, the MSDs are important for the binding and translocation of substrate and it is the
physical interactions between the TMs and the NBDs, via the cytoplasmic loops, that result in
active substrate translocation through conformational changes in a transporter (6).
In most instances, the functional core of mammalian ABC transporters is translated as a
single polypeptide with an H2N-MSD-NBD-MSD-NBD-COOH arrangement. Exceptions
include some members of the “C” subfamily ABC transporters (MRP1, 2, 3, 6 and 7) which
possess an extra MSD at the NH2-terminus, and others such as ABCG2, ABCG5 and ABCG8
where the NBD precedes the MSD and which are translated as half-transporters and homo- or
hetero-dimerize to form a functional transporter (Fig. I.1) (2).
In 2006, Locher and colleagues (7), proposed a mechanism of substrate translocation by
ABC transporters as follows: binding of substrate occurs at the TMs, during or after which the
NBDs bind ATP causing a “dimerization” of the NBDs, promoting an outward-facing
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A
B
C
Figure I.1 Topological structures of some mammalian ABC transporters. Differences are noted between the (A)
long (MRP1, 2, 3, 6, 7) and (B) short (MRP4, 5, 8) mammalian ABC “C” transporters, where the long ones have an
extra MSD (MSD0). Pgp has a typical four-domain ABC transporter structure, labeled short, above. (C) In ABCG2,
G5 and G8, the NBD precedes the MSD and the protein homodimerizes (ABCG2) or heterodimerizes (ABCG5, 8)
to make a functional transporter.
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conformation. Upon hydrolysis of ATP, the hydrolytic products are released promoting a shift to
an inward facing conformation, thus resulting in substrate extrusion (and/or intrusion in the case
of bacterial transporters) (6, 7).
There are 48 members of the human superfamily of ABC transporters which are
separated into 7 subfamilies denoted A through G (2). The role of some human ABC transporters
includes drug disposition and excretion of endo and xenobiotics, which can represent a ‘doubleedged sword’ when it comes to human health and disease. Thus, some members of the ABC
superfamily translocate important biological molecules including conjugated metabolites,
eicosanoids, bile acids and steroids (2, 8). In addition, many human ABC transporters are
directly involved in the absorption, distribution and excretion or elimination, and thus the
efficacy and toxicity, of various clinically relevant drugs (9). Some ABC transporters (notably Pglycoprotein (Pgp, ABCB1), MRP1 (ABCC1) and ABCG2 (ABCG2), discussed below) which
confer multidrug resistance (MDR) on tumour cells, have been implicated in drug resistant
malignant disease.
In addition to their physiological and pharmacological roles as transporters of
metabolites, xenobiotics and mediators / regulators of ion flow, several ABC transporters are
involved in human disease, where their altered or lack of expression results in various genetic
disorders. For example, cystic fibrosis, Dubin-Johnson syndrome (DJS), Pseudoxanthoma
elasticum and Tangier disease, are caused by mutations in the genes encoding the proteins, cystic
fibrosis transmembrane conductance regulator (CFTR) (ABCC7), MRP2 (ABCC2), MRP6
(ABCC6) and ABCA1 (ABCA1), respectively (10-13).
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1.2 An Overview of P-glycoprotein and MRP1
1.2.1 P-glycoprotein (Pgp / gene symbol ABCB1)
Of the several ABC transporters shown to transport clinically relevant therapeutics in
humans, P-glycoprotein (Pgp/ABCB1) is the most thoroughly studied. Pgp has a 4-domain
structure (MSD-NBD-MSD-NBD) (Fig. I-1B) and mediates resistance to multiple classes of
compounds with vastly differing structures and mechanisms of action; however, there are some
commonalities among the substrates. Typically, Pgp transports hydrophobic drugs which have
polyaromatic backbones, and are neutral or carry positive charges (14). Pgp can transport Vinca
alkaloids, anthracyclines, taxanes and camptothecins, among other clinically relevant agents
(15). Currently, no physiological roles have been assigned to Pgp although it has been reported
that Pgp is responsible for the transport of platelet-activating factor in porcine kidney cells (16).
However, this function has never been corroborated in a knock-out (KO) mouse model (16). Pgp
can also translocate lipid analogues, but likely does not transport major membrane lipids (17).
The apical expression of Pgp in normal polarized epithelial or endothelial cells results in
substrate being translocated across the apical membrane of the cell into the lumen of the tissue
where the cell is located (18). Pgp is found in various so-called pharmacological ‘sanctuary sites’
in the human body, such as at the blood-brain barrier (BBB), the blood-testis barrier, and the
fetal-maternal blood barrier as well as in the gut where it functions to restrict the bioavailability
of drugs and protect tissue (18-20).
Little was known of the in vivo roles of Pgp until the generation of KO mice lacking one
or both of the two drug transporting murine Pgp homologs (Mdr1a / Mdr1b). Borst and
coworkers were the first to derive and characterize mouse Pgp KO models (by generating single
Mdr1a-/- and Mdr1b-/- KO mouse as well as a double KO mouse). They then showed that the
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single and double KO mice were both viable and fertile with most histological, hematological,
serum chemical and immunologic parameters being normal (21). However, when challenged
with drugs known to be Pgp substrates, altered distribution and elevated levels of drugs were
noted in various tissues (brain, adrenal glands and ovaries) in the mice, but differences between
the single and double KOs were evident in accordance with the different tissue distribution of the
two homologs Mdr1a and Mdr1b (21).
1.2.2 Multidrug Resistance Proteins (MRP / gene symbol ABCC)
The human ABCC, or MRP/CFTR subfamily of membrane transporters, contains 13 members, 8
of which are (at least in vitro) organic anion transporters designated the MRPs (MRP1 (gene
symbol ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), MRP5 (ABCC5), MRP6
(ABCC6), MRP7 (ABCC10) and MRP8 (ABCC11)). Thus far, MRP9 (ABCC12) appears nonfunctional and ABCC13 is a pseudogene, while CFTR (ABCC7) is a cAMP-gated chloride
channel, and the sulfonylurea receptors SUR1 (ABCC8) and SUR2 (ABCC9) are regulators of
inwardly rectifying potassium channels (2). The MRP/ABCC members can be further grouped
based on their structure, where MRP1, 2, 3, 6 and 7 have an extra MSD (MSD0), and so are
called the ‘long’ MRPs (3 MSDs, 2 NBDs) while the remaining 4 members (MRP4, 5, 8 and
ABCC12) are ‘short’ MRP/ABCC transporters (2 MSDs, 2 NBDs), possessing the more typical
four-domain ABC transporter structure (Fig. I.1). Some of the MRP subfamily proteins are
implicated in the disposition of clinically used drugs in normal and malignant tissues, as
reviewed in (22).
1.2.3 Multidrug Resistance Protein 1 (MRP1 / gene symbol ABCC1)
MRP1 is the most thoroughly characterized of the MRP-related ABCC transporters with
respect to its roles in clinical drug resistance (2, 5, 19, 22-26). MRP1 was first cloned in 1992
5
from a doxorubicin-selected small cell lung cancer (SCLC) cell line (27). After doxorubicin
selection, the cell line showed a cross resistance to a wide range of structurally dissimilar
cytotoxic drugs; however, this cell line did not overexpress Pgp, the only known transporter
capable of conferring resistance on tumour cells at that time (28, 29). Ultimately, a gene
encoding a 1,531 amino acid protein was identified and shown to be a novel ABC transporter
that shared some structural features with CFTR, thus giving rise to the MRP/CFTR designation
for the ABCC subfamily of ABC proteins (27).
The substrate specificity of MRP1 is diverse, ranging from glutathionylated,
glucuronidated and sulfate-conjugated metabolites, GSH and GSSG, HIV protease inhibitors,
anthracyclines, epipodophyllotoxins and Vinca alkaloids (5, 22). Although the substrate list is
vast, MRP1 is not a completely indiscriminate transporter of organic anions. For example, 17β
estradiol-17 β(D)–glucuronide (E217βG) is a good substrate of human MRP1 in vitro, but its 3substituted glucuronide structural isomer is not (5, 30). The role of MRP1 in normal cellular
physiology will be discussed later in Section 1.3.
1.2.4 Multidrug Resistance in Cancer Mediated by MRP1
Cancer cells are under constant selection to change and adapt to their surroundings in
order to facilitate their survival and proliferation. As a consequence of these changes, many
tumour cells can develop resistance to various chemotherapeutics due to enhanced expression of
one or more of a subset of ABC transporters, a phenomenon known as MDR (31). MDR is a
barrier to effective treatment in many cancers and new methods of treating cancers expressing
ABC transporters are a long sought after therapeutic objective. Of the 48 human ABC proteins,
there are three drug transporters (Pgp, ABCG2 and MRP1) that confer drug resistance on cancer
6
cells that are considered to be clinically relevant. Numerous additional ABC transporters have
been characterized as functional drug efflux pumps in vitro, which may contribute to drug
sensitivity in vivo, and may affect distribution of drugs in normal cells (18).
Treatment of cancer patients over the course of history has entailed surgical removal of
tumour tissue, chemo- and/or radio-therapy. However, when a tumour becomes metastatic,
chemotherapy often becomes the first line of treatment. Anticancer drugs can fail to destroy
tumour cells for various reasons; however, one mechanism through which a tumour cell loses
sensitivity to chemotherapy is up-regulation and overexpression of energy-dependent efflux
transporters, such as Pgp, ABCG2 and MRP1. MDR can be inherent, or acquired. Inherent MDR
refers to a cell’s ability to be resistant to a drug without having been exposed to the drug.
Acquired MDR is different in that at first, cancer cells succumb to the drug(s); however, a small
population develops resistance and is able to grow and resist the cytotoxic action of subsequent
chemotherapy (32). In the case of MDR caused by ABC transporters, once a tumour cell
develops resistance, it is also cross resistant to various structurally similar and diverse
chemotherapeutics making effective treatment difficult (33).
While Pgp and ABCG2 have garnered much interest with respect to their roles in cancer
MDR, for the purposes of this thesis, only MRP1 will be discussed. There are several recent
reviews covering the involvement and importance of both ABCG2 and Pgp with respect to drug
response and MDR in cancer (15, 26, 33-37).
Since the original observation that MRP1 could confer drug resistance in vitro (38, 39),
many studies have tried to determine whether MRP1 is important in malignant disease in
patients. Although there is overwhelming evidence that MRP1 plays a distinct role both in vitro
7
and in in vivo animal studies with respect to cancer drug resistance, the contribution of MRP1 to
clinical drug resistance is still uncertain (15).
When ascertaining whether MRP1 (or another transporter) is expressed in tumour tissues,
one of the major confounders is the ubiquitous expression of MRP1 in normal tissues and the
degree to which its presence in clinical tumour samples can be attributed to contamination by
non-malignant tissue. Nevertheless, high levels of MRP1 have been detected in untreated and
treated non-small cell lung cancer (NSCLC), which, unlike SCLC, is inherently multidrug
resistant (40, 41). High MRP1 mRNA levels present in NSCLC tumours (as determined from
tumour specimens taken from trans-bronchial biopsy from patients treated with neo-adjuvant
therapy) are a significant negative indicator of tumour-free survival and overall survival (42).
Conversely, in a more recent study, Merk et al. (43) showed that expression of various ABC
transporters, including MRP1, in patient-derived xenograft models of NSCLC did not correlate
with treatment response (drugs tested included etoposide, carboplatin, gemcitabine, paclitaxel,
cetuximab and erlotinib) in any case except one (ABCG2 expression and etoposide response).
MRP1 protein levels in untreated NSCLC are higher compared to SCLC, and MRP1 expression
in SCLC appears to be limited to small patches of cells around the edges of the tumour tissue
(41, 44). However, a retrospective analysis of SCLC patients with limited disease who
underwent surgical resection revealed no significance between MRP1 levels and overall survival
in a multivariate analysis by immunohistochemical staining (45). Recent evidence suggests MDR
in SCLC may also occur through a MRP1 micro-RNA (miRNA) regulated mechanism. Thus,
miRNA-134 was shown to down-regulate MRP1 mRNA, and down-regulation of MRP1
correlated with elevated levels of miRNA-134. This finding may provide an indirect method of
determining whether patients with SCLC will have MRP1 positive tumours (46).
8
MRP1 expression has also been investigated in breast and prostate cancers, as well as
neuroblastoma and haematological malignancies. In certain forms of breast cancer, MRP1 is a
negative prognostic marker and increased levels of MRP1 expression are observed in tumours of
patients who have relapsed (47, 48). More recent evidence suggests that MRP1 expression at
diagnosis is not necessarily associated with a more aggressive phenotype, and no association
between MRP1 expression and estrogen receptor status was observed (49). The impact of MRP1
expression on early stage chemotherapy resistant breast cancer clinical outcome was reported by
Filipits et al. (50). MRP1 expression was found to be an independent predictive marker for
shorter relapse-free survival and overall survival in patients treated with cyclophosphamide,
methotrexate and fluorouracil, but it did not predict shorter relapse-free survival and overall
survival in patients treated with tamoxifen. This has implications in treatment methodology as
early-stage breast cancer patients whose tumours overexpress MRP1 can be stratified to an
endocrine treatment regimen (50).
With respect to prostate cancer, MRP1 protein is expressed at high levels in precursor
prostate cancer lesions (prostatic intraepithelial neoplasia) as well as in prostate adenocarcinoma
(5, 51). Furthermore, some primary cultures of prostate cancer show a low response to
chemotherapy (methotrexate and doxorubicin) as a result of elevated MRP1 (protein levels) (52).
More recently, Sanchez et al. (53) showed that pharmacological inhibition (MK571) and siRNA
knockdown of MRP1 in primary prostate cancer cell lines re-sensitized these cells to
methotrexate, and suggested a role for MRP1 in the MDR of these cell lines.
The role of MRP1 in neuroblastoma has been examined most extensively by Haber and
colleagues (54). Amplification of the NMYC gene is a major molecular indicator of outcome in
9
neuroblastoma and MRP1 expression has been shown to positively correlate with NMYC
amplification (55). MRP1 has also been shown as a stand-alone negative prognostic indicator in
neuroblastoma patients who do not overexpress NMYC (54, 56, 57). In a recent study by Oue et
al. (58), MRP1 was shown to be expressed (protein) in 3 of 8 chemotherapy naive cases of
neuroblastoma; however, following chemotherapy (indicated as “standard chemotherapy” in the
article), MRP1 levels increased in 7 of 8 cases, indicating upregulation of the protein following
treatment.
The clinical relevance of MRP1 expression in hematological malignancies is a
contentious issue with some groups reporting major clinical significance while others show no
prognostic or predictive value associated with MRP1 expression. Thus, El-Sharnouby et al. (59)
conducted a study in which leukemic blasts were collected from 34 pediatric acute lymphoblastic
leukemia (ALL) patients and analyzed for MRP1 mRNA. All eleven patients who did not have
elevated MRP1 mRNA levels achieved complete remission whereas only 9 of 19 with elevated
MRP1 mRNA did so (59). Conversely, an earlier study by Olson et al. (60) reported that levels
of MRP1 protein in pediatric ALL did not correlate with treatment failure. Furthermore, a 2008
study of blasts isolated from ALL patients (not strictly pediatric) were examined and high levels
of MRP1 mRNA did not correlate with poor clinical outcome; however, in patients with AML,
there was a significant correlation (61).
For acute myelogenous leukemia (AML), studies have been difficult to interpret with
some authors reporting a correlation between MRP1 levels (mRNA or protein) and poor
prognosis (61, 62, 63) while a number of studies report no correlation (64, 65). Studies on other
forms of leukemia (adult ALL, pediatric AML) have mainly shown no prognostic impact (66).
10
Assessing MRP1 levels in leukemic cells is complicated by the fact that MRP1 and other ABC
transporters are expressed in normal leukocytes (67). Furthermore, differences in MRP1
detection methods (protein levels vs mRNA), and the specific antibody used for protein detection
may differ, as well as the lack of strict correlation between MRP1 mRNA levels with MRP1
protein levels, may account for some inconsistencies among groups.
1.3 MRPs in Drug Disposition and Drug-Drug Interactions
1.3.1 MRP1
In addition to its observed drug resistance role in tumour cells, MRP1 contributes to the
disposition of various endo- and xenobiotics in normal tissues (19). As mentioned previously,
MRP1 transports many diverse organic anion glutathione (GSH)-conjugated metabolites as well
as glucuronate and sulfate conjugates (68) (Table I-1). MRP1 has unique interactions with GSH
in that GSH transport is stimulated by drugs such as verapamil and some bioflavonoids (69);
however, the drugs and bioflavonoids themselves are not transported, and there is no evidence
that a cotransport mechanism is implicated (69). Vincristine is able to enhance the transport of
GSH, and vice versa, indicating that these compounds are co-transported since no conjugation
occurs (70).
Like Pgp, MRP1 plays a protective role in the body through its expression at various socalled pharmacological sanctuary sites including the blood-cerebrospinal fluid barrier, bloodtestis barrier and the blood-placental barrier (71). MRP1 also appears to be playing a protective
role
11
Table I.1
A brief list of substrates of MRP1, MRP2 and MRP4
Adapted from reference (22).
a
Monophosphorylated forms are transported
12
against xenobiotics through its expression in the gut, kidney and lung as well as other tissues (41,
72-74). A physiologically relevant molecule, leukotriene C4 (LTC4), which is a proinflammatory
mediator, has been identified as a high affinity substrate for MRP1 (75). Again, little was known
of the in vivo roles of MRP1 until the Abcc1 KO mouse model was developed. Abcc1 KO mice
are viable and fertile, but their response to inflammatory stimuli is impaired (71). When
challenged with etoposide, the Abcc1 KO mice are hypersensitive to escalating doses and unable
to survive at doses >60 mg/kg (whereas wild-type mice are able to survive doses up to 100
mg/kg) (71). Murine Mrp1 has 88% amino acid sequence identity with human MRP1 (76);
however, transfection studies using murine Mrp1 do not provide cells with resistance to
anthracyclines (eg. doxorubicin, daunomycin, epirubicin) nor does Mrp1 transport E217βG (76).
Interestingly, Mrp1-mediated resistance to heavy metal oxyanions as well as vincristine and
etoposide is comparable to that of the human MRP1 (76).
Three pharmacologically distinct substrate binding sites on MRP1 have thus far been
identified, some of which involve amino acids in TM’s 6, 10-11 and 16-17 (77, 78). It is not
surprising that MRP1 has more than one binding site as it is able to identify and transport
numerous structurally diverse and unrelated compounds. Its binding pocket(s) have been
described as being more complex than being simply bipartite, but if functionally linked, they are
more likely multipartite. Using limited trypsin digestion, sensitivity assays, photolabeling and
immunoblot analysis, Qian et al. (78) determined not only that an azido-derivatized high affinity
GSH-dependent inhibitor ([125I]LY475776) of MRP1 binds to TMs 16 and 17, they also found
weak labeling of TMs 12, 13, 14 and 15 (78). Studies using mutational analysis identified Thr1242
and Trp1246 in TM17 as essential amino acids for transport of E217βG; however their mutation to
alanine did not hinder LTC4 transport activity, thus providing further evidence that these
13
substrates are interacting with MRP1 at mutually exclusive sites (79). Further evidence for
multiple binding sites was provided by the Georges group who showed that an azido-deravitized,
iodinated form of Rhodamine-123 could bind to multiple places on MRP1 (80). Although there
is evidence showing the existence of more than one binding site, it is uncertain whether specific
subsets of amino acids work in concert to transport individual or classes of conjugated and
unconjugated substrates (78).
1.3.2 MRP2 (gene symbol ABCC2)
MRP1 was the first MRP in the ABCC subfamily to be cloned in 1992; since then, 7
additional MRP proteins have been cloned, each having its own unique set of physiological and
pharmacological characteristics. MRP2, originally functionally characterized as a canalicular
multi-specific organic anion transporter (cMOAT), was first cloned from rat liver in 1996 (81)
and then from humans in the same year (82). MRP2 is a long MRP (3 MSDs) with 46% amino
acid similarity with MRP1. Unlike MRP1, however, MRP2 localizes to the apical plasma
membrane in polarized cells (23). Nonetheless, similar to MRP1, MRP2 is involved in the active
efflux of various organic anions, many of which are conjugated metabolites (23, 68). Mutations
in the ABCC2 gene results in the autosomal recessive disorder DJS, which is largely
asymptomatic; however, DJS can lead to hyperbilirubinemia and a jaundice phenotype in
patients due to elevated serum levels of conjugated bilirubin (11, 83). In contrast to MRP1,
MRP2 is highly expressed in the liver where it can transport a wide range of mostly organic
anion substrates into bile including bile acids, GSH and many GSH, glucuronide and sulfate
conjugates (68, 83, 84) (Table I-1).
14
MRP2 can also confer resistance to a wide spectrum of anticancer drugs when transfected
into polarized and certain unpolarized cells in culture, similar to MRP1 (22). However, unlike
MRP1, MRP2 can also confer resistance to cisplatin (85, 86). Despite these in vitro properties of
MRP2, its clinical role is likely modest as no convincing correlations between MRP2 levels and
clinical drug resistance have been established (87). Thus, the most important role of MRP2
appears to be as an organic anion transporter at the apical membranes of bile canalicular and
renal tubular cells in normal tissues. Furthermore, like Pgp, the presence of Mrp2 in the intestine
helps to reduce oral bioavailability of certain xenobiotics (88).
Alongside its role in bile acid efflux and restriction of oral bioavailability, MRP2
transports toxic endo- and xenobiotics including the GSH conjugate, dinitrophenyl-SG and the
glucuronide conjugated tobacco metabolite, (4-methylnitrosamino)-1-(3-pyridyl)-1-butanol-Oglucuronide (89, 90).
Mrp2-deficient rats (Eisai hyperbilirubinemic or Groninger Yellow rats) have long
provided an experimental working animal model for DJS (81, 91). Prior to identifying that it was
Mrp2-deficiency that caused this phenotype, the mutant rats were used to functionally
characterize what was then called the cMOAT (11). The cMOAT was later shown to be related
to MRP1, thus it was designated MRP2 (81, 92). These Mrp2 deficient rats are otherwise healthy
but have reduced to nonexistent hepatobiliary elimination of organic anions (83). Chu et al. (93)
created a KO mouse model of Mrp2. The KO mice were healthy and showed no overt
phenotypic abnormalities; however, levels of conjugated and unconjugated bilirubin were
significantly elevated, as expected (93). However, in contrast to the Mrp2-deficient rats,
induction of Mrp3 was not observed; in fact, strong induction of Mrp4 in the liver was noted in
15
the Mrp2 KO mice (93). In some instances, patients with DJS, where no detectable MRP2 is
present, have high levels of MRP3, as shown by liver biopsy and immunoflouorescense (94).
Thus, it appears that lack of Mrp2 in the rat liver causes a similar induction of compensatory
proteins, mainly Mrp3, as seen in humans with DJS.
Like Pgp and MRP1 discussed earlier, MRP2 contains more than one pharmacologically
distinct binding site within the protein (80, 95-97). Evers et al. (98) found that sulfinpyrazone
stimulated GSH efflux from polarized Madine-Darby canine kidney (MDCK) cells (98). This led
Borst et al. (87) to propose that MRP2 contains two different sites, one called the D site (drugbinding site) and the other the G site (GSH-binding site). However, when this stimulatory
phenomenon was observed with compounds other than GSH, and with other substrates of MRP2,
the sites were renamed the S site (substrate) and the M site (modulatory), respectively (87, 99).
Many of the compounds that stimulate MRP2 transport activity are not substrates of the
transporter; however, E217G is both a substrate and a modulator of transport (99). Thus,
E217G modulates its own transport in vitro where the plotting of the transport rate versus the
substrate concentration gives a sigmoidal curve, indicative of two E217G binding sites that
display positive cooperativity (99). Since this potentiation phenomenon was first defined, many
compounds have been identified as MRP2 stimulators, including acetaminophen-glucuronide,
sulfanitran, sulfinpyrazone, probenecid, glibenclamide and indomethacin, with some differences
in modulatory activity dependent on the concentration tested (99-103) (Fig. I.2).
16
Figure I.2. Chemical structures of known stimulators of MRP2-mediated transport activity.
17
Because one substrate can modulate the MRP2-mediated transport of another, it raises the
possibility that the clinical response to some drugs which are substrates of MRP2 can be altered
if co-administered with drugs known to be modulators of MRP2. Unexpected and potentially
deleterious drug-drug interactions could occur if the efflux activity of MRP2 was increased or
decreased, while taking other drugs that are substrates for MRP2.
1.3.3 MRP4 (gene symbol ABCC4)
Another MRP involved in drug disposition is MRP4, a short MRP with only two MSDs
and two NBDs. It was first discovered in a human T-lymphoid cell line in 1999 (104) and
subsequently was shown to efflux monophosphorylated nucleoside analog antiretroviral drugs
out of cells, thus impairing antiretroviral efficacy (104). MRP4 can also transport, both in vitro
and in vivo, several endogenous molecules that have roles in cellular signaling and
communication including some eicosanoids and cyclic nucleotides (Table I-1).
Among the first endogenous MRP4 substrates to be identified were the cyclic nucleotides
cAMP and cGMP (105). These molecules are of particular interest because of their roles as
intracellular second messengers that mediate a wide range of important cellular processes.
Although the apparent in vitro affinity for these second messengers is reported to be low (KM 10
and 45 µM for cGMP and cAMP, respectively (22)), it has been hypothesized that MRP4 may
efflux these cyclic nucleotides in various compartmentalized micro-domains of cells where their
local concentrations are higher (106).
In vitro, MRP4 can transport various eicosanoids including prostaglandin E2,
thromboxane B2 and prostaglandin F2α as well as leukotriene B4 and C4 (107, 108). MRP4 can
also efflux folate and the conjugated steroids dehydroepiandrosterone 3-sulphate and E217G
18
(105, 109, 110). Bile acids including cholate, cholytaurine and chenodeoxycholylglycine are also
transported by MRP4 and this bile acid transport has been reported to be GSH dependent (111).
MRP4 has been implicated in the transport of the purine end metabolite urate; however, a more
prominent role for ABCG2 in urate transport has been reported recently (112, 113). Thus,
polymorphisms in ABCG2 cause uric acid buildup in the blood leading to gout, indicating that
ABCG2, rather than MRP4, plays the most important role in urate transport (113).
Unlike almost all other MRPs, MRP4 can traffic to either the apical or the basolateral
plasma membranes of polarized cells depending on the tissue type in which it is expressed. For
example, MRP4 localizes apically in renal proximal tubules; however, it localizes basolaterally
in hepatocytes (114, 115). The underlying mechanism of the cell specific localization is not fully
understood; however, recent evidence suggests interactions with PSD-95/Disc Large 1/ZO-1
(PDZ)-based scaffolding proteins such as the Na+/H+ exchanger regulatory factor (NHERF)
family, specifically NHERF1, are involved in membrane localization (116, 117). Furthermore,
MRP4 has been reported to interact with CFTR through interactions with NHERF3. This
coupling has been hypothesized to play a part in mediating the ability of cAMP to regulate CFTR
(106). Similar to MRP1 and MRP2, MRP4 is thought to have regulatory substrate binding sites
that can allosterically influence its transport activity (112).
Abcc4 KO mice have provided a wealth of information with respect to the functions of
Mrp4 (118). Like Abcc1, Abcc2, Mdr1a/Mdr1b KO mice, the Abcc4 KO mice are viable, fertile
and possess no apparent histological, renal or hepatic abnormalities (119). However, it appears
that Mrp4 is an important factor not only in tissue distribution of specific xenobiotics, including
topotecan, but also with respect to toxicity towards nucleoside analogues and antiretrovirals used
19
in the treatment of cancer and viral infections as well as inflammatory diseases (104, 114, 118120). The anticancer agent topotecan accumulates in the brain and in the cerebral spinal fluid of
Abcc4 KO mice, which reflects the dual membrane localization capabilities of Mrp4 (119). Mrp4
also limits the distribution of the oseltamivir analog, [3R, 4R, 5S]-4-acetamido-5-amino-3-(1ethylpropoxy)-1-cyclohexene-1-carboxylate phosphate (Ro 64-0802), in the brain of mice (121).
The presence of MRP4 at the renal proximal tubule may have an impact on tenofovir toxicity.
Using KO mice, it was shown that loss of Mrp4 resulted in increased renal proximal tubule
toxicity, thus potentially showing why some patients treated with tenofovir present with renal
failure (122).
Sassi et al. (123) have recently proposed a role exists for MRP4 in heart physiology by
suggesting that it acts alongside phosphodiesterases to regulate cAMP-dependent signaling in
cardiac myocytes. They observed that inhibition of MRP4 (using the non-specific MRP-inhibitor
MK571 and an Abcc4 KO mouse model) prevented and reversed pulmonary hypertension in
mice (124, 125). MRP4 has also been implicated in a key control mechanism involving human
leukemia cell proliferation and differentiation through regulation of cellular cAMP levels, at
least in vitro (126). Finally, MRP4 also appears to play a role in regulating inflammatory pain
thresholds (127). Using an Abcc4 KO mouse model, Lin et al. (127) showed that lack of Mrp4
resulted in decreased peripheral prostaglandin (PGE2) levels resulting in an increased
inflammatory pain threshold (127).
1.4 Reversal of Drug Resistance Mediated by Pgp and MRP1
1.4.1 Modulators of Pgp
Since its discovery in 1976 (128), the physiological and pharmacological roles of Pgp
have been largely investigated in vitro through ATPase assays using cell lines and in vivo using
20
KO mice as well as humans. When MDR was first described by Dano in 1973, it was also shown
that these cells could be re-sensitized by exposure to certain compounds, referred to as
modulators, preventing or diminishing the export of the cytotoxic drug (129). The identification
and development of clinical Pgp modulators happened in a stepwise fashion beginning with
identification of already approved drugs as inhibitors of Pgp (130). Firstly, vincristine resistance
by P388/VCR cells was reversed by verapamil, an already U.S Food and Drug Administration
(FDA) approved cardiovascular drug (130). This allowed this agent to be fast-tracked into
studies of patient populations suffering from drug resistant cancers (130). It was less than 10
years after the discovery of Pgp that compounds like verapamil, as well as quinine (an
antimalarial) and cyclosporine A (an immunosuppressant) were approved for clinical trials in
combination with chemotherapy. However, what was originally thought to be a potential ‘magic
bullet’ was met largely with failure and disappointment because these compounds failed to
improve responses in patients, and gave rise to unacceptable toxicities in most cases (31). One of
the biggest issues encountered in using drugs for off-label targets (such as reversing Pgpmediated MDR) is that the doses needed to elicit the desired effects are often much higher than
the doses approved for the original therapeutic intention (131). Such was the case for verapamil
when used to target MDR. Cardiotoxic side effects were the largest deterrents to using this drug
as a reversal agent (132).
Second generation modulators were created, often through chemical modifications to
reduce non-specific toxicities while increasing the selectivity of the modulator for its target (133,
134). For example, in 1991, a non-immunosuppresive analog of cyclosporine A, PSC 833, was
developed (135). PSC 833 was shown to be 10-fold more potent in vitro, compared to
cyclosporine A, and significantly prolonged the survival time of Pgp-overexpressing tumour21
bearing mice treated with chemotherapy (135, 136). However, PSC 833 limited the clearance and
metabolism of the co-administered chemotherapeutic agents (daunorubicin and etoposide), thus
elevating their plasma concentrations to toxic levels (31, 136, 137).
Third generation compounds were also developed based on the data compiled from
studies using first and second generation reversing agents; as well, a proposed pharmacophore
was developed in vitro as an aid in predicting modulatory activity (136). These compounds were
designed to have a high Pgp affinity, but low pharmacokinetic interactions (31). Zosuquidar
(LY335979) is one of the most potent Pgp inhibitors to date and inhibits transport of various
chemotherapeutics (doxorubicin, etoposide, vinblastine) at nanomolar concentrations in vitro and
in mice (138). Thus, preclinical studies showed prolonged survival and marked decreases in
tumour mass in mice engrafted with human Pgp+ tumours treated with LY335979 and
chemotherapy (daunorubicin, doxorubicin and etoposide) (138). LY335979 also enhanced
paclitaxel levels in the mouse brain (139). However, in adult patients older than 60 years of age
with newly diagnosed AML, LY335979 with standard chemotherapy failed to improve their
outcome, even in the population shown to have Pgp expressing blasts (140).
Another third generation compound, elacridar (GF1210918), reverses MDR in certain
human sarcoma cell lines and increases the bioavailability of topotecan in normal mice (141,
142). However, because topotecan is a poor substrate for Pgp, it was suggested that the results
are likely due to inhibition of ABCG2 by elacridar (141). However, elacridar inhibits Pgp with
an IC50 value of 193 nM (in vitro) (142). The authors showed that coadministration of paclitaxel
and elacridar led to an increased paclitaxel concentration in the brain of Navel Medical Research
Institute (NMRI) nu/nu mice (142). Furthermore, Schinkel’s group showed recently that
22
dasatanib, a tyrosine kinase inhibitor, cannot cross the BBB because of Pgp (143). However,
when Pgp is inhibited by elacridar, the levels of dasatanib in wild-type mouse brain are increased
4.4 fold. This may provide a rationale for administration of this dual Pgp and ABCG2 inhibitor
alongside dasatanib for the treatment of central nervous system Philadelphia chromosomepositive chronic myelogenous leukemia (CML) (143). Another third generation Pgp inhibitor is
CBT-1, a bisbenzylisoquinoline plant alkaloid. During phase I clinical trials, it showed no effect
on pharmacokinetics and it is currently enrolled in phase II and III clinical trials (144).
Previous work by the Gottesman group identified a series of compounds with Pgpselectivity (145). More recent work by the same group showed increases in selectivity through
modification of the substituents of an N-linked aryl group (146).This series of isatin-thiosemicarbazones also selectively targeted MDR cells that overexpress Pgp (146). Ongoing
synthetic work is aimed at identifying more potent compounds through variations in substitution
at sites other than the N-linked aryl group; however, the clinical applicability is yet to be
evaluated.
New compounds identified either through serendipity, active screening of natural
products and/or redevelopment/reclassification of older drugs are being tested as potential fourth
generation modulators of Pgp-mediated drug efflux. Typically, compounds are initially identified
through an in vitro screen(s) and subsequently in cell lines and mouse models. New Pgp
modulators are identified through high- and low-throughput screening of newly synthesized or
isolated compounds, or using computer modeling and quantitative structure-activity relationship
(QSAR) mapping. There are many other late generation compounds developed as Pgp
modulators that show promising preclinical activity; however, for those that have been entered
23
into early stage clinical trials, most have failed due to unacceptable toxicities and insignificant
improvements in clinical response (15, 31, 136).
The addition of most Pgp modulators to chemotherapy regimens in many clinical trials
came with trial design flaws where the presence of the transporter in the patient tumours was not
confirmed (31). Determining the presence and level of activity of a targeted ABC transporter in
tumour tissue is important to verify that the observed response in the presence of a modulator
and drug is due to inhibition/modulation of that transporter. To further confound the issue of
ascertaining whether administered modulators do improve patient responses is the fact that in
many cases, the patients were under- or overdosed to compensate for the potential and observed
pharmacokinetic interactions between modulator and drug (134). Furthermore, the presence of
other ABC transporters was not controlled for in the patient groups; therefore, the modulatory
effect on Pgp might have been masked by other drug transporters present in the tumours.
In 2008, Sawada et al. (147) described a new class of compounds used as modulators of
Pgp activity in MDCKII-MDR cells, the chalcogenopyrylium dyes (CGP). The CGPs are
synthetic derivatives of rhodamines, a class of compounds designed as photosensitizers and
known to interact with Pgp based on their ability to stimulate the ATPase activity of the
transporter (148). Through slight modification of these rhodamines, analogs were synthesized
that modulated Pgp ATPase activity when exposed to light (149). However, not all of rhodamine
analogs synthesized to date require light to inhibit Pgp. Detty, Gannon and colleagues described
a series of rhodamines that could also inhibit Pgp-mediated efflux of calcein in the absence of
light (150). These rhodamines were examined for ATPase stimulation, and when the analogs
were tested on isolated, purified protein, the presence of tertiary amide groups was associated
24
with high affinity and stimulation of ATPase activity. Conversely, when a tertiary thioamide was
present, the analogs maintained their affinity, but now inhibited the Pgp ATPase activity (150).
Ultimately, the authors proposed a ‘switch’ mechanism, where a minor substitution resulted in
markedly different effects on Pgp ATPase activity. The authors proposed that the tertiary amide
was engaging in H-bond interactions that were then abolished when the tertiary thioamide was
“switched” in. In an attempt to create more efficacious inhibitors of Pgp and further improve the
interaction of these rhodamines with Pgp, the scaffolds of the rhodamine analogs were overlaid
on 3 and 4-point pharmacophore models of Pgp, and then synthetically modified to create
compounds with more flexibility and unique substitutions; these newly synthesized compounds
are the CGPs (147, 149, 151, 152).
The rhodamines and the CGPs incorporate chalocogen atoms at the 1-position of their
trisubstituted pyrylium core. However, the biggest difference between the rhodamines and the
CGPs is that the latter compounds are structurally less rigid. Thus, the tricyclic xanthylium core
of the rhodamines is very rigid when compared to the more flexible 2,4,6-trisubstituted
chalcogenopyrylium core (147) (Fig. I.3). The CGPs stimulate Pgp ATPase activity and thus
bind to the transporter. In addition, the CGPs inhibit efflux activity as indicated by their ability to
enhance the accumulation (prevent efflux) of the fluorescent cellular dye calcein in Pgp
transfected MDCKII-MDR cells (147). Enhancement of calcein accumulation is an indirect
measure of Pgp activity as calcein acetoxymethyl ester (calcein-AM) is a substrate for Pgp and
thus is normally prevented from entering cells expressing Pgp. When Pgp is inhibited, calceinAM enters the cell and nonspecific cellular esterases cleave the ester to yield calcein, which is
fluorescent, and cannot be effluxed by Pgp (153). Some of the CGPs evaluated by Sawada et al.
25
Figure I.3 Chemical structures of the rigid chalcogenorhodamines and the flexible chalcogenopyryliums
(CGPs). Both the rhodamines and the CGPs have chalcogen atoms (X = O, S, Se or Te). R and Y
represent various substitutions on the molecules. The CGPs here represent the scaffolds of the Class I – V
analogs investigated in this study.
26
(147), inhibited vinblastine transport by Pgp in both an inside-out membrane vesicle uptake
assay and from intact MDCKII-MDR cells (147).
1.4.2 Modulators of MRP1
Although MRP1 confers MDR on cancer cells in vitro, specific modulators of this protein
have yet to be tested extensively in humans (25). One reason that MRP1 inhibitors have yet to be
tested in clinical trials is because many of the modulators of Pgp tested in clinical trials have
failed, and the research and pharmaceutical community have become skeptical that MRP1
inhibitors will fare any better.
Nevertheless, the quinoline derivative MS-209 was entered into Phase I, II and III clinical
trials as a dual Pgp and MRP1 inhibitor (154-156). Although MS-209 was well tolerated in the
clinical setting (Phase I) and had no impact on pharmacokinetics (Phase II), the observed
response rate for patients with advanced or recurrent breast cancer treated with
cyclophosphamide, doxorubicin and fluorouracil was not improved significantly (Phase III)
(155). Another dual inhibitor of Pgp and MRP1, VX710, was entered into clinical trials (157159) and in the phase I trial of VX710, a steady state plasma concentration of 10 µM was
achieved. However, in a phase III trial, addition of VX710 to a doxorubicin and vincristine
regimen did not improve the response rate in patients with recurrent SCLC (160). In both
instances, the levels of both MRP1 and Pgp in the tumours were not determined, and it is not
possible to exclude the possibility that the lack of effect was due to the absence of these
transporters.
In pre-clinical studies, numerous structurally diverse compounds have been shown to
modulate MRP1 transport activity, and many of these show promise in mouse models. Similar to
27
Pgp, MRP1 modulators have been discovered by active screening of natural products and
chemical libraries, and through selective synthesis, sometimes aided by pharmacophore
modeling (25). Verapamil, the calcium channel blocker that potently inhibits Pgp, is a weak
inhibitor of MRP1 (161); however, in the presence of GSH, verapamil can inhibit LTC4 transport
>20-fold compared to verapamil alone (70). Verapamil and some of its analogs have been
reported to act as apoptogens of Abcc1-transfected baby hamster kidney-21 cells (25, 162), and it
has been proposed that apoptosis occurred because of GSH depletion through a MRP1-mediated
mechanism. Prior to the identification of MRP1, calcium channel antagonists, including
verapamil, were tested as chemosensitizers of non-Pgp MDR human tumour cells (161). The
antagonists did not significantly increase the cytotoxicity of doxorubicin on the MRP1overexpressing MDR tumour cells; however, the data suggested that verapamil and nicardipine
were able to enhance the cytotoxicity, but not in a dose-dependent manner (161).
Another interesting MRP1 inhibitor is the natural, marine sponge polyhydroxylated
sterol, Agosterol A and its analogs (163, 164). These sterols were first shown to reverse MRP1mediated vincristine resistance in a vincristine-selected KB-3-1 cell line (KB-CV60) (163, 165).
Through structure-activity relationships (SAR) and experimentally obtained data, it was
determined that certain acetoxy groups and hydroxyl groups of Agosterol A were important
determinants of chemosensitizing activity (166). Interestingly, the inhibition of MRP1 by
Agosterol A is GSH-dependent as described for LY477576 (Section 1.3.1) (166).
Flavonoids have also been investigated as modulators of MRP1 transport activity.
Genistein was one of the first shown to inhibit MRP1-mediated efflux, specifically of
daunorubicin from resistant SCLC cells (167). QSAR data between the flavonoids and MRP1
28
were compiled (168) and the three major structural characteristics proposed to be important for
inhibition included the total number of methoxylated moieties, the total number of hydroxyl
groups and the dihedral angle between aromatic rings on the inhibitor (168). The mechanism by
which flavonoids are thought to act is uncertain. Some appear to bind directly to MRP1 where
they modulate substrate transport or ATP hydrolysis (169). Furthermore, the inhibitory activity
of some flavonoids is enhanced by GSH (169) and in some cases, flavonoids can stimulate the
transport of GSH itself, which concomitantly makes the cell more sensitive to cytotoxic agents
(69). Thus, bioflavonoid-dependent GSH depletion may be, in itself, a mechanism of MRP1
reversal (69).
Another compound of interest is raloxifene, a nonsteroidal estrogen receptor mixed
agonist/antagonist that was used as a pharmacophore for creating modulators of MRP1. Several
analogs thus identified were effective both in vitro and in vivo, and reversed doxorubicin
resistance mediated by MRP1 in intact cell assays (25). One raloxifene-based inhibitor,
LY329146, potently inhibited LTC4 uptake into inside-out membrane vesicles by MRP1 (IC50
0.8 µM) (170). To date, no studies of raloxifene inhibitors have been published using a mouse
model of MRP1 MDR.
Tricyclic isoxazoles were identified as modulators of MRP1 by Norman et al. (171) at Eli
Lilly, through a large scale chemical library screen of potential MRP1 modulators. The SAR
analysis resulted in the preparation of LY402913, which reversed doxorubicin resistance in
MRP1-overexpressing HeLa-T5 cells (IC50 0.9 µM) (171). LY402913 also inhibited LTC4
transport (IC50 1.8 µM) (171). Lastly, the effects of LY402913 on MRP1-overexpressing
tumours in vivo were assessed in nude mice implanted with HeLa-T5 xenografts. The
29
combination of vincristine and LY402913 led to a statistically significant decrease in tumour
mass (171). Further studies by the Eli Lilly group identified even more potent inhibitors of
MRP1, which are analogs of the original LY402913 compound, that include a cyclohexyl linker
(172).
Quinazolinones were identified as dual inhibitors of Pgp and MRP1 and were subjected
to SAR analysis in an attempt to discover the most potent inhibitor (173). Subsequently, the
search for a specific modulator of MRP1 was undertaken and further screens of compound
libraries were completed using the previously identified quinazolinone compound as the
pharmacophore. This led to the discovery of the pyrrolopyrimidine analogues which retain high
MRP1 inhibitory potency, but are also highly selective for MRP1 versus Pgp (174). Thus, of the
59 pyrrolopyrimidine compounds encompassing various structurally similar scaffolds, yet having
differentially substituted moieties at various positions, five were shown to be highly potent (IC50
< 1 µM) against, and selective for MRP1 vs Pgp (174). However, activity against other MRP
family members was not tested. Mice bearing xenografts of a MRP1-overexpressing NSCLC cell
line were randomized to receive vincristine or pyrrolopyrimidine drug alone, or a combination of
the two. Compound 55 (XR12890) decreased tumour volume (by 400%) when co-administered
with vincristine (174). Follow-up studies on the pyrrolopyrimidines by Wang et al (174) again
identified a series of more potent and selective MRP1 inhibitors, with effects on tumour volume
more remarkable than XR12890 (500% tumour volume reduction)(175).
Tawari et al. (176) used the SAR data generated from the studies of Wang et al, (174,
175) to generate a pharmacophore model which identified five structural requirements needed for
interaction and inhibition of MRP1: two hydrogen bond acceptor sites, one
30
lipophilic/hydrophobic group, one positive ionic feature and one aromatic ring. To test their
proposed pharmacophore, various inhibitors of MRP1 (LY329146, LY402913 and others) were
overlaid on the generated pharmacophore and important moieties were overlapping with the
predicted important binding regions of the pharmacophore identified by the model. This
pharmacophore model may provide insights into the structural requirements for MRP inhibitors,
and may assist in the rationale design and synthesis of future MRP1 inhibitors (176).
Recently, cannabinoid type 1 receptor agonists were identified as modulators of not only
MRP1, but MRP2, 3 and 4 as well (177). The effects of 10 cannabinoid antagonists on
[3H]E217G uptake into inside-out membrane vesicles enriched for the transporter of interest
were differential. Most had modest, yet statistically significant inhibition of MRP1 and marked
stimulation of MRP2-mediated E217βG uptake. The effects on MRP3 were either stimulatory, or
none at all while 5 of 10 cannabinoid receptor antagonists were significant inhibitors (>25-75%)
of MRP4-mediated E217βG uptake (177).
Reversan, a pyrazolopyrimidine small molecule inhibitor of MRP1, was discovered by
screening a 2,300 compound library assembled around previously identified Pgp modulators;
however, the functional readout was designed to recover MRP1 modulators based on the
compound’s effects on an MRP1-overexpressing MCF7/VP cell line (178). Following
identification of “hits”, another library of 299 compounds was searched for active compounds
with ≥90% structural similarity to the previously discovered compounds. Using drug
accumulation and cytotoxicity assays, Reversan was shown to effectively inhibit MRP1 as
indicated by sensitization to vincristine (10 to 15-fold) and etoposide (7 to 12-fold), but not
31
cisplatin or paclitaxel. Furthermore, a mouse model of neuroblastoma was used to show that
Reversan could resensitize tumour xenografts to vincristine and etoposide (178).
Xanthones are dibenzo--pyrone heterocyclic compounds isolated from plants which
have been described previously as having anticancer properties (179). Prenylated xanthones have
been shown to bind to Pgp, but their effect on activity was not tested (180). Recently xanthones
were also shown to target MDR cancer cells which overexpress MRP1 (181). The mechanism
through which a specific group of xanthones are proposed to target MRP1-expressing cells for
apotosis is similar to that proposed for verapamil: binding of the xanthone to MRP1 stimulates
GSH efflux leading to cell death (181).
It is clear that the search for specific and potent modulators of ABC transporters that
cause MDR is still very active (Table I.2). Through chemical library screening and modified
syntheses, identification of new modulators of ABC transporters is happening continually. Some
structural requirements have been proposed for interaction with, and inhibition of, MRP1. It will
be compounds able to selectively inhibit MRP1 with the greatest potency and least toxicity and
pharmacokinetic interactions that will lead to hits most likely to be tested in humans.
32
Table I.2
MRP1 modulators and their respective effects on transport activity.
This table lists the most common, and for some most effective, inhibitors of
MRP1 that have been reported, but is not meant to be exhaustive. When
IC50’s have been reported, they are listed; otherwise, the compound is
simply identified as an inhibitor.
a
The effect the compound has on transport activity
µM
c
IC50 in the absence of GSH
d
IC50 in the presence of GSH
e
Inside out membrane vesicles prepared from stably or transiently transfected MRP1-overexpressing cells
f
Chemosensitization of MRP1-overexpressing, multidrug resistant cells
g
Efflux is evaluated by the amount of drug/substrate in the intact MRP1+ cell, compared to control (MRP1-) cells
and is an indirect measure of MRP1 transport activity in the presence of a modulator
GSH – glutathione, Dox – doxorubicin, VCR – vincristine, VP-16 – etoposide
b
33
1.5 Rationale, Hypothesis and Objectives
The CGPs identified as inhibitors of Pgp are known to interact directly with the
transporter (147) (Table I.3). Thus, it is of interest to determine whether these same CGPs can
interact with other physiologically and pharmacologically relevant ABC transporters. Many
modulators of Pgp activity have been tested to determine whether they interact with MRP1, and
in many cases, but not all, they do not. Thus, at present, it is not possible to predict a priori
whether or not a compound will interact with Pgp or MRP1 or both. Thus, this thesis was aimed
at determining if a class of CGPs, similar to those shown by Sawada et al (147) to inhibit Pgp,
could interact with MRP1. Since other MRPs, in particular MRP2 and MRP4, are established as
being clinically relevant in drug disposition and excretion, the CGPs were also examined for
their effects on these MRP1 homologs. Thus, it was hypothesized that the CGPs would interact
with and modulate the activity of MRP1, MRP2 and MRP4. This hypothesis was tested by
pursuing the following objectives:
1. To determine whether a series of CGPs interact with MRP1 to modulate it’s activity in an
in vitro vesicular transport assay using inside-out membrane vesicles prepared from
MRP1 stably transfected cells.
2. To determine the relative potency of the most effective CGPs identified in Objective 1,
by carrying out concentration-response experiments to determine relative IC50’s.
3. To determine if the CGPs affect the activity of MRP1 in a more biologically relevant
system by measuring their effects in an intact cell-based dye efflux assay using a stably
transfected MRP1 cell line.
4. To investigate the specificity of the most potent MRP1 modulators by measuring their
effects on MRP2 and MRP4 transport activity.
34
35
II. Materials and Methods
2.1 Materials
2.1.1 Synthesis and Characterization of Chalcogenopyrylium Dyes (CGPs)
The CGPs examined in this study were synthesized in the laboratory of Dr. Michael
Detty in the Department of Chemistry at the University of Buffalo (SUNY). The identities and
structures of the CGPs were verified using standard analytical methods (1H nuclear magnetic
resonance (NMR), 13C NMR, mass spectrometry, infrared spectrometry, high resolution mass
spectrometry and elemental analysis). Purity of the CGPs was determined by high pressure liquid
chromatography (HPLC) to be ≥ 94%. A first set of 34 CGPs were assigned to one of five
structural classes designated I through V (Fig. II-1). A second set of 5 CGPs were analogs of
CGP IV-1, and therefore designated IV-1-1 through IV-1-5 (Fig. II-2). Care was taken at all
times to minimize exposure of compounds to light. Predicted logP values were generated using
Molinspiration Interactive logP Calculator, http://www.molinspiration.com/services/logp.html.
2.1.2 Other Reagents
[6,7-3H]E217βG (45 Ci/mmol) and Western Lighting® Plus-Enhanced
Chemiluminescence blotting substrate were purchased from PerkinElmer Life and Analytical
Sciences (Woodbridge, ON). AMP, ATP, E217βG, benzamidine, bovine serum albumin (BSA),
Dulbecco’s Modified Eagle’s Medium (DMEM), poly-L-lysine and 2-mercaptoethanol were
purchased from Sigma-Aldrich (Oakville, ON). Protease inhibitors (EDTA-free) were purchased
from Roche (Mississauga, ON). Film for enhanced chemiluminescence (ECL) detection was
purchased from Ultident Inc. (St. Laurent, QC) and the Bradford protein assay kit was purchased
from Bio-Rad Laboratories (Hercules, CA). Lipofectamine 2000™ transfection reagent, Opti-
36
37
38
Mem® and fetal bovine serum (FBS) were obtained from Gibco-Invitrogen (Burlington, ON).
G418 was purchased from ONBIO (Richmond Hill, ON). Mouse monoclonal antibody (MAb)
M2I-4 specific for MRP2 and MAb M4I-10, specific for MRP4, were purchased from Alexis
Laboratories (San Diego, CA). Mouse MAb QCRL-1 specific for human MRP1 was derived in
this laboratory and detects an epitope defined as amino acids 918-924 (182, 183). Rabbit antiNa+/K+ ATPase antibody (sc-28800) was purchased from Santa Cruz Biotechnology Inc. (Santa
Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse secondary antibody for detection
of MRP1 and MRP2 and goat anti-rabbit secondary antibody for detection of Na+/K+ ATPase
were purchased from Pierce Biotechnology (Rockford, IL) while goat anti-rat secondary
antibody for detecting MRP4 was purchased from Chemicon International Inc. (Billerica, MA).
Polyvinylidene fluoride membranes were from Pall Corporation (Ville St. Laurent, QC). Calcein
acetoxymethyl ester (Calcein AM) was purchased from Cedarlane (Burlington, ON). All other
chemicals and reagents were of analytical grade.
2.2 Cell Culture and Transfection
The human embryonic kidney (HEK) cell line and the SV40-transformed HEK293T cell
line were maintained in DMEM supplemented with 4 mM L-glutamine and 7.5% FBS. A stably
transfected MRP1 overexpressing HEK cell line was generated in this laboratory by Dr. G.
Conseil using a pcDNA3.1(-) expression vector containing the entire human MRP1 cDNA
transcript (184). Following transfection using Lipofectamine 2000™, HEK cells were selected in
G418 (1000 µg/ml) and clones of surviving cells were isolated using colony rings and trypsin
digestion. Clonal cell lines were then expanded and screened for MRP1 by immunoblotting. Cell
lines expressing high levels of MRP1 were then analyzed for homogeneity of MRP1 expression
by flow cytometry using MAb QCRL-3 (183) on an Epics Altra HSS flow cytometer (Beckman
39
Coulter). A stable cell line thus obtained was designated HEK-MRP1 and maintained in
DMEM/7.5% FBS supplemented with 500 µg/ml G418. All cultures were grown at 37oC in 5%
CO2 / 95% air. For membrane vesicle preparations, MRP1 expressing cells were seeded onto 150
mm plates at 18 X 106 cells per 20 ml and maintained in DMEM / 7.5% FBS until confluent.
Cells transiently expressing MRP2 and MRP4 were generated as follows: HEK 293T
cells were seeded at approximately 18 X 106 cells in 150 mm plates in 20 ml DMEM / 7.5% FBS
at 37oC. Twenty-four h later, when cells were 90-95% confluent, pcDNA3.1(-) expression
vectors containing either human MRP2 or human MRP4 cDNA (20 µg per plate) were
transfected into the HEK293T cells using Lipofectamine 2000™ following the manufacturer’s
instructions (103, 117). After 6 h at 37oC, the medium was removed and replaced with fresh, prewarmed DMEM / 7.5% FBS. After a further 48 h of incubation, cells were collected and
centrifuged at 1800 x g for 5 min at 4oC, washed with phosphate buffered saline (PBS), and then
homogenization buffer consisting of 250 mM sucrose/50 mM Tris pH 7.4/0.25 mM CaCl2. The
cells were again collected by centrifugation and the cell pellet overlaid with homogenization
buffer (10 ml) containing 10 µl benzamidine (200 mg/ml), 1 ml protease inhibitor cocktail and 9
ml homogenization buffer. The cell pellets were snap-frozen in liquid nitrogen and kept at -80oC
until needed.
2.3 Preparation of Membrane Vesicles
The protocol for preparation of membrane vesicles was adapted from Loe et al. (75). Cell
pellets were thawed slowly on ice, resuspended in homogenization buffer and then the cells were
disrupted by argon cavitation (350 psi, 4oC, 5 min). After centrifugation at 1400 x g at 4oC for 10
min, the supernatant was transferred to high speed centrifuge tubes and the remaining pellet was
40
resuspended in homogenization buffer with 0.5 mM EDTA. Following a second centrifugation at
1400 x g, the supernatants from the first and second centrifugations were combined and overlaid
onto a 35% (w:v) sucrose/1 mM EDTA/50 mM Tris, pH 7.4 cushion and centrifuged for 1-2 h at
100,000 x g at 4oC in an Optima™ L-90K Ultracentrifuge (Beckman Coulter, Fullerton, CA).
The interface layer containing cellular membranes was removed, placed in 25 mM sucrose/50
mM Tris, pH 7.4 buffer and centrifuged at 100,000 x g at 4oC for 30 min. The isolated
membranes were then washed with 1 ml Tris-sucrose buffer (TSB) (250 mM sucrose, 50 mM
Tris, pH 7.4) and centrifuged at 55,000 x g at 4oC for 25 min in a TL-100 Ultracentrifuge
(Beckman Coulter). The supernatant was removed by aspiration and the pellet resuspended in
TSB (100 µl/150 mm plate of cells) by passage through a 1 ml syringe with a 27-gauge needle
until homogeneous. An additional 15 passages through the needle were completed to ensure
proper resuspension and revesicularization of membranes. The membrane vesicles were
aliquoted and stored at -80oC until needed. Vesicular protein concentrations were determined
using the Bradford method with BSA as a standard.
2.4 Immunoblotting for MRP1, MRP2 and MRP4 Proteins
The presence of MRP1, MRP2 and MRP4 protein in the membrane vesicles was
confirmed by immunoblot analysis, as follows: proteins were loaded onto a 1.0 mm thick, 10 or
15-well, SDS polyacrylamide gel (7% acrylamide resolving gel, 4% acrylamide stacking gel) and
separated by electrophoresis at 75-135 V for 1.5-2.5 h. The proteins were then electrotransferred
to polyvinylidene fluoride membranes using a BioRad Mini-Gel apparatus at 400 mA for 2 h on
ice. After transfer, the membranes were washed in TBS plus 0.1% Tween-20 (TBST) and then
blocked in 4% (w:v) skim milk powder in TBST for 1 h. Following blocking, the membranes
were incubated overnight at 4oC with the human MRP1-specific MAb QCRL-1 (diluted
41
1:10000), MRP2-specific MAb M2I-4 (diluted 1:5000), or MRP4-specific MAb M4I-10 (diluted
1:5000), in blocking solution (103, 117, 185). After 5 washes in TBST (5 min each),
immunoblots were incubated with horseradish peroxidase-conjugated secondary antibody in
blocking solution for 1-2 h at room temperature. Immunoblots were then washed 5 times in
TBST, each for 5 min. ECL blotting substrate was then applied and the membranes exposed to
film for times ranging from 10 s to 2 min depending on the antibody. As a protein loading
control, the membranes were probed with rabbit anti-Na+/K+ ATPase antibody (diluted 1:5000 in
4% skim milk/TBST) overnight at 4oC, followed by incubation in horseradish peroxidaseconjugated goat anti-rabbit IgG (diluted 1:10000) for 1 h at room temperature. The blot was
washed and ECL solution added and the film exposed as described above. Relative levels of
MRP1, MRP2 and MRP4 proteins were then determined by densitometric analysis of the
immunoblots using ImageJ 1.36b software (http://rsb.info.nih.gov/ij/).
2.5 MRP-mediated Uptake of [3H]E217βG by Membrane Vesicles
ATP-dependent uptake of 3H-labeled E217βG by MRP1-, MRP2- and MRP4-enriched
membrane vesicles was measured using a 96-well rapid filtration method previously described
(116, 186). Reactions were carried out in duplicate in 96-well round bottomed plates (Starstedt,
Newton, NC) in a final reaction volume of 30 µl in TSB at 37oC.
Stock solutions of CGPs in DMSO were prepared and were kept at -20oC in the dark until
needed, and when transported in the lab, they were covered with aluminum foil. Stock solutions
of CGPs were diluted as needed in TSB and then added to both the reaction mix (containing
either AMP or ATP (4 mM), MgCl2 (10 mM) and E217βG / [3H]E217βG) and the membrane
vesicle preparations. The reaction plate was allowed to acclimatize to 37oC for 3 min prior to
42
initiating the uptake reaction by adding the reaction mix (24 µl) to the membrane vesicles (6 µl).
The final concentration of DMSO never exceeded 1% and the reactions were performed in
duplicate.
For MRP1-mediated E217βG uptake, 2 µg of vesicle protein were incubated with reaction
mix containing [3H]E217βG (400 nM, 20 nCi) for 3 min at 37oC (186). For MRP2-mediated
uptake of E217βG, 6 µg of membrane vesicle protein were incubated with [3H]E217βG (400 nM,
40 nCi) for 4 min at 37oC (185). To measure MRP4-mediated E217βG uptake, 5 µg of vesicle
protein were incubated with [3H]E217βG (1 µM, 60 nCi) for 10 min at 37oC (116). Uptake of
[3H]E217βG was stopped by rapid dilution by transferring the entire reaction into a Dynablock
2000™ 96-deep-well plate (VWR International, Mississauga, ON) containing 1 ml of ice-cold
TSB followed by rapid filtration of the wells’ contents onto a Unifilter-96 GF/B filter plate using
a 96-well Filtermate Harvester apparatus (Packard BioScience, Meriden, CT). Radioactivity was
measured by liquid scintillation counting on a TopCount NXT™ Microplate Scintillation and
Luminescence Counter (Perkin-Elmer) and uptake was expressed in pmol [3H]E217βG per mg of
protein per min (pmol/mg/min) using Microsoft® Excel™ software. To determine ATPdependent uptake, uptake in the presence of AMP was subtracted from uptake in the presence of
ATP. IC50 values were computed using GraphPad Prism 3.0 software (GraphPad Software Inc.,
San Diego, CA) and represent the concentration of CGP needed to inhibit [3H]E217βG uptake
activity by 50%. To determine if IC50’s were statistically different from one another, one way
Anova’s with ad hoc Dunnett’s tests or unpaired students t-tests were performed using Graph
Pad Prism 3.0, P < 0.05 was considered statistically significant.
43
2.6 MRP1-mediated Calcein Efflux from Intact HEK-MRP1 Cells
The effect of the CGPs on MRP1-mediated calcein efflux from HEK-MRP1 cells was
measured using a protocol adapted by Dr. G. Conseil and Mrs. K. Sparks as follows: Stably
transfected HEK-MRP1 cells were plated into wells of a 96-well plate pre-coated with 0.01%
poly-L-lysine (Sigma) (1 X 105cells per well in 100 µl). After 1.5 h at 37oC, stock solutions of
the CGPs in DMSO were diluted in OptiMem® as needed and then added to the plates in a
volume of 50 µl such that their final concentration was 10 µM. The plates were incubated for 30
min at 37oC. As a vehicle control, 50 µl of TSB were added in place of the CGPs. Fifty µl of
calcein-AM (24 µM in OptiMem® with 0.1% BSA) was then added to the wells to a final
concentration of 6 µM. Reactions were carried out in triplicate. After 15 min at 37oC, the
medium was aspirated and replaced with 200 µl pre-warmed OptiMem® with 10% FBS. After 10
min at 37oC to allow calcein-AM hydrolysis and calcein efflux to occur, the plate was placed on
ice and the medium was aspirated and replaced with 200 µl ice-cold PBS. Fluorescent calcein
remaining in the cells was immediately measured on a Spectramax Gemini XS (Molecular
Devices, Sunnyvale, CA) (excitation wavelength – 495 nm; emission wavelength – 515 nm).
Efflux activity was calculated from the raw fluorescence values as follows: The calcein
fluorescence (arbitrary units; AU) remaining in the vehicle control HEK-MRP1 cells (no CGP)
were subtracted from the AU remaining in the HEK-MRP1 cells after incubation with a CGP,
and then expressed relative to the maximal value of calcein efflux which was calculated as the
difference between the AU remaining in the HEK cells (no MRP1, no CGP) minus the AU
remaining in the vehicle control HEK-MRP1 cells (no CGP). To determine if the effects of the
CGPs were statistically different from one another, unpaired students t-tests were performed
using Graph Pad Prism 3.0. P < 0.05 was considered statistically significant.
44
III. Results
3.1 Modulation of MRP1 Transport Activity by the CGPs
3.1.i Initial screen of CGPs on MRP1-mediated vesicular uptake of [3H]E217βG
To determine whether an initial series of 34 CGPs could modulate MRP1 transport
activity, their effect on MRP1-mediated uptake of [3H]E217G into inside-out membrane
vesicles was assessed. For these studies, a stably transfected HEK-MRP1 cell line was used as a
source for membrane vesicles. Following immunoblot analysis to confirm the expression of
MRP1 (Fig. III-1A), the vesicles were initially tested for their [3H]E217βG uptake activity to
assess the integrity of the vesicles as reflected by their baseline transport levels.
Having confirmed the transport competence of the MRP1-enriched membrane vesicles,
the 34 CGPs were first screened at a single concentration of 30 µM with the exception of II-1,
III-2, -4 and V-2, which were tested at 1 µM, and I-7, II-2, III-5, -6, -12 and -15, which were
tested at 5 µM. Testing these CGPs at a lower concentration was necessary because of either
insufficient solubility and/or because they generated an unacceptably high background, which
interfered with accurate scintillation counting in the transport assay. As summarized in Tables
III-1 to III-5, the modulatory effects of the 34 CGPs on MRP1-mediated uptake of [3H]E217βG
varied significantly. Almost half of them (16/34) inhibited MRP1-mediated uptake >50% (7096%). Nine of the remaining 18 (II-1, III-2, -4, -5, -6, -10, -11, -12, and V-2) inhibited
[3H]E217βG uptake by <30%. Inhibition by the other nine (I-2, III-1, -3,-7, -8, -9, -15, IV-3 and
-4) ranged from 30 to 50%. However, it should be noted that CGPs II-1, III-2, -4, -12 and V-2
could only be tested at a maximum of 1 µM, while III-5, -6 and -15 could only be tested at a
maximum of 5 µM. Therefore, the efficacy of these latter 8 CGPs (six of which were from Class
III) may be underestimated.
45
46
Class I CGPs were generally the most effective group of MRP1 inhibitors (Table III-1).
Thus, the mean inhibition of [3H]E217G uptake by 6 of 7 Class I CGPs at 30 µM ranged from
82 to 96%. The seventh, CGP I-2, inhibited MRP1 transport activity only 33% (30 µM). CGP I-7
precipitated out of solution at 30 µM; however, at 5 µM, it inhibited [3H]E217G uptake by 83%.
Four of five Class II CGPs were also effective inhibitors of MRP1-mediated [3H]E217G uptake
with mean inhibition values ranging from 70 to 96% when tested at 30 µM (Table III-2).
Because of unacceptable background scintillation levels, CGP II-1 could only be tested at 1 µM.
At this concentration, II-1 inhibited uptake of [3H]E217G by only 15%.
In contrast to Class I and II compounds, Class III CGPs were relatively ineffective
inhibitors of MRP1 transport activity. Only 2 of 15 Class III CGPs inhibited [3H]E217G uptake
by >50% (Table III-3). Thus, the inhibition values in the presence of the structurally similar III13 and III-14 (at 30 µM) were 71% and 81%, respectively. Inhibition by CGP III-15 (the Tecontaining analog of III-13 (S) and III-14 (Se)) was 50%. For the remaining 12 Class III CGPs,
inhibition ranged from 0-45%. However, for reasons stated earlier, six of these had to be tested at
< 30 µM; therefore, their true efficacy may be underestimated.
Two of the four S-containing Class IV CGPs (IV-1 and IV-2) were effective inhibitors of
[3H]E217G uptake with mean inhibition values of 95% and 96% at 30 µM, respectively (Table
III-4). CGP IV-3 and -4 were moderate inhibitors at 30 µM (43% and 50%, respectively). Two of
three Te-containing Class V CGPs were quite effective (mean inhibition by V-1 and V-3 was
96% and 89% at 30 µM, respectively) (Table III-5). The efficacy of CGP V-2 may be
underestimated because it could not be tested at concentrations > 1 µM; however, at this lower
concentration, it inhibited [3H]E217G uptake by just 8%.
47
Table III-1
Inhibition of MRP1-mediated uptake of [3H]E217βG by
Class I CGPs
Results shown are means of two independent experiments.
CGP
X
Y
n
R
MW
LogPa
% Inhibitionb
(30 µM)
I-1
Te
Te
0
Ph
845.74
6.9
86
I-2
Se
Se
0
tBu
668.50
7.6
33
I-3
Se
O
0
tBu
609.64
8.9
94
I-4
Te
O
0
tBu
654.18
9.2
82
I-5
Te
S
0
tBu
670.24
10
96
I-6
Se
Se
1
tBu
585.02
8.2
92
I-7
Te
O
1
tBu
570.50
9.7
83c
a
Predicted using Molinspiration interactive logP calculator
% inhibition of [3H]E217βG uptake relative to vehicle control (1% DMSO)
c
tested at 5 µM
b
R
Y
+
R
R
48
X
+
R
Table III-2
Inhibition of MRP1-mediated uptake of [3H]E217βG by
Class II CGPs
Results shown are means of two independent experiments.
CGP
X
R
LogPa
MW
% Inhibitionb
(30 µM)
II-1
Se
Me
431.86
4.0
15c
II-2
S
Bu
518.50
5.5
70d
II-3
S
tBu
518.50
5.4
94
II-4
Se
tBu
473.94
5.3
96
II-5
Te
tBu
567.03
5.3
96
a
Predicted using Molinspiration interactive logP calculator
% inhibition of [3H]E217βG uptake relative to vehicle control
(1% DMSO)
c
tested at 1 µM
d
tested at 5 µM
b
N
X
N
49
+
R
Table III-3
Inhibition of MRP1-mediated uptake of [3H]E217βG by
Class III CGPs
Results shown are means of two independent experiments.
CGP
X
R
Y
MW
LogPa
% Inhibitionb
(30 µM)
III-1
S
tBu
Ph
365.37
5.4
33
III-2
Se
tBu
Ph
412.27
5.3
0c
III-3
Te
tBu
Ph
460.91
5.3
32
III-4
S
tBu
HOOC
480.47
4.9
8c
III-5
Se
tBu
S
527.36
4.9
14d
III-6
Te
tBu
576.00
5.4
24d
III-7
S
tBu
529.56
6.0
34
III-8
Se
tBu
576.46
5.9
31
III-9
Te
tBu
625.10
6.5
45
III-10
Se
tBu
Me
415.26
4.7
0
III-11
O
tBu
Me
352.30
4.6
0
III-12
S
Ph
Me
408.34
4.1
0c
III-13
S
tBu
S
563.67
6.6
71
III-14
Se
tBu
610.56
6.5
81
III-15
Te
tBu
659.20
7.0
50d
NEt2
O
N
S
a
Predicted using Molinspiration interactive logP calculator
% inhibition of [3H]E217βG uptake relative to vehicle control
(1% DMSO)
c
tested at 1 µM
d
tested at 5 µM
Y
b
2+
R
X
+
50
R
Table III-4
Inhibition of MRP1-mediated uptake of [3H]E217βG by
Class IV CGPs
Results shown are means of two independent experiments.
CGP
LogPa
MW
R
% Inhibitionb
(30 µM)
S
IV-1
N
626.72
4.8
95
543.53
4.9
96
527.56
6.0
43
428.43
5.4
50
S
COOH
IV-2
S
IV-3
NEt2
O
IV-4
a
b
Ph
Predicted using Molinspiration interactive logP calculator
% inhibition of [3H]E217βG uptake relative to vehicle control
(1% DMSO)
R
S
+
+
N
51
Table III-5
Inhibition of MRP1-mediated uptake of [3H]E217βG by
Class V CGPs
Results shown are means of two independent experiments.
CGP
R1
R2
MW
R3
LogPa
% Inhibitionb
(30 µM)
O
V-1
-
NH2
666.06
3.4
96
NH2
NH2
681.08
2.7
8c
O
O
N
N
736.15
3.5
89
N
O
V-2
N
V-3
a
Predicted using Molinspiration interactive logP calculator
% inhibition of [3H]E217βG uptake relative to vehicle control (1% DMSO)
c
tested at 1 µM
b
R1
Te
+
R2
R3
52
3.1.ii Relative Inhibitory Potencies of CGPs on MRP1-mediated [3H]E217βG Uptake
The 16 of 34 CGPs that inhibited MRP1-mediated [3H]E217G uptake by >50% were
selected for further concentration-dependent inhibition studies to determine their relative
potencies. Thus, the IC50 values of the 16 CGPs identified in the first screen were determined to
range from 0.7 to 7.6 µM and the results are summarized in Table III-6. The concentrationresponse curves for the CGPs showed a classic sigmoid shape, and representative graphs for
CGPs I-3, I-4, I-6, II-5 and IV-1 are presented in Fig. III-2.
Of the 16 CGPs tested, 9 were quite potent with IC50’s < 2 µM. These included 5 of 6
Class I CGPs which all had comparable IC50 values (0.9 – 1.4 µM) while the IC50 of I-1 was 4
µM. Similarly, the two Class IV CGPs (IV-1, -2) had comparable IC50 values of 1.6 and 2.0 µM,
respectively. Only two of four Class II CGPs had IC50 values < 2 µM (II-3, 1.8 µM; II-4, 0.7
µM), with II-4 being significantly more potent than II-3 (P < 0.05). On the other hand, the IC50’s
for II-2 and II-5 were comparable at 3.9 µM and 2.5 µM, respectively (P > 0.05). The two Class
III CGPs (III-13, -14) tested had comparable IC50 values of 7.6 and 4.9 µM (P > 0.05),
respectively, while the respective IC50 values of V-1 and V-3 were 5.3 and 3.3 µM, with V-3
being significantly more potent than V-1 (P < 0.05). In summary, of the 9 CGPs that had IC50’s ≤
2 µM, five belonged to Class I (I-3, -4, -5, -6, -7), two belonged to Class II (II-3, -4), and two to
Class IV (IV-1, -2).
Of the CGPs with IC50’s < 2 µM, there is no inhibitor whose potency is significantly
greater than the rest; however between classes, there are statistically significant differences in
potency. CGP I-3, I-6 are more potent than II-3 (P < 0.05) while II-4 is more potent than IV-1 (P
< 0.05).
53
Table III-6
Summary of IC50 Data
The values shown represent the mean ± SD of the IC50 values obtained in
3-4 independent experiments (indicated in parentheses). When the
experiment was repeated just once, both values are displayed.
IC50 (µM)a
CGP
Class I
Class II
Class III
Class IV
Class V
LogPb
[3H]E217βG uptake
I-1
4.7, 3.2
6.9
I-3
1.1 ± 0.4 (4)
8.9
I-4
1.2 ± 0.2 (3)
9.2
I-5
1.2, 0.9
10
I-6
0.9 ± 0.1 (3)
8.2
I-7
1.4, 1.4
9.7
II-2
3.9 ± 1.9 (3)
5.5
II-3
1.8 ± 0.1 (3)
5.4
II-4
0.7 ± 0.3 (3)
5.3
II-5
2.5 ± 0.5 (4)
5.3
III-13
7.6 ± 3.1 (3)
6.6
III-14
4.9 ± 3.9 (3)
6.5
IV-1
1.6 ± 0.4 (3)
4.8
IV-2
2.0, 1.8
4.9
V-1
5.3 ± 0.7 (3)
3.4
V-3
3.3 ± 0.6 (3)
3.5
a
Within Class I, no CGP was significantly more potent than another.
CGP II-4 was more potent than II-2, -3 and -5 (P < 0.05). CGP V-3
was more potent than V-1 (P < 0.05). The IC50’s of Class III CGPs
were not statistically different (P > 0.05). For Class IV, the sample size
was too small for statistical analyses.
b
Predicted using Molinspiration interactive logP calculator
54
55
3.1.iii Modulation of MRP1-mediated calcein efflux from intact HEK-MRP1 cells by CGPs
To further examine the efficacy of the 16 CGPs identified as inhibitors of MRP1mediated E217βG transport, an intact cell calcein efflux assay was employed using the HEKMRP1 cell line and an untransfected HEK cell line as a control. Of the 16 CGPs tested, just 5
inhibited MRP1-dependent efflux of calcein > 25% at 10 µM (Fig. III-3). Three of these were
from Class I (I-3, -4 and -6) and inhibited calcein efflux by 66 ± 19%, 46 ± 10% and 59 ± 23%,
respectively, while CGP IV-1 inhibited efflux by 52 ± 9%. None of the four were significantly
more effective than any one (P > 0.05). On the other hand, CGP II-5 inhibited calcein efflux by
just 23 ± 4% and was significantly less effective than CGP I-3, -4, -6 and IV-1 (P < 0.05). The
structures of these 5 CGP modulators of MRP1-mediated calcein efflux are shown in Fig. III-4.
When the inhibitory potency of these 5 CGPs in the vesicular transport assay was
compared to their efficacy in the calcein efflux assay, the three Class I CGPs and CGP IV-1 were
similar. On the other hand, CGP II-5 was the least effective inhibitor of MRP1-mediated calcein
efflux and was also the least potent inhibitor of MRP1-mediated [3H]E217G uptake.
3.1.iv Modulation of MRP1-mediated [3H]E217βG Uptake by Class IV CGP Analogs
To determine how specific substituents of the Class IV CGPs could impact their efficacy,
a second series of five CGP IV-1 analogs was synthesized with different substituents on the
chalcogenopyrylium ring at positions 2 and 6, and at the connecting carbon atom between the
56
57
58
thiophene and the N-substituted cyclohexane (thioketone vs ketone) from the 4-position of the
chalcogenopyrylium ring. These new analogs were designated IV-1-1, -2, -3, -4 and -5, and their
structures are shown in Fig. II-2. The new series of CGPs was tested for its effects on MRP1mediated [3H]E217G uptake as before, and the results are shown in Fig. III-5. At 1 µM, IV-1-1
and IV-1-3 inhibited MRP1-mediated uptake of [3H]E217G very modestly with mean inhibition
values of 25% and 18%, respectively, while IV-1-2, IV-1-4 and IV-1-5 at 1 µM showed no effect
at all. At 10 µM, when compared to the parent compound IV-1, all five of the analogs were also
less efficacious (Fig. III-5). Thus, CGP IV-1-1 was the most comparable to the parent CGP IV-1
with a mean inhibition value of 81% for IV-1-1 compared with 93% for IV-1. CGP IV-1-3 and
IV-1-4 inhibited MRP1-mediated uptake of [3H]E217G by 78% and 59%, respectively, at 10
µM. IV-1-2 and IV-1-5 (10 µM) appeared to be the least effective at 32% and 11% inhibition,
respectively (Fig. III-5).
3.2 Modulation of MRP2-mediated [3H]E217G uptake by CGP I-3, I-4, I-6, II-5 and IV-1
To determine whether the 5 CGPs selected from the first series of transport and efflux
experiments were specific inhibitors of MRP1, or could modulate the activity of other MRP
transporters, membrane vesicles were prepared from transiently transfected HEK293T cells
expressing human MRP2 (confirmed by immunoblotting; Fig. III-1B). MRP2-mediated uptake
of [3H]E217G into membrane vesicles was then determined in the presence of a single
concentration (30 µM) of CGPs I-3, I-4, I-6, II-5 and IV-1. Two of the five CGPs (I-4, -6)
inhibited MRP2-mediated [3H]E217G uptake by >90%, two (I-3, IV-1) stimulated transport >
2-fold, and one (II-5) was ineffective (Fig. III-6).
59
60
61
To further investigate the relative potency of the five CGPs on MRP2-mediated
[3H]E217G uptake, concentration response experiments were conducted in the presence of five
different concentrations of CGP, to a maximum of 30 µM. For CGPs I-4 and I-6, the IC50’s were
2.0 and 7.1 µM, respectively (Fig. III-7, Panels B and C). In contrast, CGPs I-3 and IV-1
stimulated [3H]E217G uptake activity to a maximum of approximately 2.3-fold at 30 µM (Fig.
III-7, Panels A and E). CGP II-5 remained ineffective over the entire concentration range tested
(0.1 to 30 µM) (Fig. III-7, Panel D).
The five second generation CGP Class IV-1 analogs were also tested at 1 and 10 µM for
their effects on MRP2-mediated [3H]E217G uptake (Fig. III-8). At 1 µM, IV-1-1, -2 and-3 had
no effect on MRP2-mediated [3H]E217G uptake whereas CGP IV-1-4 and -5 stimulated uptake
by 1.5-fold. When tested at 10 µM, four of the five CGP IV-1 analogs stimulated MRP2mediated uptake of [3H]E217G to varying degrees. The exception was CGP IV-1-1 which had
no effect. Stimulation by IV-1-2 was weak (1.2-fold at 10 µM), while IV-1-3 and IV-1-5 were
slightly more effective (1.4-fold simulation) at the same concentration. CGP IV-1-4 appeared to
be the most effective of the five analogues (1.7-fold stimulation) and was comparable to the
parent CGP IV-1 at 10 µM (1.8-fold stimulation). Taken together, the data indicate that although
some of the CGP Class IV-1 analogs retained the ability to stimulate MRP2-mediated transport,
the efficacy of only one was comparable to the parent CGP IV-1.
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63
64
3.3 Modulation of MRP4-mediated uptake of [3H]E217βG
The 5 CGPs (I-3, I-4, I-6, II-5, IV-1) were also tested for their ability to modulate
[3H]E217G uptake by MRP4. Following transient transfection of a human MRP4 cDNA
expression vector into HEK293T cells, membrane vesicles were prepared and the presence of the
transporter confirmed by immunoblotting (Fig. III-1C). In contrast to the immunoblots for MRP1
and MRP2, two bands were detected with the MRP4-specific MAb M4I-10. The reason for the
two bands is currently unknown, but they have been occasionally observed previously in HEK
cells but not HeLa cells (116). It may be caused by variable glycosylation of MRP4 when
expressed in HEK cells (116). When tested at 1 µM, none of the five CGPs (I-3, I-4, I-6, II-5 or
IV-1) inhibited uptake of [3H]E217G by MRP4 by >20%. However, when tested at 10 µM,
[3H]E217G uptake was inhibited by >75% (77-86%) by all 5 CGPs (Fig. III-9).
The second generation Class IV-1 analogs were also tested at 1 and 10 µM for their
effects on MRP4-mediated uptake of [3H]E217G (Fig. III-10). Little or no modulation was seen
at 1 µM. However, at 10 µM, three of five Class IV-1 analogs (IV-1-1, -3 and -4) inhibited
[3H]E217G uptake by MRP4 by approximately 60%. The other two (IV-1-2 and -5) inhibited
uptake by < 20% at this concentration. Thus, none of the class IV-1 analogs were as effective as
the parent IV-1 compound which inhibited MRP4-mediated [3H]E217G uptake by 77% at 10
µM.
65
66
67
IV. Discussion
In a previous study, a group of structurally similar, but differently substituted CGPs were
shown to modulate Pgp transport activity (147). Here, a group of novel CGPs was tested for their
effects on MRP1, MRP2 and MRP4 transport activity. Specifically, the ability of 34 CGPs to
modulate uptake of [3H]E217G into inside-out membrane vesicles enriched with one of MRP1,
MRP2 or MRP4 was tested. In addition, the CGPs were tested for their ability to modulate
MRP1 activity in intact cells using a calcein efflux assay. As described in Section III, CGPs I-3,
I-4, I-6, II-5 and IV-1 modulated MRP1-, 2- and 4-mediated uptake of [3H]E217G as well as
calcein efflux by MRP1, thus demonstrating that these CGPs can modulate MRP transporters.
Among the 5 classes tested, there were differences in efficacy and potency. Initially, the
34 CGPs were screened at a single concentration (30 µM) which differed for some CGPs
discovered to have limited solubility, or those which generated unacceptably high background
levels during scintillation counting (1, 5 µM). Thus, the CGPs which inhibited MRP1 by 50% or
greater were selected for further concentration-response studies; however, efficacy of some
CGPs (II-1, III-2, -3, -4, -5, -12, -15 and V-2) may be largely underestimated due to the
aforementioned necessary decreases in concentration. The 16 CGPs identified to be efficacious
were then tested to determine their rank order potency, and another cutoff was set, < 2 µM,
which defined the CGPs that would be described as potent.
The Class I CGPs were the most efficacious and potent class of CGPs, since six of seven
inhibited MRP1-mediated uptake of [3H]E217G, and five of six Class I CGPs tested had IC50’s
< 2 µM despite variations in their size. CGP I-2 is structurally similar to I-6, with the exception
of two additional carbon atoms in the alkyl chain of CGP I-6 which connects the
68
chalcogenopyrylium ring to the Se-substituted benzyl group. CGP I-2’s effects were unlike those
of CGP I-6 which was one of the most potent inhibitors of MRP1-mediated uptake of
[3H]E217G (IC50 0.9 µM). Thus, the 2-carbon difference between CGPs I-2 and I-6 appears to
account for major differences in the modulatory activities of these compounds. The effect of I-6
may come about through favourable interactions between the Se group (H-bond) or the bulk of
the tertiary butyl (tBu) group at either end of the molecule and MRP1. When the length of the
alkyl chain is decreased, as is seen with I-2, the inhibitory effect is also decreased, indicating,
perhaps, that the interaction is no longer happening due to a decrease in length.
CGPs I-4 and I-7 also share a 2-carbon difference in chain length and are similarly
substituted; however, the chalcogen atom is tellurium rather than selenium. Despite the 2-carbon
difference in alkyl chain length between these two compounds, both I-4 and I-7 modulate MRP1
to the same extent and have comparable IC50’s (1.2 and 1.4 µM, respectively) and predicted
LogP values.
It is not uncommon for differences in alkyl length to cause differences in modulatory
ability of otherwise similar compounds. Wong et al. (187) observed that by adding additional
carbons to the alkyl chain connecting two apigenin homodimers, a stepwise decrease in
inhibitory activity was observed for each carbon added beyond the optimal length of 6-carbons
(187). Also, various S-alkyl GSH compounds inhibited MRP1-mediated uptake of GSHconjugated aflatoxin in an inside-out membrane vesicular assay (188). Increases in alkyl-chain
length from S-methyl to S-decyl led to stepwise increases in inhibitory effect (188). In the
present study, differences in alkyl chain length also led to differences in modulatory capacity;
however, these differences could be attenuated by substitution with an alternate chalcogen atom.
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The chalcogen atoms (O, S, Se, Te) of previously reported chalcogenorhodamines
(introduced in Chapter I) are effective H-bond acceptors (150). The chalcogen in the xanthylium
core of the rhodamines is conducive to accepting H-bonds due to efficient delocalization of the
positive charge through conjugated double bonds to the N-atom outside of the tricyclic core.
Thus, it was proposed that the chalcogen atoms may be interacting with H-bond donating amino
acids on Pgp (Fig. I.3) (150, 189). The compounds used in the present study can delocalize the
positive charge about the chalcogen atom through the aromaticity of the CGP core. This allows
for acceptance of H-bonds by the chalcogen atom; however, they are weaker than those observed
with the rhodamines (personal communication, Dr. Michael Detty). Thus, similar to Pgp, the
chalcogen atom at the core of the CGPs investigated in this study may be interacting with Hbond donors in the substrate binding pockets of MRP1, 2 or 4.
Four of five Class II CGPs were effective inhibitors of MRP1 activity with IC50 values
from 0.7 to 3.9 µM. CGP II-2 was the least effective inhibitor (IC50 3.9 µM). Some of the IC50’s
from Class II are higher than most Class I CGPs and may indicate that the abundance of large,
bulky tBu groups found in the Class I CGP structures accounts for the interaction between the
CGP and MRP1 (I-3, -4 and -6 IC50’s are significantly lower than II-2 and -5; P < 0.05)
Furthermore, the predicted LogP values of Class I are higher than those predicted for II-2 and II5, perhaps indicating that hydrophobicity is an important property of the Class I CGPs.
The ineffective fifth, CGP II-1, is less bulky than the rest of the Class II CGPs. This may
indicate that the presence of a butyl or tBu group at the R-position of Class II CGPs helps favour
interactions with MRP1. Substitution of the butyl group at the R-position on CGP II-2 with a
bulky tBu group (II-3, -4, -5) appeared to increase the inhibitory activity. CGP II-3, II-4 and II-5
70
only differed with respect to the chalcogen atom substituted at the 1-position of the
chalcogenopyrylium ring, yet they have similar activities. This indicates that the tBu groups
influence the modulatory activity more than the chalcogen atom in this class of CGPs.
CGPs II-3 and II-4 were previously tested for their ability to inhibit Pgp using a calcein
efflux assay (147). As observed for MRP1, their IC50’s were similar, which may indicate that the
difference in the chalcogen atom at the 1-position is not as important as the bulkiness around the
CGP ring for inhibition of both MRP1 and Pgp.
The Class III CGPs were the least effective and least potent CGPs with only two of
fifteen (III-13 and -14) causing > 50% inhibition of MRP1-mediated uptake of [3H]E217G
(IC50’s 7.6 and 4.9 µM, respectively). CGP III-13 is significantly less potent than some of the
other CGPs (I-3, -4, -6, II-3, -4, -5, IV-1 and V-3), many of which had IC50’s < 2 µM (P < 0.05).
The structural nuances of this class of CGPs are many with some compounds having complex
substitutions at the 4-position of the chalcogenopyrylium ring. When the substitution at position
Y is very small (eg. Me), as seen in III-10, -11 and -12, there is no inhibitory activity at all. By
contrast, CGP III-13, -14 and -15 all have the same moiety (thiophene-thioketone-N-substituted
cyclohexane) at the Y-position and it appears important for inhibitory activity. Furthermore, a
similar inhibitory effect was observed when the substituted chalcogen was either S (III-13) or Se
(III-14). Therefore, the Y-substituent appears to be important for imparting inhibitory activity to
this class of compounds.
Of the four Class IV CGPs tested at 30 µM, two (IV-1 and IV-2) inhibited MRP1mediated uptake by >95% while the other two (IV-3 and IV-4) inhibited uptake by 43% and
50%, respectively. The IC50’s of both Class IV CGPs included in concentration-dependent
71
experiments were comparable (IV-1 1.6 µM, IV-2 1.9 µM) as were their predicted LogP values.
The predicted LogP of IV-3 is higher and its inhibitory effect lower than that of IV-1 and -2.
This may indicate that the less hydrophobic nature of both IV-1 and -2 is better for inhibitory
activity. All Class IV CGPs have a sulfur atom at the 1-position of the CGP ring and a Ph-NMe2
substitution at the 6-position of the CGP ring. CGPs with thiophene groups as part of the Rsubstituent of the CGP ring (IV-1, 95% and IV-2, 96%) appear to be more effective inhibitors
than those where R was Ph-CO-NEt2 (IV-3, 43%) or Ph (IV-4, 50%). CGP IV-2 has a polar
COOH substituent capable of relatively strong H-bond interactions with various residues in
MRP1. Thus, the thiophene-COOH substituent of IV-2, and perhaps the thioketone of IV-1, may
H-bond with MRP1.
The Class V CGPs contain the most aromatic character with each R- substituent attached
to an aryl group which is attached to the CGP ring. Interestingly, this Class also has the lowest
predicted LogP values of all other classes. CGP V-1 was the most effective inhibitor in Class V
(at 30 µM), and it is the most potent as well. Structurally, V-3 is larger than V-1 due to the
presence of substituted morpholino groups and this difference in size may account for its
decreased potency compared to V-1. The Class I CGPs and II-4 are significantly more potent
than V-1 (P < 0.05). Due to the technical difficulties in using CGP V-2 (discussed in Chapter
III), it is not possible to conclude whether the addition of the NH2 group at the R2-position led to
a decrease in activity (as CGP V-2 could only be tested at 1 µM), or whether the molecule is
inherently just a poor inhibitor.
To determine whether some of the CGPs could inhibit MRP1 in intact cells, a calcein
efflux assay was employed. Of the 16 CGPs shown to potently inhibit MRP1-mediated uptake of
72
[3H]E217βG (IC50 0.7 – 7.6 µM), only 5 inhibited the efflux of calcein appreciably, and a cutoff
of >25% inhibition was set to describe CGPs evaluated in the calcein assay as being efficacious.
According to their IC50’s, the 5 CGPs could be ranked I-6 < I-3 < I-4 < IV-1 < II-5. A similar
ranking for the calcein efflux data was observed I-3 > I-6 > IV-1 > I-4 > II-5, with I-3, -4, -6 and
IV-1 being significantly more effective than II-5 (P < 0.05) but not significantly more effective
than one another (P > 0.05). Of the two methods used to determine the modulatory effect of
certain CGPs on MRP1 transport activity, the most important data is obtained from the intact cell
efflux assay as the applicability of these CGPs to whole cells provides a rationale for testing
them in higher order biological systems.
As expected, the levels of inhibition of MRP1 transport activity were lower in the intact
cell system, and in no case was complete inhibition observed. These results are similar to those
reported by the Borst group (190) who examined a series of peptidomimetic glutathione
conjugates for their ability to inhibit MRP1-mediated uptake of [3H]E217βG (190). After
demonstrating that [3H]E217βG uptake was inhibited by analog VII (IC50 0.3 µM), the authors
then assessed its efficacy using an intact cell calcein efflux assay. Because of its limited
solubility, the authors esterified VII to VII-dimethyl ester to enhance its cell permeation
properties. Nevertheless, the IC50 of the VII-dimethyl ester was still 100-fold greater (36 µM)
than observed in the vesicular transport assay (190). CGP II-5 was the weakest inhibitor of
MRP1-mediated calcein efflux and also had the highest IC50 (E217βG transport) of the 5 CGPs
tested.
Following investigation of the CGPs effects on MRP1 transport activity, there were no
definitive insights into what structural features impart the inhibitory activity. Clearly, the bulky
73
functional groups appear to be important for some of the inhibition; however, in some instances,
the effect may be a result of the chalcogen atom present in the ring system. Thus far, the
molecular triggers of MRP1 inhibition for these CGPs are yet to be determined.
After identifying 5 CGPs as modulators of MRP1 transport in vitro and in intact cells,
their effects on MRP2 and MRP4 were assessed. The modulatory effects of the 5 selected CGPs
on MRP2-mediated uptake were largely differential, and minor differences in substitution about
the 1-position of certain CGPs (I-3 and I-4) accounted for markedly different effects. CGP I-3
which has a Se substituted at the 1-position of the CGP ring stimulated transport by ~2.5-fold
which is similar to the effects of various well documented MRP2 stimulators (100). Strikingly,
CGP I-4, which has a Te at the 1-position, rather than Se, as in CGP I-3, is a potent inhibitor of
MRP2-mediated uptake (IC50 2.0 µM). To the best of our knowledge, this is the first time such a
marked difference in effect has been observed with the substitution of one atom. However, small
changes in structure can alter the stimulatory effect of certain compounds on MRP2 transport
activity. For example, analogs of the prototypical cannabinoid type 1 receptor antagonist
rimonabant were developed and tested for their effects on MRP1, 2, 3 and 4 transport activities
(177). An analog with a tertiary amine as the altered substituent (R-group) markedly stimulated
MRP2 transport activity, even more so than the parent compound rimonabant. Consequently,
further analogs were created which altered the exposure of this tertiary amine to its surroundings
(NMe2, NEt2, etc.) and were shown to also stimulate MRP2 transport activity. However, the
more substitutions around this tertiary amine, the less effective the compound was at stimulating
MRP2 transport activity (177). This decrease in effect was observed more so when the amine
was cyclized which resulted in a 2-fold reduction in stimulatory effect. Unlike the results of their
study, all of the analogs in this study still stimulated MRP2 transport activity, albeit to varying
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degrees, where minor structural changes in the CGPs I-3 and I-4 resulted in markedly different
effects (simulation versus inhibition).
Structural characteristics required for stimulation of MRP2 transport activity have
previously been proposed by Pedersen et al. (100) using experimentally derived modulatory data
of MRP2-mediated uptake of [3H]E217βG (100). From the data interpretation, eight molecular
descriptors were assigned and the stimulators differed from the ‘non-interacting’ group of
compounds tested by having: a greater negative charge, larger number of functional groups
involved in H-bonding, lower lipophilicity and a smaller molecular weight, among others (100).
Another interesting point was that the charge characteristics of the stimulators were similar to
those of MRP2 substrates, suggesting that the stimulatory binding site may be similar to the
substrate binding site.
One of the molecular descriptors is contrary to the observations made in this study in that
both CGPs that stimulated MRP2 (I-3 and IV-1) possess a positive charge at the chalcogen atom
in the aromatic ring; however, it is delocalized across the entire chalcogenopyrylium ring. It may
be that the CGPs are binding to an allosteric site on MRP2, rather than to a substrate binding site,
as most stimulators tested by Pedersen were negatively charged. It is also possible that CGPs I-3
and IV-1 stimulate MRP2 transport activity through a different mechanism. Interestingly,
although CGP I-3 and IV-1 both stimulate MRP2-mediated transport activity, they are
structurally different. CGP I-3 is more symmetric, while IV-1 has a much larger substitution at
the 4-position and an asymmetric core.
The 5 selected CGPs were also effective inhibitors of MRP4-mediated uptake of
[3H]E217βG at 10 µM. However, not one of the 5 selected CGPs inhibited MRP4 better than the
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next, similar to their effects on MRP1 activity, but distinct from their effects on MRP2. Although
the amino acid sequence homology and substrate specificity of MRP2 is more similar to MRP1,
the effects of these 5 CGPs on MRP4 were more similar to MRP1. This observation is similar to
that of Wittgen et al. (177) who showed that certain cannabinoid type 1 receptor antagonists
inhibited MRP4 more so than MRP1; however, their effects were similar on both proteins
(inhibition) whereas their effects on MRP2 and 3 were stimulatory.
To further explore the chemical properties responsible for the inhibitor activity of one
Class IV CGP, IV-1, a series of 5 analogs were generated and tested as modulators of MRP1-, 2and 4-mediated E217βG uptake activity. Overall, these Class IV-1 CGP analogs modulated
transport similarly or less effectively than the parent compound.
CGP IV-1-4 differs from its parent compound in that it has a ketone at the connecting
carbon between the thiophene and N-substituted cyclohexane where IV-1 has a thioketone. Its
effects on MRP1-mediated uptake are similar to that of the parent compound IV-1, but the
inhibition, albeit noteworthy (59% inhibition at 10 µM), was different than the parent compound
CGP IV-1 (93% inhibition at 10 µM). One of the most effective MRP1 inhibitors was IV-1-1
(81% inihibition at 10 µM). CGP IV-1-1 is structurally larger than IV-1 with a Ph-NMe2 in place
of the tBu at the 2-position of the CGP ring. The functionality of this class of analogs appears to
come from both the substitution about the CGP ring and the differing ketone/thioketone at the
connecting carbon between the thiophene and N-substituted cyclohexane. CGPs IV-1-1 and IV1-3 both have Ph-NMe2 groups at the 2- and 6- position, but differ with respect to the
ketone/thioketone, yet their effects on transport activity are nearly identical. CGPs IV-1-2 and
IV-1-5 both have tBu groups at the 2- and 6-position, and neither inhibited MRP1-mediated
76
uptake by more than 50% at 10 µM. The presence of the thioketone appears to impart the
inhibitory ability to CGP IV-1 when compared to IV-1-4, which does not have the thioketone
functionality.
Thus, the Class IV-1-X CGPs may interact differentially with MRP1 and the substituents
at differing parts of the molecule may have effects on their ability to inhibit transport. This result
is commonly observed when testing various analogs for effects on transport activity by MRPs.
For example, Genoux-Bastide et al. (181) showed that differences in substitution about
xanthones resulted in markedly different effects on MRP1-expressing cells (181). Slight
modifications on the compound (changing H to OH to OMe, for example) resulted in marked
differences in MRP1-mediated GSH efflux (181). Thus, similar to the results presented here,
minor structural changes can elicit differences in effect of compounds interacting with MRP1.
The CGP IV-1-X analogs’ effects on MRP2 were also investigated and were differential,
with only two of them having a stimulatory effect like the parent compound IV-1. CGP IV-1-4
stimulated transport most closely to the parent CGP at 10 µM (1.7-fold vs 1.8-fold, respectively).
The ketone/thioketone substituent of CGP IV-1 and IV-1-4 may be H-bonding with a region of
MRP2, and this H-bonding may induce changes to the protein conducive to stimulated transport.
Similar to MRP1, but in contrast to MRP2, most of the Class IV-1-X analogs showed
some inhibition of MRP4. The 3 Class IV-1 analogs (IV-1-1, IV-1-3 and IV-1-4) that inhibited
MRP1 most effectively also inhibited MRP4 most effectively. Another pan-specific MRP
inhibitor is MK571, which inhibits not only MRP1, but also MRP4, albeit with different
potencies (191, 192). MK571 also interacts with MRP2; however, unlike the Class IV-1 analogs
investigated here, MK571 inhibits MRP2 (193). In contrast to MK571, there are several
77
inhibitors of MRP1 and MRP4 that stimulate MRP2 as effectively as the CGPs reported here.
For example, sulfinpyrazone, a uricosuric agent used to treat gout, inhibits MRP1 and MRP4, but
is a potent stimulator of MRP2 (194, 195). Similarily, probenecid inhibits both MRP1 and
MRP4, but stimulates MRP2 transport activity, at least during MRP2-mediated efflux of
paclitaxel, docetaxel and vinblastine (196).
Structurally, MK571 contains aromaticity, H-bond acceptors and donors, sulfur groups
and 2 tertiary nitrogens, all capable of transient interaction with the MRPs. One major difference
between MK571 and CGP I-3 is size, with I-3 being much smaller and having little chemical
functionality beyond tBu groups and the substituted chalcogen atoms Se and O. These
differences may account for the varying effects each compound has on MRP2 transport activity.
In contrast, IV-1 is more chemically similar to MK571 in that it has 2 tertiary N groups, sulfur
groups and aromaticity. However, unlike MK571, IV-1 stimulates MRP2 transport activity; thus,
these two CGPs may be binding to different sites on the protein.
78
V. Conclusions and Future Directions
This study showed that a group of novel CGPs modulated MRP1-, MRP2- and MRP4mediated E217βG uptake with varying effects on each transporter. Most notably, the differential
effects included marked stimulation of E217βG uptake by MRP2 at concentrations of CGPs I-3
and IV-1 that inhibited MRP1 and MRP4. This observation was not entirely unexpected, as
MRP2 potentiation is a well documented phenomenon; however, the minor differences in
structure accounting for significant differences in effect on MRP2-mediated uptake are
unexpected.
Although some CGPs investigated in this study differentially modulated E217βG uptake
by MRP1, MRP2 and MRP4, no conclusions can be drawn as to whether they, or other
structurally similar CGPs, would interact with and modulate other MRPs. Furthermore,
predicting the effect CGPs will have on MRP-mediated uptake of [3H]E217βG is difficult, if not
impossible, as minor changes about the CGP ring were observed to alter the modulatory effects,
at least for MRP2. Furthermore, the higher predicted LogP values appear to trend with potency;
however, without actual LogP values, this observation may be meaningless.
Importantly, the mechanism of modulation is unknown and future studies with the select
CGPs shown to modulate MRP1, 2 and 4 should focus on the kinetics of inhibition and
stimulation. Following identification of their inhibitory properties, their effects on MRP2- and 4mediated efflux from whole cells (similar to their characterization here, for MRP1) is an
important avenue of study.
Further syntheses of various analogs encompassing various aspects of the “hits”
described here are a valuable tool for discovering other, perhaps more potent modulators of
79
MRP1, MRP2 and MRP4. Future studies should investigate modifications about the CGP ring in
the Class I CGPs, as they were the most efficacious class.
The applicability of these CGPs in vivo is another unexplored avenue which needs to be
examined to determine if they could ever have clinical utility. Studies using non-tumour
burdened mice may provide valuable data as to their disposition in healthy mice and might help
determine if sensitization of MRP-overexpressing tumours using CGPs is a feasible goal.
However, due to the differential effects of these CGPs on the 3 MRPs studied, careful
deliberations are necessary when considering these compounds for in vivo applications.
80
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