824 Biochemical Society Transactions (2016) Volume 44, part 3
The multidrug transporter ABCG2: still more
questions than answers
Aaron J. Horsey*, Megan H. Cox*, Sunehera Sarwat* and Ian D. Kerr*1
*School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, U.K.
Abstract
ABCG2 is one of at least three human ATP binding cassette (ABC) transporters which can facilitate the export
from cells of a wide range of chemically unrelated drug molecules. This capacity for multidrug transport
is not only a confounding factor in chemotherapy, but is also one of the more perplexing phenomena in
transporter biochemistry. Since its discovery in the last decade of the 20th century much has been revealed
about ABCG2’s localization, physiological function and its broad substrate range. There have also been many
investigations of its structure and molecular mechanism. In this mini review article we take a Rumsfeldian
approach to ABCG2 and essentially ask what we do know about this transporter, and what we will need to
know about this transporter if we wish to use modulation of ABCG2 activity as a therapeutic approach.
Multidrug transporters in biology that comprise the family in humans. Three of these, ABCB1
Multidrug resistance (MDR) can be defined as the ability (P-glycoprotein), ABCC1 (multidrug resistance protein 1)
of cells or organisms to resist the cytotoxic effects of a and ABCG2 (breast cancer resistance protein) have been the
diverse range of chemical structures, often with different subject of many decades of investigation, and there are already
intracellular targets. MDR is observed across the spectrum numerous reviews on many facets of their structure, function,
of infectious disease therapy, as well as being documented regulation and role in disease [2,3]. The aim here is not to
in cancer chemotherapy. In many cases, the acquisition provide an exhaustive overview of one of this triumvirate
of a MDR phenotype is a poor prognostic indicator. For (ABCG2) but to present a personal view about what we know,
example, the emergence of drug resistant pathogenic bacteria and what we need to know about this transporter if we are to
and the subsequent lack of effective therapies is one of the develop rational interventions in chemotherapy.
global challenges for the 21st century. MDR may manifest
through many mechanisms, often operating with what one
presumes to be a degree of synergy, including chemical
modification of the drug molecule rendering it ineffective (or
ABCG2 localization and physiological
less effective) and the alteration of gene expression to enable function: an incomplete picture
cellular metabolism to proceed through other pathways. The Although first isolated from multidrug resistant cancer cells,
mechanism of interest here is the export of the drug from ABCG2’s expression and distribution pattern in normal
the cell, thus preventing it from achieving clinically effective tissues implies that it must fulfil important physiological
concentrations. MDR by drug export is catalysed by at roles, such as protecting the organism as a first line of defence
least 5 (and almost certainly more) families of membrane against environmental toxins. Schinkel’s original study
proteins with examples well documented in antimicrobial, on ABCG2-null mice confirmed this role, demonstrating
antiparasitic and anticancer chemotherapy [1]. that these animals are more susceptible to diet-induced
In humans, MDR is most widely associated with the failure protoporphyria and phototoxicity, caused by accumulation
of cancer chemotherapy. A number of cancers have been of pheophorbide A [4], a chlorophyll degradation product
shown to have poorer prognosis if there is either a pre- and a confirmed ABCG2 transport substrate [5].
existing expression of an MDR pump, or if expression of an Extensive studies have continued to affirm that in humans
MDR pump develops as a result of chemotherapy (see section and rodents, the localization of ABCG2 can play a vital
below). Several human ATP binding cassette (ABC) proteins role in limiting absorption (in the small intestine), mediating
have been well described to be capable of MDR, although distribution (e.g. in the blood–brain and blood–placental
doubtless there will be more from the 48 ABC proteins barriers) and facilitating elimination and excretion (in the
liver and kidney) of drugs or xenobiotics that are ABCG2
transport substrates. This specific ABCG2 distribution
Key words: ABCG2, ABC transporter, ATPase, multidrug pump, pharmacology. profile among the different types of tissues is closely related
Abbreviations: ABC, ATP binding cassette; ALL, acute lymphoblastic leukaemia; EM, electron
to the physiological role it assumes in the body. For instance,
microscopy; MDR, multidrug resistance; NBD, nucleotide binding domain; NSCLC, non-small cell
lung cancer; SNP, single nucleotide polymorphism; TMD, transmembrane domain. in the blood–brain barrier, ABCG2 is expressed highest on
1
To whom correspondence should be addressed (email ). the luminal side of brain endothelial cells. Here, it serves
Biochem. Soc. Trans. (2016) 44, 824–830; doi:10.1042/BST20160014
c 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence
4.0 (CC BY-NC-ND).
, Membrane Proteins From A to Z 825
as a crucial barrier to drug access, significantly limiting the flavopiridol and tyrosine kinase inhibitors (imatinib, gefitinib
penetration of drugs or xenobiotics into the brain [6,7], and nilotinib) [17].
potentially in a synergistic manner with ABCB1 [8], with At present, over 200 transport substrates of ABCG2
implications for brain tumour therapy and imaging [9]. have been identified, with some attempts made to analyse
In addition, ABCG2 is expressed in the apical membrane of structure–activity relationships (SAR). One such study
epithelial cells in the gastrointestinal (GI) tract, with highest demonstrated the impact of polarity on transport in a series of
expression in the duodenum and a gradual decrease along the novel camptothecin analogues, which were actively extruded
GI tract to the rectum. Here, ABCG2 plays an important role at a greater rate dependent on their higher polarity [18].
in limiting the absorption of orally administered anticancer Additionally, small chemical libraries of ABCG2 inhibitors
drugs and ingested toxins [10,11]. In the liver canalicular have been investigated to determine important functional
membranes, where ABCG2 is constitutively expressed, a groups. For example, inhibitors of casein kinase II were
role in toxin and metabolite excretion is apparent [11,12]. repurposed into a series of ABCG2 inhibitors by an
In the human kidney ABCG2 is expressed on the apical overall increase in hydrophobicity and aromaticity [19]. This
membrane of proximal tubular cells, however with a lower argument for polarity being associated with transport, and
expression level than that in the liver and the small hydrophobicity with inhibition is interesting when taken
intestine, and is responsible for urate export. Indeed, ABCG2 alongside the most thorough attempt to determine the typical
activity as a urate transporter is known to be abrogated in features of ABCG2 interacting compounds, presented in 2015
gout [13]. by Anna Seelig’s group [20,21]. In two papers they employed
Among normal human tissues, ABCG2 is expressed functional ATPase activity assays, physicochemical data and
highest on the apical membrane of the placental syncytio- molecular modelling to attempt to distinguish between the
trophoblasts. Here, ABCG2 expels drugs or xenobiotics from chemistries of ABCB1 and ABCG2 transport substrates.
the foetal compartment back to the maternal circulation, The authors argued that ABCG2 transports chemistries with
limiting foetal exposure of the toxic substances and playing a higher hydrophilicity than ABCB1. However, in common
major role in protecting foetus against maternally derived with ABCB1, it does so subsequent to the partitioning of
toxins [14]. ABCG2 is also expressed in the mammary compounds from the aqueous to the lipid phase. A loose set
gland being induced strongly during the lactation phase of rules incorporating hydrophobicity, amphipathicity and
in mice, cows, and humans. It has been shown that ionization state was derived from analysis of a test series
ABCG2 actively transports not only beneficial vitamins (e.g. of chemicals to predict ABCG2’s likely interaction with or
riboflavin) but also, seemingly paradoxically, toxic drugs inhibition by other compounds [21]. Given the hundreds
and xenobiotics (e.g. topotecan, cimetidine and 2-amino-1- of compounds within the repertoire of ABCG2’s chemical
methyl-6-phenylimidazo [4,5-b] pyridine) into breast-milk interactome it would be interesting to see how these rules
[15,16]. evolve with the acquisition of further data.
How do we develop a greater understanding of the tissue- What do we know about how and where these drugs bind?
specific roles of ABCG2? This question remains important The short answer: surprisingly little! Initial studies aimed to
to answer, particularly considering the impact of ABCG2 understand ABCG2 pharmacology, and included equilibrium
function on multiple drug pharmacokinetics and the potential and kinetic radioligand binding assays to study the interaction
for ABCG2 polymorphisms to have differential effects on of radiolabelled daunomycin with ABCG2 expressed in insect
transporter function (see below). (Sf9) cell membranes [22]. This study was performed with an
R482G mutant version of ABCG2 due to its broader substrate
range (see below). The data demonstrated an affinity (K d ) of
ABCG2 for daunomycin of approximately 100 nM, which
ABCG2 transport substrate diversity and is still the only quantitative demonstration of an affinity
pharmacology: all mapped out? of ABCG2 for a transport substrate. Relative potencies for
As discussed above, the exact physiological roles of ABCG2 other drugs (mitoxantrone, Hoechst 33342, doxorubicin and
remain somewhat enigmatic. The distribution of the trans- prazosin) to displace daunomycin binding were obtained
porter within tissues that have a predominantly secretory and led to a picture of multiple drug binding sites showing
or barrier function leads to ABCG2 being recognised for a complex network of allosteric communications [22].
a role in controlling the disposition and tissue exposure Changes in transport substrate affinity were demonstrated
of endobiotics and xenobiotics, confirmed in numerous in subsequent studies upon the binding, rather than the
in vitro and in vivo studies. These include antibiotics, hydrolysis of ATP [23], but other direct, quantitative
sterols, immune-suppressants (including anti-HIV drugs), data for ABCG2:transport substrate interaction remain
fluorescent dyes (e.g. Hoechst 33342), photosensitizers scarce.
(pheophorbide A and protoporphyrin IX). The increased In the absence of such data a great deal of time and
expression of ABCG2 has been linked to MDR in cancer effort has been put into in vitro site-directed mutagenesis
(see below) and there have also been numerous studies studies of ABCG2. Arginine 482 to glycine/threonine (TM3;
describing ABCG2 mediated transport of chemotherapeutic Figure 1) is a classic example of a mutation which impacts
drugs including mitoxantrone, methotrexate, topotecans, substrate binding and/or transport resulting in the ability
c 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence
4.0 (CC BY-NC-ND).
The multidrug transporter ABCG2: still more
questions than answers
Aaron J. Horsey*, Megan H. Cox*, Sunehera Sarwat* and Ian D. Kerr*1
*School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, U.K.
Abstract
ABCG2 is one of at least three human ATP binding cassette (ABC) transporters which can facilitate the export
from cells of a wide range of chemically unrelated drug molecules. This capacity for multidrug transport
is not only a confounding factor in chemotherapy, but is also one of the more perplexing phenomena in
transporter biochemistry. Since its discovery in the last decade of the 20th century much has been revealed
about ABCG2’s localization, physiological function and its broad substrate range. There have also been many
investigations of its structure and molecular mechanism. In this mini review article we take a Rumsfeldian
approach to ABCG2 and essentially ask what we do know about this transporter, and what we will need to
know about this transporter if we wish to use modulation of ABCG2 activity as a therapeutic approach.
Multidrug transporters in biology that comprise the family in humans. Three of these, ABCB1
Multidrug resistance (MDR) can be defined as the ability (P-glycoprotein), ABCC1 (multidrug resistance protein 1)
of cells or organisms to resist the cytotoxic effects of a and ABCG2 (breast cancer resistance protein) have been the
diverse range of chemical structures, often with different subject of many decades of investigation, and there are already
intracellular targets. MDR is observed across the spectrum numerous reviews on many facets of their structure, function,
of infectious disease therapy, as well as being documented regulation and role in disease [2,3]. The aim here is not to
in cancer chemotherapy. In many cases, the acquisition provide an exhaustive overview of one of this triumvirate
of a MDR phenotype is a poor prognostic indicator. For (ABCG2) but to present a personal view about what we know,
example, the emergence of drug resistant pathogenic bacteria and what we need to know about this transporter if we are to
and the subsequent lack of effective therapies is one of the develop rational interventions in chemotherapy.
global challenges for the 21st century. MDR may manifest
through many mechanisms, often operating with what one
presumes to be a degree of synergy, including chemical
modification of the drug molecule rendering it ineffective (or
ABCG2 localization and physiological
less effective) and the alteration of gene expression to enable function: an incomplete picture
cellular metabolism to proceed through other pathways. The Although first isolated from multidrug resistant cancer cells,
mechanism of interest here is the export of the drug from ABCG2’s expression and distribution pattern in normal
the cell, thus preventing it from achieving clinically effective tissues implies that it must fulfil important physiological
concentrations. MDR by drug export is catalysed by at roles, such as protecting the organism as a first line of defence
least 5 (and almost certainly more) families of membrane against environmental toxins. Schinkel’s original study
proteins with examples well documented in antimicrobial, on ABCG2-null mice confirmed this role, demonstrating
antiparasitic and anticancer chemotherapy [1]. that these animals are more susceptible to diet-induced
In humans, MDR is most widely associated with the failure protoporphyria and phototoxicity, caused by accumulation
of cancer chemotherapy. A number of cancers have been of pheophorbide A [4], a chlorophyll degradation product
shown to have poorer prognosis if there is either a pre- and a confirmed ABCG2 transport substrate [5].
existing expression of an MDR pump, or if expression of an Extensive studies have continued to affirm that in humans
MDR pump develops as a result of chemotherapy (see section and rodents, the localization of ABCG2 can play a vital
below). Several human ATP binding cassette (ABC) proteins role in limiting absorption (in the small intestine), mediating
have been well described to be capable of MDR, although distribution (e.g. in the blood–brain and blood–placental
doubtless there will be more from the 48 ABC proteins barriers) and facilitating elimination and excretion (in the
liver and kidney) of drugs or xenobiotics that are ABCG2
transport substrates. This specific ABCG2 distribution
Key words: ABCG2, ABC transporter, ATPase, multidrug pump, pharmacology. profile among the different types of tissues is closely related
Abbreviations: ABC, ATP binding cassette; ALL, acute lymphoblastic leukaemia; EM, electron
to the physiological role it assumes in the body. For instance,
microscopy; MDR, multidrug resistance; NBD, nucleotide binding domain; NSCLC, non-small cell
lung cancer; SNP, single nucleotide polymorphism; TMD, transmembrane domain. in the blood–brain barrier, ABCG2 is expressed highest on
1
To whom correspondence should be addressed (email ). the luminal side of brain endothelial cells. Here, it serves
Biochem. Soc. Trans. (2016) 44, 824–830; doi:10.1042/BST20160014
c 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence
4.0 (CC BY-NC-ND).
, Membrane Proteins From A to Z 825
as a crucial barrier to drug access, significantly limiting the flavopiridol and tyrosine kinase inhibitors (imatinib, gefitinib
penetration of drugs or xenobiotics into the brain [6,7], and nilotinib) [17].
potentially in a synergistic manner with ABCB1 [8], with At present, over 200 transport substrates of ABCG2
implications for brain tumour therapy and imaging [9]. have been identified, with some attempts made to analyse
In addition, ABCG2 is expressed in the apical membrane of structure–activity relationships (SAR). One such study
epithelial cells in the gastrointestinal (GI) tract, with highest demonstrated the impact of polarity on transport in a series of
expression in the duodenum and a gradual decrease along the novel camptothecin analogues, which were actively extruded
GI tract to the rectum. Here, ABCG2 plays an important role at a greater rate dependent on their higher polarity [18].
in limiting the absorption of orally administered anticancer Additionally, small chemical libraries of ABCG2 inhibitors
drugs and ingested toxins [10,11]. In the liver canalicular have been investigated to determine important functional
membranes, where ABCG2 is constitutively expressed, a groups. For example, inhibitors of casein kinase II were
role in toxin and metabolite excretion is apparent [11,12]. repurposed into a series of ABCG2 inhibitors by an
In the human kidney ABCG2 is expressed on the apical overall increase in hydrophobicity and aromaticity [19]. This
membrane of proximal tubular cells, however with a lower argument for polarity being associated with transport, and
expression level than that in the liver and the small hydrophobicity with inhibition is interesting when taken
intestine, and is responsible for urate export. Indeed, ABCG2 alongside the most thorough attempt to determine the typical
activity as a urate transporter is known to be abrogated in features of ABCG2 interacting compounds, presented in 2015
gout [13]. by Anna Seelig’s group [20,21]. In two papers they employed
Among normal human tissues, ABCG2 is expressed functional ATPase activity assays, physicochemical data and
highest on the apical membrane of the placental syncytio- molecular modelling to attempt to distinguish between the
trophoblasts. Here, ABCG2 expels drugs or xenobiotics from chemistries of ABCB1 and ABCG2 transport substrates.
the foetal compartment back to the maternal circulation, The authors argued that ABCG2 transports chemistries with
limiting foetal exposure of the toxic substances and playing a higher hydrophilicity than ABCB1. However, in common
major role in protecting foetus against maternally derived with ABCB1, it does so subsequent to the partitioning of
toxins [14]. ABCG2 is also expressed in the mammary compounds from the aqueous to the lipid phase. A loose set
gland being induced strongly during the lactation phase of rules incorporating hydrophobicity, amphipathicity and
in mice, cows, and humans. It has been shown that ionization state was derived from analysis of a test series
ABCG2 actively transports not only beneficial vitamins (e.g. of chemicals to predict ABCG2’s likely interaction with or
riboflavin) but also, seemingly paradoxically, toxic drugs inhibition by other compounds [21]. Given the hundreds
and xenobiotics (e.g. topotecan, cimetidine and 2-amino-1- of compounds within the repertoire of ABCG2’s chemical
methyl-6-phenylimidazo [4,5-b] pyridine) into breast-milk interactome it would be interesting to see how these rules
[15,16]. evolve with the acquisition of further data.
How do we develop a greater understanding of the tissue- What do we know about how and where these drugs bind?
specific roles of ABCG2? This question remains important The short answer: surprisingly little! Initial studies aimed to
to answer, particularly considering the impact of ABCG2 understand ABCG2 pharmacology, and included equilibrium
function on multiple drug pharmacokinetics and the potential and kinetic radioligand binding assays to study the interaction
for ABCG2 polymorphisms to have differential effects on of radiolabelled daunomycin with ABCG2 expressed in insect
transporter function (see below). (Sf9) cell membranes [22]. This study was performed with an
R482G mutant version of ABCG2 due to its broader substrate
range (see below). The data demonstrated an affinity (K d ) of
ABCG2 for daunomycin of approximately 100 nM, which
ABCG2 transport substrate diversity and is still the only quantitative demonstration of an affinity
pharmacology: all mapped out? of ABCG2 for a transport substrate. Relative potencies for
As discussed above, the exact physiological roles of ABCG2 other drugs (mitoxantrone, Hoechst 33342, doxorubicin and
remain somewhat enigmatic. The distribution of the trans- prazosin) to displace daunomycin binding were obtained
porter within tissues that have a predominantly secretory and led to a picture of multiple drug binding sites showing
or barrier function leads to ABCG2 being recognised for a complex network of allosteric communications [22].
a role in controlling the disposition and tissue exposure Changes in transport substrate affinity were demonstrated
of endobiotics and xenobiotics, confirmed in numerous in subsequent studies upon the binding, rather than the
in vitro and in vivo studies. These include antibiotics, hydrolysis of ATP [23], but other direct, quantitative
sterols, immune-suppressants (including anti-HIV drugs), data for ABCG2:transport substrate interaction remain
fluorescent dyes (e.g. Hoechst 33342), photosensitizers scarce.
(pheophorbide A and protoporphyrin IX). The increased In the absence of such data a great deal of time and
expression of ABCG2 has been linked to MDR in cancer effort has been put into in vitro site-directed mutagenesis
(see below) and there have also been numerous studies studies of ABCG2. Arginine 482 to glycine/threonine (TM3;
describing ABCG2 mediated transport of chemotherapeutic Figure 1) is a classic example of a mutation which impacts
drugs including mitoxantrone, methotrexate, topotecans, substrate binding and/or transport resulting in the ability
c 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence
4.0 (CC BY-NC-ND).
