Analysis of ABCG2 pharmacology and oligomerisation by solution based fluorescence correlation spectroscopy

ABCG2 is a human ATP-binding cassette (ABC) transporter. It has a broad substrate range that includes a number of small molecule drugs as well as endogenous metabolites. With its expression in barrier tissues (e.g. kidney epithelium) ABCG2 has a strong influence on pharmacokinetics in addition to other physiological roles (e.g. urate excretion). ABCG2 is also expressed in several tumours and confers resistance to various chemotherapy agents. The detailed interactions and mechanisms that support such polyspecificity are not well-established and progress in understanding ABCG2 pharmacology has been limited. The aim of this project was to develop novel, solution-based analysis methods to investigate ABCG2 pharmacology. Styrene-maleic acid copolymer lipid particles (SMALPs) were used to isolate ABCG2 in nanodiscs from a mammalian expression system (HEK293T). SMALP-ABCG2 particles were then characterised by fluorescence correlation spectroscopy (FCS) to investigate the stoichiometry of ABCG2 complexes and the pharmacology of their substrate binding. ABCG2 tagged with GFP or SNAP-tag was expressed in HEK293T cells. The appropriate plasma membrane localisation was demonstrated by confocal microscopy and the activity of the fusion proteins were validated using mitoxantrone efflux assays. ABCG2 fusion proteins were extracted from HEK293T cell membrane preparations using SMALPs. This process yielded solubilised ABCG2 that was analysed by dynamic light scattering and FCS. These analysis confirmed ABCG2 was extracted into single SMALP particles of 10-20nm diameter with <5% aggregation. Photon counting histogram analysis was performed on FCS data gathered from SMALP-ABCG2 as well as monomeric (CD86) and dimeric (CD28) control proteins also extracted in SMALPs. The brightness of ABCG2 (51000cpms-1) was compared to that of the monomeric control (27000cpms-1) and the dimeric control (60000cpms-1). This revealed that ABCG2 was extracted as a dimer; the minimum functional unit. Subsequent extraction of ABCG2 from larger scale HEK293T membrane preparations facilitated purification of SMALP-ABCG2 by immobilised metal ion affinity chromatography at concentrations of up to 2μM. Binding of a fluorescent drug substrate of ABCG2 (BODIPY-FL-prazosin) to SMALP-ABCG2 was also analysed using FCS. In the presence of SMALP-ABCG2 (initially 20-30nM) both fast-moving free drug (700μm2s-1) and a much slower protein-bound drug (37μm2s-1) species could be quantified. This was extended into assessing substrate and inhibitor interactions where FCS was used in competition binding assays. Inhibition of BODIPY-FL-prazosin binding by unlabelled prazosin was observed, but not by the known transport inhibitor Ko143. Overall, the techniques developed in this project represent an improved workflow for the purification and assessment of protein complex stoichiometry. This extends to a novel platform for investigating substrate and inhibitor binding and recognition mechanisms

Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2

© 2020 The Authors ABCG2 is one of a trio of human ATP binding cassette transporters that have the ability to bind and transport a diverse array of chemical substrates out of cells. This so-called “multidrug” transport has numerous physiological consequences including effects on how drugs are absorbed into and eliminated from the body. Understanding how ABCG2 is able to interact with multiple drug substrates remains an important goal in transporter biology. Most drugs are believed to interact with ABCG2 through the hydrophobic lipid bilayer and experimental systems for ABCG2 study need to incorporate this. We have exploited styrene maleic acid to solubilise ABCG2 from HEK293T cells overexpressing the transporter, and confirmed by dynamic light scattering and fluorescence correlation spectroscopy (FCS) that this results in the extraction of SMA lipid copolymer (SMALP) particles that are uniform in size and contain a dimer of ABCG2, which is the predominant physiological state. FCS was further employed to measure the diffusion of a fluorescent ABCG2 substrate (BODIPY-prazosin) in the presence and absence of SMALP particles of purified ABCG2. Autocorrelation analysis of FCS traces enabled the mathematical separation of free BODIPY-prazosin from drug bound to ABCG2 and allowed us to show that combining SMALP extraction with FCS can be used to study specific drug: transporter interactions

The multidrug transporter ABCG2: still more questions than answers

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

Analysis of ABCG2 pharmacology and oligomerisation by solution based fluorescence correlation spectroscopy

ABCG2 is a human ATP-binding cassette (ABC) transporter. It has a broad substrate range that includes a number of small molecule drugs as well as endogenous metabolites. With its expression in barrier tissues (e.g. kidney epithelium) ABCG2 has a strong influence on pharmacokinetics in addition to other physiological roles (e.g. urate excretion). ABCG2 is also expressed in several tumours and confers resistance to various chemotherapy agents. The detailed interactions and mechanisms that support such polyspecificity are not well-established and progress in understanding ABCG2 pharmacology has been limited. The aim of this project was to develop novel, solution-based analysis methods to investigate ABCG2 pharmacology. Styrene-maleic acid copolymer lipid particles (SMALPs) were used to isolate ABCG2 in nanodiscs from a mammalian expression system (HEK293T). SMALP-ABCG2 particles were then characterised by fluorescence correlation spectroscopy (FCS) to investigate the stoichiometry of ABCG2 complexes and the pharmacology of their substrate binding. ABCG2 tagged with GFP or SNAP-tag was expressed in HEK293T cells. The appropriate plasma membrane localisation was demonstrated by confocal microscopy and the activity of the fusion proteins were validated using mitoxantrone efflux assays. ABCG2 fusion proteins were extracted from HEK293T cell membrane preparations using SMALPs. This process yielded solubilised ABCG2 that was analysed by dynamic light scattering and FCS. These analysis confirmed ABCG2 was extracted into single SMALP particles of 10-20nm diameter with <5% aggregation. Photon counting histogram analysis was performed on FCS data gathered from SMALP-ABCG2 as well as monomeric (CD86) and dimeric (CD28) control proteins also extracted in SMALPs. The brightness of ABCG2 (51000cpms-1) was compared to that of the monomeric control (27000cpms-1) and the dimeric control (60000cpms-1). This revealed that ABCG2 was extracted as a dimer; the minimum functional unit. Subsequent extraction of ABCG2 from larger scale HEK293T membrane preparations facilitated purification of SMALP-ABCG2 by immobilised metal ion affinity chromatography at concentrations of up to 2μM. Binding of a fluorescent drug substrate of ABCG2 (BODIPY-FL-prazosin) to SMALP-ABCG2 was also analysed using FCS. In the presence of SMALP-ABCG2 (initially 20-30nM) both fast-moving free drug (700μm2s-1) and a much slower protein-bound drug (37μm2s-1) species could be quantified. This was extended into assessing substrate and inhibitor interactions where FCS was used in competition binding assays. Inhibition of BODIPY-FL-prazosin binding by unlabelled prazosin was observed, but not by the known transport inhibitor Ko143. Overall, the techniques developed in this project represent an improved workflow for the purification and assessment of protein complex stoichiometry. This extends to a novel platform for investigating substrate and inhibitor binding and recognition mechanisms