Cholesterol on the rescue: HT screening of ABCG2 transporter - drug interactions

June, 2007

Cholesterol on the rescue: improved membrane assay for HT screening of ABCG2 transporter - drug interactions

Written by Judit Janossy MSc, Senior Scientific Associate, Solvo ABCG2, one of the most important efflux transporters at the major pharmacological barriers ABCG2 also known as BCRP or MXR is located on the apical side of the epithelium on several pharmacological barriers (reviewed in Sarkadi et al. 2006). It is a homodimer half-transporter with broad substrate specificity transporting hydrophobic, anionic, as well as cationic drugs (Mao and Unadkat, 2005). It is involved in intestinal absorption (Polli et al., 2004) and in excretion of xenobiotics and metabolites (Ebert et al., 2005), like biliary excretion of sulphate conjugates (Zamek-Gliszczynski et al. 2006). ABCG2 plays an important protective role at the blood-brain (Breedveld et al., 2005) and blood-placental (Jonker et al., 2000) barriers. ABCG2 knock-out models shed light on some special functions of ABCG2, such as secretion of drugs and other xenobiotics into milk (Jonker et al., 2005) as well as protection of stem cells from hypoxia-induced protoporphyrin accumulation and damage (Krishnamurthy and Schuetz, 2005).

Membrane based high throuphput studies

Drug-transporter interactions are commonly screened by high throughput (HT) systems using transfected insect and/or human cell lines. (for reviews see Glavinas et al. 2004; The determination of ABCG2-ATPase activity is a suitable method to identify ABCG2 substrates and inhibitors. The membrane assay system allows for transport studies as the transported substrates accumulate in the inside-out vesicles in an ATP-dependent manner. For most applications the transporter is expressed in insect cell lines (e.g. Sf9) taking advantage of the robust baculovirus - insect cell system. (Ozvegy et al., 2001). Alternatively, membranes are prepared from human cell lines (Han and Zhang, 2004, Glavinas et al. 2007) overexpressing the transporter.

Development of a reliable HT in vitro model that mimic the physiological phenotype

In the recent publication of SOLVO we have shown that the ATPase activities of the ABCG2 transfected Sf9 cell membranes (MXR-Sf9) and ABCG2-overexpressing human cell membranes (MXR-M) differ (Pal et al., 2007). Known substrates showed different ATPase profiles in the two membranes. While the glycosylation level of the protein had no effect on the transporter, cholesterol loading potentiated drug-induced ATPase stimulation of ABCG2 expressing MXR-SF9 membranes. Differences in cholesterol content in the insect and human cell membranes, showed by cholesterol loading and depletion experiments, conferred the difference in stimulation of basal ABCG2-ATPase of the two cell membranes (see Fig. 1). Basal ABCG2-ATPase activity could be stimulated by sulfasalazine, prazosin, and topotecan, known substrates of ABCG2 in cholesterol-loaded MXR-Sf9 and untreated MXR-M cell membranes. In contrast, ABCG2-ATPase could not be stimulated in MXR-Sf9 or in cholesterol-depleted MXR-M membranes (Fig. 1). In cholesterol-loaded Sf9 membranes all substrates, (Fig. 2, A-C ), but Hoechst 33342 (Fig. 2D) stimulated the basal ATPase activity and inhibited the activated ATPase in a concentration-dependent manner. Hoechst 33342, a known substrate of WT human ABCG2, similarly to Ko134 (Fig. 3E), a specific inhibitor of ABCG2 inhibited the basal as well as the stimulated ATPase activity of ABCG2. Moreover, cholesterol loading significantly improved the drug transport into inside-out membrane vesicles prepared from MXR-Sf9 cells (Fig. 3). It increased dramatically the maximal velocity of methotrexate transport without affecting Km (Fig 3A). For prazosin, little ATP-dependent transport was seen in the MXR-Sf9 vesicles whereas significant transport was detected in the cholesterol-loaded MXR-Sf9 vesicles (Fig 3B). MXR-M and cholesterol-loaded MXR-Sf9 cell membranes displayed similar ABCG2-ATPase activity and vesicular transport. The study highlights the importance of a meticulous approach in validation of in vitro system. It also shows that the cholesterol-loaded Sf-9-HAM membrane based assay are an excellent in vitro HT technology to predict drug-transporter interactions. Solvo has filed a patent application to protect this new technology.

Fig. 1 Cholesterol loading of MXR-Sf9 membranes makes their ABCG2-ATPase activity profile similar to the ABCG2-ATPase activity profile of MXR-M membranes.

MXR-Sf9 cells were loaded with cholesterol using cholesterol@RAMEB (1 mM total cholesterol) for 30 min at 37 °C. Cholesterol depletion of MXR-M membranes were carried out at 37 °C, for 30 min using 8 mM RAMEB. The ABCG2-ATPase activation of cholesterol loaded (▲) and control (●) MXR-Sf9 membranes (panel A, C, and D) as well as cholesterol depleted (▲) and control (●) MXR-M membranes (panel B, D and F) upon sulfasalazine (panel A and B), topotecan (panel C and D) and prazosin (panel E and D) treatment (40 min, 37 °C) was determined. Data represent mean S.D.of triplicates.

Figure 2 Effect of activators and inhibitors on basal (▲) and sulfasalazin stimulated (●) ABCG2-ATPase activity. Panel A: Sulfasalazin. Panel B: Prazosin. Panel C: Topotecan.

Panel D: Hoechst 33342 Panel E: Ko143. The ABCG2-ATPase activity measurements were carried out using the PREADEASY ATPase Kit according to the manufacturers suggestions. Data represent mean Âą S.D.of duplicates

Figure 3. Effect of cholesterol on ABCG2 vesicular transport and ABCG2-ATPase correlates in both the MXR-Sf9 and MXR-M membranes.

Estrone-3-sulfate transport was carried out at 32 °C for 1 min and monitored by measuring the amount of membrane associated 3H-estrone-3-sulfate (panel A and B). In estrone-3-sulfate stimulated ABCG2-ATPase activity measurements membranes were incubated for 40 min at 37 °C with estrone-3-sulfate at concentrations indicated in the figure (panel C and D). Cholesterol loaded (●) and control (▲) MXR-Sf9 membranes (panel A and C) and cholesterol depleted (▲) and control (●) MXR-M membranes (panel B and C) were studied. Data represent mean Âą S.D.of duplicates.


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