Written by Judit Janossy PhD, Senior Scientific Associate, SOLVO Biotechnology Bile acids are oxidation products of cholesterol, synthesized by hepatocytes, and transported as bile salts by the Bile Salt Export Pump (BSEP, ABCB11), an ABC efflux transporter, to the bile. In humans, inhibition of BSEP by drug molecules leads to clinical cholestasis. As a result, there is an increasing need for reliable, validated screening tools suited to test drug candidates for BSEP interaction potential during the drug development process. Human BSEP protein shares high sequence identity with its rodent orthologs. As a complimentary tool to in vivo mouse models, in vitro expression of the mouse Bsep (mBsep) protein is essential for the analysis of functional interspecies differences and enables validation and prediction of in vivo results. SOLVO has developed a new cholesterol-enriched mouse Bsep membrane (mBsep-HAM). This patented proprietary technology enables the high-throughput screening of the cholestatic potential of drugs. The recently published article by Kis et al. (J Biomol Screen. 2009 Jan;14(1):10-5) describes this novel tool. The paper presents the effect of membrane cholesterol content on the transporter function of mouse Bsep expressed in a baculovirus infected insect cell (Sf9) system. It was found that cholesterol loading enhances the mouse Bsep-mediated transport (Fig. 1A) and substrate-induced ATPase activity of mBsep-Sf9 vesicles (Fig. 1B). Compared to untreated mBsep-Sf9 vesicles cholesterol loading led to increased maximal velocity of the vesicular transport of taurochenodeoxycholate (TCDC), a known BSEP substrate (967±8 versus 525±5 pmol/mg/min) with a slight shift in the KM from 8.6±0.2 to 18.1±0.3 µM (Fig. 1A). The accumulation of the radiolabeled substrate in the inside-out vesicles was ATP, concentration and time dependent. In parallel with TCDC transport, the ATPase activity of the transporter showed TCDC concentration dependence both in mBsep-Sf9 and in mBsep-HAM-Sf9 membranes (Fig. 1B). Cholesterol loading of the insect membrane decreased the basal vanadate sensitive activity and increased the maximal activation with the same EC50 value (16.5 µM) in both systems.
Figure 1. A) ATP-dependent transport of TCDC into mBsep and mBsep-HAM-Sf9 inside-out vesicles, B) Stimulation of the vanadate-sensitive ATPase activity of mBsep-Sf9 and mBsep-HAM-Sf9 by TCDC. The inhibitory effect of BSEP interactors on probe substrate transport was tested both in vesicular transport and ATPase assays using cholesterol-loaded membrane vesicles. A good rank order correlation was found between IC50 values measured in TCDC-stimulated mBsep ATPase assay and in the human BSEP vesicular transport assay utilizing taurocholate (TC) as probe substrate (see Table 1). In summary, the upgraded form of the mouse Bsep-HAM ATPase assay is a user-friendly, sensitive, non-radioactive method for early high throughput screening of drugs with BSEP-related cholestatic potential. It may complement the human BSEP-mediated taurocholate vesicular transport inhibition assay. Table 1: Correlation between TCDC-stimulated ATPase inhibition, TCDC transport inhibition of the mouse Bsep transporter and human BSEP transporter mediated TC transport inhibition data (IC50 values)
|Compound||IC50 values (µM)|