Development of Novel, 384-Well High-Throughput Assay Panels for Human Drug Transporters: Drug Interaction and Safety Assessment in Support of Discovery Research

Tang, Huaping; Shen, Ding Ren; Han, Yong‐Hae; Kong, Yan; Balimane, Praveen; Marino, Anthony M.; Gao, Mian; Wu, Sophie; Xie, Dan; Soars, Matthew G.; O’Connell, Jonathan C.; Rodrigues, A. David; Zhang, Litao; Cvijic, Mary Ellen

doi:10.1177/1087057113494807

Cited by 16 publications

(6 citation statements)

References 28 publications

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“…An elementary consequence of standard enzyme kinetics is that molecules using the same protein may compete with or inhibit each other, in this case each other's transport. This is a simply vast topic, so (notwithstanding earlier critiques of summarizing via the enormous review literature), we here simply point out several useful and recent reviews (from the last 3 years only) that describe in detail the many named and genetically identified transporters that are involved in DDI (Han, 2011; Kido et al, 2011; Klatt et al, 2011; König, 2011; Maeda et al, 2011; Marzolini et al, 2011; Müller and Fromm, 2011; Riches et al, 2011; Shitara, 2011; Zhang et al, 2011b; Bi et al, 2012; Elsby et al, 2012; Feng et al, 2012, 2013, 2014; Fromm, 2012; Grandvuinet et al, 2012; Karlgren et al, 2012; Keogh, 2012; Lepist and Ray, 2012; Nies et al, 2012; Sissung et al, 2012; Sprowl and Sparreboom, 2012, 2014; Takanohashi et al, 2012; Varma et al, 2012; Yeo et al, 2012, 2013; Yoshida et al, 2012, 2013; Kis et al, 2013; König et al, 2013; Maeda and Sugiyama, 2013; Sugiyama and Steffansen, 2013; Tang et al, 2013; Zamek-Gliszczynski et al, 2013; Goswami et al, 2014; Tannenbaum and Sheehan, 2014; Vildhede et al, 2014). We are not aware of any papers that showed such DDI based on any measured competition for transport via the phospholipid bilayer.…”

Section: Some Further Areas Where the Hypothesis Of Dominant Transpormentioning

confidence: 99%

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

Kell¹,

Oliver²

2014

Front. Pharmacol.

146

169

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One approach to experimental science involves creating hypotheses, then testing them by varying one or more independent variables, and assessing the effects of this variation on the processes of interest. We use this strategy to compare the intellectual status and available evidence for two models or views of mechanisms of transmembrane drug transport into intact biological cells. One (BDII) asserts that lipoidal phospholipid Bilayer Diffusion Is Important, while a second (PBIN) proposes that in normal intact cells Phospholipid Bilayer diffusion Is Negligible (i.e., may be neglected quantitatively), because evolution selected against it, and with transmembrane drug transport being effected by genetically encoded proteinaceous carriers or pores, whose “natural” biological roles, and substrates are based in intermediary metabolism. Despite a recent review elsewhere, we can find no evidence able to support BDII as we can find no experiments in intact cells in which phospholipid bilayer diffusion was either varied independently or measured directly (although there are many papers where it was inferred by seeing a covariation of other dependent variables). By contrast, we find an abundance of evidence showing cases in which changes in the activities of named and genetically identified transporters led to measurable changes in the rate or extent of drug uptake. PBIN also has considerable predictive power, and accounts readily for the large differences in drug uptake between tissues, cells and species, in accounting for the metabolite-likeness of marketed drugs, in pharmacogenomics, and in providing a straightforward explanation for the late-stage appearance of toxicity and of lack of efficacy during drug discovery programmes despite macroscopically adequate pharmacokinetics. Consequently, the view that Phospholipid Bilayer diffusion Is Negligible (PBIN) provides a starting hypothesis for assessing cellular drug uptake that is much better supported by the available evidence, and is both more productive and more predictive.

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Section: Some Further Areas Where the Hypothesis Of Dominant Transpormentioning

confidence: 99%

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

Kell¹,

Oliver²

2014

Front. Pharmacol.

146

169

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“…16 Screening assays include the ATPase activity assay and inverted plasma membrane vesicles, which can be conducted in multi-well plate formats. [17][18][19][20] In both assays, the human BCRP gene (ABCG2) is overexpressed in Spodoptera frugiperda (Sf9) cells and the plasma membrane is isolated as fragments for the ATPase assay or inverted into membrane vesicles. Cell-based screening models include cultured cells that overexpress transfected BCRP or endogenously express BCRP.…”

Section: Introductionmentioning

confidence: 99%

In vitro screening of environmental chemicals identifies zearalenone as a novel substrate of the placental BCRP/ABCG2 transporter

et al. 2015

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The BCRP (ABCG2) transporter is responsible for the efflux of chemicals from the placenta to the maternal circulation. Inhibition of BCRP activity could enhance exposure of offspring to environmental chemicals leading to altered reproductive, endocrine, and metabolic development. The purpose of this study was to characterize environmental chemicals as potential substrates and inhibitors of the human placental BCRP transporter. The interaction of BCRP with a panel of environmental chemicals was assessed using the ATPase and inverted plasma membrane vesicle assays as well as a cell-based fluorescent substrate competition assay. Human HEK cells transfected with wild-type BCRP or the Q141K genetic variant, as well as BeWo placental cells that endogenously express BCRP were used to further test inhibitor and substrate interactions. To varying degrees, the eleven chemicals inhibited BCRP activity in activated ATPase membranes and inverted membrane vesicles. Further, genistein, zearalenone, and tributyltin increased the retention of the fluorescent BCRP substrate, Hoechst 33342, between 50–100% in BeWo cells. Additional experiments characterized the mycotoxin and environmental estrogen, zearalenone, as a novel substrate and inhibitor of BCRP in WT-BCRP and BeWo cells. Interestingly, the BCRP genetic variant Q141K exhibited reduced efflux of zearalenone compared to the wild-type protein. Taken together, screening assays and direct quantification experiments identified zearalenone as a novel human BCRP substrate. Additional in vivo studies are needed to directly determine whether placental BCRP prevents fetal exposure to zearalenone.

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“…29 Others reported values of 0.8 for OATP1B1, 0.7 for OATP1B3, and 0.8 for NTCP. 45 The high signal strength provides an excellent dynamic range relative to control, a characteristic that would facilitate the detection of the inhibitory effect of test compounds and calculation of their corresponding IC 50 values.…”

Section: Discussionmentioning

confidence: 99%

Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay

Wang

Wilkerson

et al. 2020

Int J Toxicol

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Sodium taurocholate cotransporting polypeptide (NTCP), which is highly expressed in the sinusoidal membrane of hepatocytes, maintains bile acid homeostasis and participates in the hepatic disposition of a variety of endogenous substances as well as xenobiotics. Manifested by the involvement of organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) in the hepatic uptake of statin drugs, sinusoidal membrane transporters play an important role in the pharmacokinetics and pharmacodynamics of these agents. It has been speculated that NTCP may function as an alternative pathway for statin hepatic uptake, complementary to OATP1B1 and OATP1B3. In the current study, we produced stable NTCP-expressing human embryonic kidney 293 (HEK293) cells and developed a fluorescence-based assay using flow cytometry for measuring NTCP transport with chenodeoxycholyl-(Nε-7-nitrobenz-2-oxa-1,3-diazole)-lysine (CDCA-NBD) as the substrate. NTCP-mediated CDCA-NBD transport was time-dependent and exhibited typical Michaelis–Menten kinetics, with a K m of 6.12 µM. Compounds known to interact with NTCP, including chenodeoxycholic acid and taurocholic acid, displayed concentration-dependent inhibition of NTCP-mediated CDCA-NBD transport. We report here a systematic evaluation of the interaction between statins and the NTCP transporter. Utilizing this system, several statins were either found to inhibit NTCP-dependent transport or act as substrates. We find a good correlation between the reported lipophilicity of statins and their ability to inhibit NTCP. The objective was to develop a higher-throughput system to evaluate potential inhibitors such as the statins. The in vitro assays using CDCA-NBD as fluorescent substrate are convenient, rapid, and have utility in screening drug candidates for potential drug–NTCP interactions.

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Development of Novel, 384-Well High-Throughput Assay Panels for Human Drug Transporters: Drug Interaction and Safety Assessment in Support of Discovery Research

Cited by 16 publications

References 28 publications

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

In vitro screening of environmental chemicals identifies zearalenone as a novel substrate of the placental BCRP/ABCG2 transporter

Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay

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