Membrane Assays to Characterize Interaction of Drugs with ABCB1

Abstract

ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions.

Crown ethers reverse P-glycoprotein-mediated multidrug resistance in cancer cells

“…The nature of interaction between compound and transporter can be assessed by an in vitro P-gp-ATPase assay. This assay measures two different modes: ATPase activation and ATPase inhibition 27 . Both DAC-2Amide and -3Amide inhibited ATPase activity in a concentration dependent manner (Fig.…”

“…However, both substrates and inhibitors can activate ATPase. For example, verapamil, in addition to being an inhibitor of P-gp, stimulates P-gp ATPase activity 27 .…”

“…Specifically, the increase of ATPase activity was observed at lower compound concentrations, and a decrease at higher concentrations. In addition, the majority of compounds classified as inhibitors (except cyclosporine A) do activate P-gp ATPase 27 , 50 . We observed a similar trend upon treatment with effective crown ether compounds.…”

Respuesta transcripcional de Populus a bifenilos policlorados: Aspectos aplicados a la fitorremediación

“…Esta última tiene relación con el mecanismo de transporte para la exclusión de xenobióticos y de sus metabolitos (Fekete et al, 2015). Lo cual sugiere que este transporte es parte integral del mecanismo de detoxificación de plantas.…”

“…Las plantas no tienen sistema eficiente para excretar compuestos para el medio ambiente, tienen que almacenar los compuestos potencialmente tóxicos en vacuolas que tienen una actividad metabólica muy limitada. Además existen numerosas investigaciones que revelan que las MDR no sólo están implicados en la desintoxicación, sino también en la regulación celular de almacenaje (Gaedeke et al, 2001;Fekete et al, 2015;Cummins et al, 2015). Se ha encontrado que AT3G59140 (AtMRP14; MRP14) está inducido más de 2,5 veces (fold change) en respuesta a Benzoxazolinona en Arabidopsis (Baerson et al, 2005).…”