Characterization of drug translocation pathway in multidrug resistance proteins: P-glycoprotein and ABCG2 using structural approaches

Abstract

P-glycoprotein (ABCB1) and ABCG2 are transmembrane, ATP dependent exporters that transport structurally diverse compounds across the membrane. They are present at the blood tissue barriers and control substrate movement with roles in xenobiotic protections. Additionally, overexpression of P-glycoprotein and ABCG2 in tumour cells is associated with efflux of drugs out of cells, a phenomenon called multidrug resistance. Although both proteins have been studied for decades, the drug translocation pathways still require understanding. Towards this goal, a mixed approach of computational docking and thermostability assays was used. Further, cryo-EM was used to study the interaction of ivacaftor with mouse P-glycoprotein. Our results suggested that ivacaftor bind to mouse P-glycoprotein with a Kd of 1μM. further, docking results suggested the binding of ivacaftor to a novel binding site between TM9, 10 and 11 near the entry portal. This site was also observed in cryo-EM data of mouse P-glycoprotein with ivacaftor. Small-angle X-ray scattering analysis showed aggregation as a confounding factor in the scattering profiles. however, mouse P-glycoprotein showed improved protein properties in the presence of substrates. Docking of lapatinib analogues with ABCG2 showed binding of the kinase inhibitors between the paired and evolutionary conserved residue Phe439. Mutation of this residue in collaborating laboratory to a lipophilic residue reduces the affinity of the molecule for ABCG2. However, mutation to an aromatic residue did not affect the binding which suggests the key role of π-π interaction in ligand-protein interaction. This observation was supported with pharmacophore models where the presence of aromatic moiety was common. Virtual screening of drug libraries showed kinase inhibitors as a major class of interactors for both P-glycoprotein and ABCG2. Ultimately, this project sheds light on the ligand-protein interaction of drugs with P-glycoprotein and ABCG2. Also, we proposed a novel ‘single residue clamp’ model as a mechanism of drug translocation in ABCG2.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Robert Ford (Supervisor) & Stephen Prince (Supervisor)