Structure of ABCB1/P-glycoprotein bound to the CFTR potentiator ivacaftor

Barbieri, Alessandro; Thonghin, Nopnithi; Shafi, Talha; Prince, Stephen M.; Rf, Collins; Ford, Robert C.

doi:10.1101/2021.06.11.448073

Cited by 3 publications

(1 citation statement)

References 54 publications

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“…More recently, an ivacaftor binding site in the neighborhood of the CFTR intracellular loop 4 (ICL4) (between NBD1 and MSD2) has been proposed [28], but further investigations are required to determine whether this site is present in other ABC transporters. Another ivacaftor binding site was identified in the substrate-binding site of ABCB1 in the large central cavity that communicates with the cytoplasm and the lipid bilayer [29]. This site appears to be absent in CFTR, favoring the hypothesis that ivacaftor is a substrate of ABCB1 [30].…”

Section: Discussionmentioning

confidence: 99%

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators

Mareux

Lapalus

Saad

et al. 2022

IJMS

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ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin–Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.

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Section: Discussionmentioning

confidence: 99%

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators

Mareux

Lapalus

Saad

et al. 2022

IJMS

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Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases

Delaunay

Elbahnsi

Bruneau

et al. 2023

IJMS

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ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4.

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Drug-Induced Conformational Dynamics of P-Glycoprotein Underlies the Transport of Camptothecin Analogs

Mensah,

Schaefer,

Bartlett

et al. 2023

IJMS

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P-glycoprotein (Pgp) plays a pivotal role in drug bioavailability and multi-drug resistance development. Understanding the protein’s activity and designing effective drugs require insight into the mechanisms underlying Pgp-mediated transport of xenobiotics. In this study, we investigated the drug-induced conformational changes in Pgp and adopted a conformationally-gated model to elucidate the Pgp-mediated transport of camptothecin analogs (CPTs). While Pgp displays a wide range of conformations, we simplified it into three model states: ‘open-inward’, ‘open-outward’, and ‘intermediate’. Utilizing acrylamide quenching of Pgp fluorescence as a tool to examine the protein’s tertiary structure, we observed that topotecan (TPT), SN-38, and irinotecan (IRT) induced distinct conformational shifts in the protein. TPT caused a substantial shift akin to AMPPNP, suggesting ATP-independent ‘open-outward’ conformation. IRT and SN-38 had relatively moderate effects on the conformation of Pgp. Experimental atomic force microscopy (AFM) imaging supports these findings. Further, the rate of ATPase hydrolysis was correlated with ligand-induced Pgp conformational changes. We hypothesize that the separation between the nucleotide-binding domains (NBDs) creates a conformational barrier for substrate transport. Substrates that reduce the conformational barrier, like TPT, are better transported. The affinity for ATP extracted from Pgp-mediated ATP hydrolysis kinetics curves for TPT was about 2-fold and 3-fold higher than SN-38 and IRT, respectively. On the contrary, the dissociation constants (KD) determined by fluorescence quenching for these drugs were not significantly different. Saturation transfer double difference (STDD) NMR of TPT and IRT with Pgp revealed that similar functional groups of the CPTs are accountable for Pgp-CPTs interactions. Efforts aimed at modifying these functional groups, guided by available structure-activity relationship data for CPTs and DNA-Topoisomerase-I complexes, could pave the way for the development of more potent next-generation CPTs.

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Structure of ABCB1/P-glycoprotein bound to the CFTR potentiator ivacaftor

Cited by 3 publications

References 54 publications

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators

Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases

Drug-Induced Conformational Dynamics of P-Glycoprotein Underlies the Transport of Camptothecin Analogs

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