Structural basis of small-molecule inhibition of human multidrug transporter ABCG2

Abstract

ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.

Cryo-EM sample preparation method for extremely low concentration liposomes

“…One method is to use lipid nanodiscs, where the membrane protein resides in a small patch of lipid bilayer encircled by an amphipathic scaffolding protein (Bayburt et al, 2002). The nanodisc method has been employed to study the anthrax toxin pore at 22-Å resolution (Katayama et al, 2010), TRPV1 ion channel in complex with ligands at 3-4 Å resolution (Gao et al, 2016), and other membrane proteins (Autzen et al, 2018;Bayburt et al, 2002;Chen et al, 2017;Dang et al, 2017;Gao et al, 2016;Jackson et al, 2018;Katayama et al, 2010;McGoldrick et al, 2018;Roh et al, 2018;Srivastava et al, 2018;Taylor et al, 2017). Another method, called "random spherically constrained" (RSC) single-particle cryo-EM, where the membrane protein is reconstituted into liposomes, was developed and employed to study the large conductance voltage-and calcium-activated potassium (BK) channels reconstituted into liposomes at 17-Å resolution (Wang and Sigworth, 2009).…”

Epigenetic Regulation of Multidrug Resistance Protein 1 and Breast Cancer Resistance Protein Transporters by Histone Deacetylase Inhibition

“…BCRP is a 72kDa half-transporter that is 655 amino acids in length. It has one N-terminal NBD and one C-terminal six-segment TMD (Allikmets et al, 1998;Taylor et al, 2017;Jackson et al, 2018). The half-transporter forms a homodimer through disulfide bond formation, an event required for efflux function (Henriksen et al, 2005;Wakabayashi et al, 2006;Khunweeraphong et al, 2017).…”

The in vitro toxicity of nitrile and epithionitrile derivatives of glucosinolates from rutabaga in human and bovine liver cells

“…As high levels of ABCG2 is 9 closely correlated with multi-drug resistance in cancer cells, numerous studies have 10 investigated the structural requirements for inhibition of this protein. The protein structure of 11 the ABCG2-FTC complex has recently been published (19). This shows that FTC binds into 12 the active site (competitive inhibition) and prevents conformational changes required for the 13 transportation activity (19).…”

“…The protein structure of 11 the ABCG2-FTC complex has recently been published (19). This shows that FTC binds into 12 the active site (competitive inhibition) and prevents conformational changes required for the 13 transportation activity (19). It is presumed that this is the primary mode of inhibition due to 14 the fact that the most potent ABCG2 inhibitors contain several key structural similarities 15 which resemble the FTC molecule (20).…”

“…18 the best model. A Basic Local Alignment Search Tool (BLAST) of the Uniprot database19 found the human ABCG2 gene to be 84.5% identical to bABCG2, whereas of the other 20 experimental options, mouse was 79.9% and rat was 79.6% identical. The important 21 functional domains of ABCG2 are the nucleotide binding domain, and the integral 22 transmembrane domain(16).…”