3-(6-isobutyl-9-methoxy-1-4-dioxo-1-2-3-4-6-7-12-12a-octahydropyrazino(1--2--1-6)pyrido(3-4-b)indol-3-yl)propionic-acid-tert-butyl-ester and tryptoquivaline

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.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily G, Member 2; Binding Sites; Cholesterol; Cryoelectron Microscopy; Diketopiperazines; Drug Design; Drug Resistance, Multiple; Drug Screening Assays, Antitumor; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrolysis; Indoles; Kinetics; Lipids; Molecular Structure; Neoplasm Proteins; Phospholipids; Protein Binding; Protein Multimerization; Quinolines; Structure-Activity Relationship; Substrate Specificity

A new class of specific breast cancer resistance protein (BCRP) inhibitors was identified, showing no inhibition of the ATP binding cassette (ABC) transporters P-gp and MRP1. Some of these modulators inhibit BCRP with high potency; they are only slightly less potent than Ko143 and could serve as promising lead structures for the design of novel effective BCRP inhibitors. These inhibitors are structurally related to tariquidar (XR9576) and belong to a library of multidrug-resistance modulators synthesized by our research group. The absence of the tetrahydroisoquinoline substructure appears to play a crucial role for specificity; we found that the presence of this substructure is not essential for interaction with BCRP. To determine the type of interaction between pheophorbide A and compounds with and without the tetrahydroisoquinoline substructure, various substrate pheophorbide A concentrations were used in enzyme kinetics assays. The resulting data show that these compounds share a noncompetitive-type interaction with pheophorbide A. Experiments with imatinib and pheophorbide A revealed a mixed-type interaction. The combination of imatinib and compounds with and without the tetrahydroisoquinoline substructure resulted in a positive cooperative effect, indicating that imatinib engages a binding site distinct from that of the new compounds on one side and distinct from that of pheophorbide A on the other side as well. The results of this study suggest that the category of BCRP-specific inhibitors, which includes only fumitremorgin C, Ko143 and analogues, and novobiocin needs to be extended by this new class of inhibitors, which possess three key characteristics: specificity, potency, and low toxicity.

Topics: Adenosine; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzamides; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Diketopiperazines; Drug Resistance, Multiple; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Imatinib Mesylate; Indoles; Neoplasm Proteins; Novobiocin; Piperazines; Pyrimidines; Quinolines; Structure-Activity Relationship