pheophorbide-a and tryptoquivaline

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

The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments.. RT-PCR, western blot and fluorescent immunocytochemical analyses were performed on ABCG2-overexpressing human breast cancer (MCF-MX100) cells, human and rat brain microvessel endothelial (HBEC and RBE4, respectively), and rat glial cells.. RT-PCR analysis detected ABCG2 mRNA in all the cell culture systems. Western blot analysis with anti-ABCG2 monoclonal BXP-21 antibody detected a robust band at approximately 72 kDa in the ABCG2-overexpressing MCF-MX100 cell line, whereas low expression was found in human and rat brain cell systems. Immunofluorescence microscopy detected predominant plasma membrane localization of ABCG2 in MCF-MX100 cells but weak signal in all brain cellular compartments. In the presence of ABCG2 inhibitors, the accumulation of (3)H-mitoxantrone and pheophorbide A, two established ABCG2 substrates, was significantly increased in MCF-MX100 cells but not in the human and rodent brain cell culture systems.. Our data show low endogenous ABCG2 protein expression, localization and activity in cultures of human and rat brain microvessel endothelial and glial cells.

Topics: Animals; Animals, Newborn; Astrocytes; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Brain; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Endothelial Cells; Female; Fluorescent Antibody Technique, Indirect; Humans; Indoles; Microcirculation; Microglia; Mitoxantrone; Neoplasm Proteins; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection; Tritium