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Assessing the Biochemical Mechanism of Reversers of Multidrug Resistance in Cancer
Author(s) -
Jensen Madeline Kay,
de Oliveira Maisa Correa,
Okwuone Dakota D. D.,
Wise John G.,
Vogel Pia D.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04411
Subject(s) - abcg2 , multiple drug resistance , atp binding cassette transporter , chemistry , transporter , biochemistry , efflux , in silico , drug resistance , biology , computational biology , gene , genetics , antibiotics
The breast cancer resistance protein (BCRP) is an ABC transporter that uses energy derived from ATP hydrolysis to export toxins from the cell. In cancer cells, these transporters are overexpressed and remove chemotherapeutic drugs from the cell. This confers multidrug resistance. We have previously conducted high‐throughput in silico ligand docking studies to identify potential inhibitors of BCRP. These drug‐like molecules inhibit ATP hydrolysis by targeting the nucleotide binding domain. Biochemical and biophysical analyses of these inhibitors are necessary to study their inhibition mechanisms. Large amounts of purified protein are required to carry out these analyses. Our lab previously generated a gene for human BCRP that was codon optimized for protein expression in the yeast, PichiaPink TM . Here we report our attempts to purify BCRP from this strain as well as from a second, BCRP over‐expressing Pichia Pastoris strain. Growth conditions were previously optimized using methanol to induce BCRP expression. We also report our efforts to purify BCRP into detergent mixed micelles using Ni‐NTA affinity and ion exchange chromatography. Attempts to assemble BCRP containing nativelike membrane nanodiscs to achieve maximum ATP hydrolysis are also reported. Obtaining purified BCRP in a native‐like environment with high activity is crucial for the assessment of BCRP inhibition mechanisms of our newly found drug‐like compounds. Support or Funding Information This work is supported by the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D., of Naples, FL.