Paula B Salazar, Megumi Murakami, Nandhini Ranganathan, Stewart R Durell, Suresh V Ambudkar
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引用次数: 0
Abstract
Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.
期刊介绍:
Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution.
Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics.
The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication.
Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).