Structural and Functional Analysis of Substrate Recognition and Inhibition of the Multidrug Transporters MRP1 and MRP2

Author

Harlan Linver Pietz

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Chen Laboratory

Abstract

Neuroblastoma is the most common extracranial tumor of young children, and the five-year overall survival of children with high-risk disease is less than 50%. Overexpression of multidrug resistance protein 1 (MRP1), a plasma membrane ATP-binding cassette (ABC) transporter, is associated with high-risk neuroblastoma. While it is known that MRP1 transports multiple neuroblastoma chemotherapeutic agents out of the cell in a glutathione (GSH)-dependent manner, the mechanism of GSH-dependent MRP1 substrate transport has previously not been understood. Additionally, over the past three decades, multiple small-molecule inhibitors of MRP1 transport have also been identified, with the activity of several inhibitors also determined to be GSH-dependent. The mechanism of GSH-dependent MRP1 inhibition, however, has also previously not been understood. This thesis consists of two parts. In part one, which covers GSH-dependent MRP1 substrate transport and inhibition (Chapter 2), I determine the mechanism of GSH-dependent MRP1 substrate transport by solving the structure of MRP1 bound to GSH and the chemotherapeutic vincristine. The cysteine thiol of GSH, which is bound in the relatively polar P-pocket of the MRP1 substrate-binding site, forms a key intermolecular interaction with vincristine, which is bound in the relatively hydrophobic H-pocket. I also determine the mechanism of GSH-dependent MRP1 inhibition by solving the structure of MRP1 bound to GSH and the small molecule Reversan, a lead compound with therapeutic potential. As with vincristine, the cysteine thiol of GSH forms a key intermolecular interaction with Reversan across the substrate-binding site, and Reversan competes directly with vincristine binding in the H-pocket. In part two, I investigate the mechanism and selectivity of a novel MRP1 cyclic peptide inhibitor, CPI1, developed with a collaborator (Chapter 3). The cross-inhibition of other ABC transporters beyond the intended target – MRP1 – may alter the pharmacokinetics of clinically-relevant substrates of other ABC transporters, thus resulting in toxicity. CPI1 binding arrests MRP1 in a conformation incompatible with substrate transport or ATP hydrolysis, but also inhibits the activity of MRP2 (Chapter 4). To begin to understand the mechanism of CPI1 cross-inhibition of MRP2, I solve the structure of MRP2 in a ligand-free state. Unlike previously resolved structures of MRP1, the ligand-free structure of MRP2 features a segment of its own cytoplasmic linker sequence bound in the substrate-binding site. This linker sequence likely functions as an “affinity gate” for MRP2 substrate transport.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Pietz, Harlan Linver, "Structural and Functional Analysis of Substrate Recognition and Inhibition of the Multidrug Transporters MRP1 and MRP2" (2023). Student Theses and Dissertations. 729.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/729