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

Harlan Linver Pietz

OAI: oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1733

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.

Life Sciences