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.
","abstract_html":"<p>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.</p>","abstract_has_math":false,"creators":["Pietz, Harlan Linver"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Thesis","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Jue Chen"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-01-01T08:00:00Z","date_published":"2023-01-01T08:00:00Z","updated_at":"2026-07-24T04:11:45Z","subjects":["neuroblastoma","MRP1","glutathione","ABC transporters","chemotherapeutic resistance","structural biology","Life Sciences"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/729","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Jue Chen"]},{"key":"dc:creator","label":"Author","values":["Pietz, Harlan Linver"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["neuroblastoma","MRP1","glutathione","ABC transporters","chemotherapeutic resistance","structural biology","Life Sciences"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/729"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Structural and Functional Analysis of Substrate Recognition and Inhibition of the Multidrug Transporters MRP1 and MRP2"]}]}],"canonical_facts":{"dc:contributor":["Jue Chen"],"dc:creator":["Pietz, Harlan Linver"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/729"],"dc:subject":["neuroblastoma","MRP1","glutathione","ABC transporters","chemotherapeutic resistance","structural biology","Life Sciences"],"dc:title":["Structural and Functional Analysis of Substrate Recognition and Inhibition of the Multidrug Transporters MRP1 and MRP2"],"thesis:degree_level":["Thesis"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T04:11:45Z"}