Structure and Function of Multidrug Transporters

Multidrug resistance is defined as the simultaneous resistance to a variety of structurally and functionally unrelated chemotherapeutic agents. It is present at the time of diagnosis in some tumor types, or occurs as a consequence of chemotherapy in other cancers. As a result of this laboratory`s focus on mechanisms which mediate resistance, we have examined resistance mediated by membrane efflux transporters. The most widely studied mechanism of multidrug resistance is that, which is due to the overexpression of membrane transporters that mediate active outward efflux of antineoplastic agents. We have studied the role of one of these transporters, P-glycoprotein (P- gp, ABCB1) in drug resistance and have identified agents that are able to block the transport and so overcome resistance. An understanding of drug transporter function and availability of a protein structure at atomic resolution represent an important prerequisite for the hypothesis driven design of highly active pump inhibitors and of structurally modified anticancer drugs that are not recognized as pump-substrates any longer . Both the structure and function of this pump and of homologous microbial efflux pumps are still incompletely understood. A combinatorial library of more than 200 compounds, related to propafenone, which is in clinical use as a class 1c antiarrhythmic agent, has been designed and synthesized. These compounds are both, inhibitors and substrates of P-gp and some highly active modulators lack cardiac activity. Some analogues are photoactivatable substrates for P-gp and are used to study the molecular architecture and function of this drug efflux pump. Affinity labeling of P- gp in combination with high resolution mass spectrometry is used to characterize the substrate binding domain of P- gp. Bidentate ligands containing two photoactivatable moieties will be used as molecular rulers in crosslinking studies to approximate distances of protein regions that contribute to the drug binding domain. Site directed mutagenesis will provide a tool to characterize substrate-interacting amino acid residues of the protein and the novel approach of methionine scanning mutagenesis (i.e. replacement of amino-acid residues in transmembrane- segments by methionine) in combination with the design and synthesis of methionine selective ligands will allow to probe for substrate accessibility of defined amino acid positions. This information will be related to the individual steps of the catalytic cycle of substrate (allocrite) transport and is expected to provide constraints for a homology modeling approach using the X-ray structure of the lipidA flippase MsbA of E.coli, which has been published recently.

Multidrug resistance is defined as the simultaneous resistance to a variety of structurally and functionally unrelated chemotherapeutic agents. It is present at the time of diagnosis in some tumor types, or occurs as a consequence of chemotherapy in other cancers. As a result of this laboratory`s focus on mechanisms which mediate resistance, we have examined resistance mediated by membrane efflux transporters. The most widely studied mechanism of multidrug resistance is that, which is due to the overexpression of membrane transporters that mediate active outward efflux of antineoplastic agents. We have studied the role of one of these transporters, P-glycoprotein (P- gp, ABCB1) in drug resistance and have identified agents that are able to block the transport and so overcome resistance. An understanding of drug transporter function and availability of a protein structure at atomic resolution represent an important prerequisite for the hypothesis driven design of highly active pump inhibitors and of structurally modified anticancer drugs that are not recognized as pump-substrates any longer . Both the structure and function of this pump and of homologous microbial efflux pumps are still incompletely understood. A combinatorial library of more than 200 compounds, related to propafenone, which is in clinical use as a class 1c antiarrhythmic agent, has been designed and synthesized. These compounds are both, inhibitors and substrates of P-gp and some highly active modulators lack cardiac activity. Some analogues are photoactivatable substrates for P-gp and are used to study the molecular architecture and function of this drug efflux pump. Affinity labeling of P- gp in combination with high resolution mass spectrometry is used to characterize the substrate binding domain of P- gp. Bidentate ligands containing two photoactivatable moieties will be used as molecular rulers in crosslinking studies to approximate distances of protein regions that contribute to the drug binding domain. Site directed mutagenesis will provide a tool to characterize substrate-interacting amino acid residues of the protein and the novel approach of methionine scanning mutagenesis (i.e. replacement of amino-acid residues in transmembrane- segments by methionine) in combination with the design and synthesis of methionine selective ligands will allow to probe for substrate accessibility of defined amino acid positions. This information will be related to the individual steps of the catalytic cycle of substrate (allocrite) transport and is expected to provide constraints for a homology modeling approach using the X-ray structure of the lipidA flippase MsbA of E.coli, which has been published recently.