Related Experiment Videos
P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish
A Di Pietro1, G Dayan, G Conseil
1Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, Lyon, France. a.dipietro@ibcp.fr
Abstract:
Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.
Insights
Flavonoids show promise in combating cancer multidrug resistance by binding to P-glycoprotein (Pgp) nucleotide-binding domains (NBDs). This bifunctional modulation offers a new strategy to overcome drug resistance in cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that mediates chemotherapy resistance by extruding drugs from cancer cells.
- The broad substrate specificity and complex mechanism of Pgp hinder the development of effective multidrug resistance (MDR) reversal agents.
- Studying full-length Pgp is challenging due to difficulties in obtaining high-resolution structures, limiting mechanistic understanding.
Purpose of the Study:
- To investigate the potential of Pgp nucleotide-binding domains (NBDs) as a model for understanding Pgp function and developing MDR modulators.
- To characterize the binding sites of ATP, drug modulators, and flavonoids on Pgp NBDs.
- To identify novel classes of compounds that can modulate Pgp activity and reverse MDR.
Main Methods:
- Biochemical and biophysical characterization of recombinant Pgp NBDs.
- Assessment of ATP and analogue binding to NBDs.
- Investigation of binding interactions with known MDR modulators (steroids) and flavonoids.
Main Results:
- Recombinant Pgp NBDs bind ATP, analogues, and hydrophobic steroids near the ATP-binding site.
- Flavonoids exhibit high-affinity binding to Pgp NBDs.
- The binding site for flavonoids overlaps with both the ATP-binding site and the steroid-interacting region on the NBDs.
Conclusions:
- Pgp NBDs serve as a valuable model for studying structure-function relationships and identifying Pgp modulators.
- Flavonoids represent a novel class of bifunctional modulators of Pgp, targeting both ATP binding and steroid interaction sites.
- Flavonoids hold significant potential for developing new therapeutic strategies to reverse multidrug resistance in cancer.