P-Glycoprotein conformational changes detected by antibody competition

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• P-glycoprotein (Pgp) is a key efflux pump involved in multidrug resistance.
• Conformational changes in Pgp during drug transport can be detected using specific monoclonal antibodies (mAbs).

Purpose of the Study:

• To investigate the relationship between Pgp conformational changes and the binding of different mAbs in the presence of drug substrates or modulators.
• To develop an assay that distinguishes between classes of Pgp modulators based on their effect on mAb binding.

Main Methods:

• Utilized UIC2 and MM12.10 mAbs to assess Pgp conformation on Pgp-expressing cells.
• Performed competitive binding assays with various Pgp substrates and modulators (e.g., vinblastine, cyclosporin A, verapamil).

Main Results:

• UIC2 mAb binding increased in the presence of Pgp substrates/modulators.
• Pre-incubation with UIC2 reduced MM12.10 binding, indicating distinct binding sites or conformational states.
• Certain modulators (vinblastine, cyclosporin A, valinomycin) abolished MM12.10 reactivity upon UIC2 pre-incubation, while others (verapamil, Tween-80, nifedipine) did not.
• This assay effectively differentiated two classes of Pgp modulators.

Conclusions:

• mAb competition assays can distinguish Pgp modulator classes based on their effects on Pgp conformation.
• The conformational changes observed during mAb competition are closely related, but not identical, to those detected by the UIC2-shift.
• This method provides a novel approach to characterizing Pgp modulators and understanding Pgp-mediated drug transport.