Quantification and characterization of P-glycoprotein-substrate interactions.

Area of Science:

• Biochemistry
• Membrane Transport
• Drug Discovery

Background:

• P-glycoprotein (P-gp) is a key efflux transporter involved in multidrug resistance.
• Understanding the precise binding interactions of substrates with P-gp within the cell membrane is critical for developing effective therapies.
• Previous studies have debated the exact mechanism and quantification of the lipid-transporter binding step.

Purpose of the Study:

• To quantify and characterize the binding constants of diverse drug substrates to P-glycoprotein from both aqueous and lipid phases.
• To determine the free energy changes associated with drug partitioning into the membrane and subsequent binding to the transporter.
• To investigate the relationship between drug structure, membrane partitioning, and P-gp activation.

Main Methods:

• Selected 15 structurally diverse drugs and measured their lipid-water partition coefficients (K_lw) using surface activity measurements.
• Quantified binding constants from water to P-gp's activating region (K_tw(1)) by measuring half-maximum activation concentrations (K_1) in living cells using a Cytosensor microphysiometer.
• Calculated free energies of partitioning (ΔG°_lw) and binding (ΔG°_tw(1), ΔG°_tl(1)) to elucidate the thermodynamics of drug-transporter interactions.

Main Results:

• Determined drug membrane concentrations at half-maximum P-gp activation (C_b(1)) were significantly higher than aqueous concentrations (K_1).
• Calculated free energies of drug binding from the lipid phase to the transporter (ΔG°_tl(1)) were less negative than lipid partitioning free energies (ΔG°_lw) but showed greater variability.
• Observed that ΔG°_tl(1) values were consistent with a modular binding concept, suggesting specific structural features like hydrogen bond acceptors are key.

Conclusions:

• Drug binding to P-glycoprotein occurs within the lipid membrane phase, with partitioning into the membrane being a significant thermodynamic factor.
• The binding affinity to the transporter in the lipid phase (ΔG°_tl(1)) is highly dependent on drug structure and exhibits significant variability.
• A modular binding model, where specific functional groups like hydrogen bond acceptors contribute most effectively to binding energy, is supported by these findings.