Analysis of P-Glycoprotein Transport Cycle Reveals a New Way to Identify Efflux Inhibitors

Abstract

P-glycoprotein (P-gp) is found to be of considerable interest for the design of drugs capable of treating chemoresistant tumors. This transporter is an interesting target for which an efficient approach has not yet been developed in terms of computer simulation. In this work, we use a combination of docking, molecular dynamics, and metadynamics to fully explore the states that occur during the capture of a ligand and subsequent efflux by P-gp. The proposed approach allowed us to substantiate a number of experimentally established facts, as well as to develop a new criterion for identifying potential P-gp inhibitors.

Figure 1

P-gp structure (PDB ID: 6Q81, 6C0V).

Figure 2

Three docking areas as exemplified by bis-benzimide.

Figure 3

Considered compounds: high-affinity P-gp substrates (A); third generation P-gp inhibitors (B).

Figure 4

Docking results. Energetics of ligand interactions with different docking areas (A); ligand interactions with P-gp alpha helices, as exemplified by bis-benzimide (B).

Figure 5

Flexibility and distance between P-gp NBDs: yellow, apo form of P-gp (efflux requires the binding of two ATP molecules); green, each NBD is bound by one ATP molecule (corresponds to the active form ready for substrate transfer).

Figure 6

Spatial structure of P-gp: red, XRD data collected in the absence of ATP (PDB ID: 4Q9H); green, dynamic snapshot of homologously extended P-gp in the presence of ATP (ATP not shown); light green, cryo-electron microscopy data collected in the presence of 9 mM Mg²⁺/ATP (PDB ID: 6C0V, ATP not shown). View I, location of NBDs; view II, formation of a cavity between TMDs.

Figure 7

P-gp metadynamics in the absence of a substrate. Amino acids used in the construction of the free-energy surface of the system are indicated: red ○—S430, orange ○—K1208, blue ○—E93.

Figure 8

Energy profiles of P-gp conformational changes depending on the bound nucleotides: dark gray—Apo-P-gp; yellow—P-gp-ATP/ADP; light gray—P-gp-ADP/ADP; green—P-gp-ATP/ATP.

Figure 9

Energetically favorable states of the transporter in the presence of the nucleotides: green—P-gp-ATP/ATP; yellow—P-gp-ATP/ADP.

Figure 10

Alternative transport cycles of P-gp.

Figure 11

P-gp metadynamics in the presence of a substrate.

Figure 12

Energy profiles of P-gp conformational changes depending on the bound nucleotides and compounds: green—P-gp-ATP/ATP; blue—P-gp-ATP/ATP + bis-benzimide; orange—P-gp-ATP/ATP + tariquidar.

Figure 13

Inhibitor binding evenly increases the energy advantage of all intermediate states (A), while substrate binding promotes the transition to the outward-facing state (B).

Figure 14

Energetically favorable states of the P-gp-ATP/ATP complex in the presence of bis-benzimide (blue) and tariquidar (orange). In the case of bis-benzimidine, a channel for the release of the substance is formed, while the release of tariquidar is not possible due to the absence of a channel.