Efflux mechanism and pathway of verapamil pumping by human P-glycoprotein

Highlights

• Verapamil pumping by P-glycoprotein studied by molecular dynamics simulations.

• Energetics analysis identifies the efflux mechanism and pathway.

• The pumping pathway of verapamil mainly involves eight amino acid residues.

• Only two residues shared by the pumping pathway of verapamil and doxorubicin.

Abstract

Multidrug resistance (MDR) caused by overexpressed permeability–glycoprotein (P-gp) in cancer cells is the main barrier for the cure of cancers. P-gp can pump many chemotherapeutic drugs, which is a viable target to overcome P-gp-mediated MDR by efficient inhibitors of P-gp. However, limited understanding of the efflux mechanism by human P-gp hinders the development of efficient inhibitors. Herein, the transport of a P-gp inhibitor, verapamil, by human P-gp has been investigated using targeted molecular dynamics simulations and energetics analysis based on our previous research on the transport of a drug (doxorubicin). The energetics analysis identifies that the driving forces for the transport of verapamil are electrostatic repulsions contributed by the positively charged residues in the initial stage and then hydrophobic interactions contributed by the important residues in the later stage. This scenario is generally consistent with that in the transport of doxorubicin. However, the positively charged residues and the important residues for the transport of verapamil are incompletely consistent with the relative residues for the transport of doxorubicin. Moreover, the binding free energy contributions of the positively charged residues for the transport of verapamil are generally higher than them for the transport of doxorubicin, while the important residues constitute significantly different binding free energy compositions in the transports of the two substrates. Consequently, the pathway for the transport of verapamil is identified, which shares only two residues (F336 and M986) with the pathway of doxorubicin. This may imply the weak competitiveness of verapamil with doxorubicin in the substrate efflux. Taken together, this work provided new insights into the efflux mechanisms by human P-gp and would be beneficial in the design of potent P-gp inhibitors.

Introduction

Cancer is the leading cause of death, and the failure of chemotherapy for curing cancer is mainly due to multidrug resistance (MDR) [1]. Although many causes contribute to MDR, overexpressed permeability-glycoprotein (P-gp) in cancer cells is a key factor [2]. As the most characterized MDR transporter, P-gp can pump more than 300 compounds [3] including hundreds of chemotherapeutic drugs [2]. Therefore, P-gp has been taken as a viable target to the treatment of cancer [4], and it is considered as a promising strategy to develop efficient inhibitors of P-gp to overcome P-gp-mediated MDR. However, limited success has been achieved because of the low potency and specificity of these inhibitors [4], mostly due to the substrate promiscuity and unclear atomic details of the transport process.

P-gp is a member of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter proteins, which is composed of two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) [5,6]. The sequences of NBDs of different ABC transporters are homologous and the structures are highly conserved. Different from NBDs, however, the sequence of TMDs is variable among the different ABC transporters, which is beneficial for the binding of extremely diverse substrates. Although the atomic details of the transport process are unclear, the general transport mechanism of P-gp is recognized by different conformations form crystallographic data of P-gp and other ABC exporters [7]. The substrate efflux of P-gp depends on the conformational transition from the inward- to outward-facing. TMDs provide specific substrate binding sites (also named “drug binding pocket”) containing many hydrophobic or aromatic amino acid residues [8], and the pathway for the transport by conformational transition, while NBDs provide the energy needed for the transportation by binding and hydrolyzing ATP [9,10].

Since the first P-gp inhibitor, verapamil, was found [11], development of small molecular P-gp inhibitors or modulators started as a hopeful strategy to overcome the P-gp-mediated MDR. To date, three generations of P-gp inhibitors have been developed [12], but none of them have been approved in clinical trials due mostly to their high toxicities. It is noted that the low potency and specificity of these inhibitors are also the major defects. Therefore, a clear explanation of the transport process by P-gp would be helpful for the effective inhibitor design.

To date, many efforts have been made to explore the molecular details that are essential for understanding the transport process of P-gp. Experimental efforts including mutagenesis studies and pharmacological studies identified the relative contributions of different residues on substrate binding and transportation and multiple substrate binding sites, respectively [7]. Computational methods have also been playing important roles in the investigations because they can reveal the molecular details on an atomic level [13]. To date, most of the computational researches have focused on the characterization of the binding sites. For example, Ferreira et al. [14] performed a docking study of the crystal structure of murine P-gp comprising a database of 68 molecules aiming to describe three putative drug-binding sites; Subramanian et al. [15,16] used umbrella sampling techniques combined with potential of mean force (PMF) to identify the transport-competent minimum free energy binding locations of several compounds in murine P-gp; Zhang et al. [17] studied the mechanism of substrate promiscuity of P-gp by the interactions with paclitaxel and doxorubicin spontaneously approaching the binding site in human P-gp during 100 ns molecular dynamics (MD) simulations; Zhang et al. [18] explored the possible portals and pathways of paclitaxel and doxorubicin to access the binding site in murine P-gp using random accelerated molecular dynamics (RAMD) simulations. Of the MD simulation methods, targeted MD simulations, sometimes combined with umbrella sampling techniques [19], are useful in the investigation of conformational changes of proteins. The targeted MD simulations have been popular in the investigation of the conformational changes of human P-gp from the inward- to outward-facing in the absence [2,20] or presence of different substrates [21,22]. They were also proven capable of presenting the transport pathways of daunorubicin and verapamil and evaluating the important residues [22]. Recently, we reported the transport process of doxorubicin in human P-gp using targeted MD simulations and the driving forces and important residues were identified by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis [23], which explained the transport of daunorubicin by energetics analysis.

The three-dimensional structures of P-gp in different conformations during the transport cycle have been obtained from different organisms. For example, Caenorhabditis elegans P-gp [24], murine P-gp [25], Escherichia coli MsbA [26], and human P-gp (PDB ID: 6QEX) [27] are in the inward-facing conformations, while Staphylococcus aureus vancomycin intermecdiate resistance 1866 transporter (Sav 1866) [28], human P-gp (PDB ID: 6C0V) [3], and Salmonella typhimurium MsbA [26] are in the outward-facing conformations, which show openings for access to the cytoplasm in different sizes. These structures have often been used for homology modeling to obtain different conformations of human P-gp [29], especially murine P-gp [30], which are considered as an ideal template for the homology modeling of human P-gp because of its 87% sequence identity and 100% identity of the residues inside the drug binding pocket to the human P-gp [14].

Although previous studies mentioned above focused on the molecular details for the transport process of P-gp, the exact description of the efflux mechanism in the molecular level was scarcely reported [23], especially the differences of efflux mechanisms of drug and inhibitor. Verapamil is a classic chemosensitizer [31], which could enhance the intracellular accumulation of many anticancer drugs, including doxorubicin in numerous cell lines [32]. Verapamil competitively inhibits the transporting function of P-gp without disrupting ATP hydrolysis [33]. Therefore, in this work, we investigated the transport process of verapamil in human P-gp using the targeted MD simulations based on our previous work on the transport process of doxorubicin [23]. Then the driving forces and the important residues contributing to the transport were identified through binding free energy between P-gp and verapamil calculated by MM-PBSA analysis [34]. The similarities and differences of molecular details between the transports of doxorubicin and verapamil were focused on to reveal the efflux mechanism. The research is expected to advance the research of understanding the efflux mechanism and to contribute to the design of potent inhibitors against P-gp-mediated MDR.