Elsevier

Biochemical Pharmacology

Volume 101, 1 February 2016, Pages 40-53
Biochemical Pharmacology

Drug–protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein

https://doi.org/10.1016/j.bcp.2015.12.007Get rights and content

Abstract

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50’s ranging from 10 to 30 nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp.

Introduction

P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily. It is a single, glycosylated polypeptide of about 170 kDa, expressed at the apical surface of epithelial cells in the intestines, kidney, liver, adrenal gland, blood-brain barrier, and placenta. This transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs [1], [2]. It plays an important role in the pharmacokinetics of many drugs, altering absorption, distribution, metabolism, and excretion. The expression of P-gp at the surface of tumor cells is also a contributing factor in the development of multidrug-resistant cancer. P-gp consists of two homologous halves, each containing six transmembrane helices and a nucleotide-binding domain (NBD). There is consensus among researchers that P-gp alternates between an inward-facing conformation with separated NBDs (inverted V-shape) competent for substrate binding, and an outward-facing conformation with united NBDs (V-shape) consistent with substrate release outside the cell. The X-ray crystal structures of mouse and Caenorhabditis elegans P-gp are representative structures of the inward-facing conformation [3], [4], [5], although the extent of domain separation in physiological conditions is a matter of debate. The X-ray structure of bacterial SAV1866 with bound ADP is representative of the outward-facing conformation [6]. Using this alternating access mechanism, substrate translocation is powered by ATP hydrolysis. Hence, most substrates and modulators stimulate the basal ATPase activity of P-gp [7]. Interestingly, a few drugs (zosuquidar, elacridar and tariquidar) have been reported to inhibit the basal ATP hydrolysis of P-gp. These drugs also happen to be potent inhibitors of P-gp transport [8], [9], [10]. They are third generation modulators of P-gp that inhibit drug transport and ATPase activity at nanomolar concentrations [11]. Further, it has been recently demonstrated that the oral co-administration of paclitaxel and docetaxel (anticancer agents) with elacridar increases plasma levels of the taxanes, thus supporting the therapeutic strategy of co-administration of drugs with a potent inhibitor of P-gp [12].

We found that mutation of polar residues that are capable of establishing hydrogen bond (H-bond) interactions with inhibitors at the drug-binding pocket of P-gp dramatically changes the typical biochemical behavior of P-gp. Drugs that usually inhibit basal ATP hydrolysis switch to stimulation when two tyrosines and one glutamine are mutated (Y307A/Q725A/Y953A). Two phenylalanine residues (F728 and F978) were also found to be essential to the inhibition profile. Molecular modeling studies revealed that the phenylalanine residues orient the aromatic ring of the tyrosine residues (Y310 and Y953) in a manner such that effective H-bond interactions are established between the protein and the drugs. Transport data showed the inhibition of the P-gp function by these drugs depends on their ability to inhibit ATP hydrolysis. When drugs lose the ability to inhibit ATP hydrolysis, they also lose the ability to reverse transport with high affinity [IC50 (cysless WT) = 5–10 nM while IC50 (Y307A/Q725A/Y953A) > 200 nM]. Based on these results, we propose that screening compounds for their ability to inhibit basal ATP hydrolysis with high affinity is a reliable method to identify high affinity modulators of P-gp and possibly of other ABC drug transporters.

Section snippets

Chemicals

The chemical compounds under investigation, zosuquidar, tariquidar and elacridar were purchased from Selleck Chemicals (Houston, TX), MedKoo Biosciences (Chapel Hill, NC), and Sigma–Aldrich Chemical Co. (St. Louis, MO), respectively. Cyclosporine A was obtained from Alexis Corporation (Lausen, Switzerland). The radioactive compound [125I]iodoarylazidoprazosin (IAAP) (2200 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). The fluorescent compounds calcein-AM, bodipy-FL-prazosin

Inhibition switches to stimulation of basal ATPase activity of P-gp when polar residues are mutated

We previously showed that residues Y307 and Q725 form part of the primary drug-binding site of human P-gp for several drugs [21]. Cakil et al. showed that Y953 is part of the drug-binding site for propafenones and rhodamine 123, although this tyrosine seems to not be part of the preferential site for rhodamine 123. They also showed that Y953 interacts with these compounds specifically through hydrogen-bonding [24]. All three residues, Y307, Q725 and Y953 have been recently shown to be part of

Discussion

In P-gp, substrates and modulators bind in the transmembrane region, 50–60 Å from the site where ATP hydrolysis takes place, and the coordination of these two events remains poorly understood at the molecular level. Ligand–protein interactions at the drug-binding pocket of P-gp are expected to consist of van der Waals and hydrogen-bond (H-bond) interactions. The H-bonds are the most electrostatic because no charge residues are present in the drug-binding pocket to form ionic bonds [3], [5].

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgments

We thank Joshua Sitler and Paola Uscanga for technical assistance with ATP hydrolysis measurements, Dr. Stewart Durell for assistance with the molecular modeling studies, and George Leiman for editorial assistance. We acknowledge Drs. Tanaji Talele (St. John’s University, Queens, NY) and John Golin (The Catholic University of America, Washington DC) for critical review of the manuscript. This research was supported by the Intramural Research Program of the National Institutes of Health,

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