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.2013 Sep;84(3):361-71.
doi: 10.1124/mol.113.086967. Epub 2013 Jun 17.

Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity

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Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity

Yu Fukuda et al. Mol Pharmacol.2013 Sep.

Abstract

Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV-associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside-based antiretroviral medications and cancer chemotherapeutics.

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Figures

Fig. 1.
Fig. 1.
Nelfinavir and ritonavir modulate MRP4 ATPase activity. (A) The beryllium fluoride (BeFx)–sensitive ATPase activity of ABCC4/MRP4 was determined using the Pi release assay in the presence of various concentrations of NFV, ritonavir (RTV), amprenavir (APV), saquinavir (SQV), or indinavir (IDV). PGE2, a known MRP4 substrate (Reid et al., 2003) that stimulates ATPase activity (Sauna et al., 2004), was used as a positive control. (B) The effect of indicated compounds on PGE2-stimulated ATPase activity (left) and quercetin (QCE)-stimulated ATPase activity (right) was evaluated.
Fig. 2.
Fig. 2.
Among common HIV PIs, only NFV is an inhibitor. (A) Immunoblot analysis of MRP4, P-glycoprotein (P-gp), or ABCG2 expression in either HEK293 or Saos2 cells programmed with either empty vector of an MRP4 expression vector. (B) Bis(POM)-PMEA uptake by Saos2 or HEK293 cells containing either empty vector or an MRP4 expression vector was determined in the presence of the indicated PIs.
Fig. 3.
Fig. 3.
NFV is a substrate and inhibitor of MRP4. (A) Bis(POM)-PMEA uptake was determined in Saos2 or HEK293 cells containing either empty vector or an MRP4 expression vector in the presence of various NFV concentrations. (B) NFV strongly increased Bis(POM)-PMEA cytotoxicity in MRP4-expressing cells (left). NFV uptake was strongly reduced in MRP4-expressing cells (middle). MRP4 expression reduced NFV cytotoxicity (right). (C) The presence of 50μM NFV (right) increased MTX cytotoxicity in MRP4-expressing Saos2 cells compared with MTX alone (left).
Fig. 4.
Fig. 4.
Mrp4/Abcc4 absence reveals nelfinavir is a substrate and inhibitor. (A) Immunoblot analysis of three independent clones for each genotype revealed no upregulation of Abcc1, Abcc5, and Abcg2 in Mrp4 KO MEFS (left). MEFs expressed Mrp4 at levels comparable with normal tissues containing functional Mrp4 (middle). Bcrp/Abcg2 was only minimally functional in Mrp4 KO MEFs (right). (B) NFV blocks Mrp4/Abcc4-mediated export of PMEA. KO and WT MEFs were incubated with Bis(POM)-PMEA under energy-depleted conditions. Subsequently, energy-containing media were restored and export of PMEA was determined (left). The proportion of PMEA in the media and cells was determined and expressed as the percentage of the of total 120 minutes after PMEA export was initiated (right). (C) Mrp4 KO MEFs were more sensitive to the cytotoxic effects of Bis(POM)-PMEA (left) and NFV (middle) but not to etoposide (right). Etop, etoposide; FTC, fumitremorgin C.
Fig. 5.
Fig. 5.
A pharmacophore model of MRP4 reveals distinct substrate properties. (A) PI MRP4 substrate common features pharmacophore showing nelfinavir mapped. Features include hydrogen bond acceptors (green) with vectors, hydrogen bond donor (purple), and vectors and hydrophobic features (cyan). (B) Mapping PGE2 (left) and quercetin (middle) onto the common pharmacophore with NFV shown in yellow. (Right) NFV (yellow), PGE2 (red), and quercetin (gray) are mapped to show that quercetin does not overlap with other two compounds.
Fig. 6.
Fig. 6.
Schematic showing two potential drug binding pockets in MRP4 and possible drug–drug interactions. (Left) Quercetin (QCE) and PGE2 have distinct binding sites. (Right) PMEA, MTX, and NFV compete for the same binding pocket as PGE2. The black arrow in the left panel shows the direction of transport. NBD, nucleotide binding domain.
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