MRP2 (ABCC2) is a member of the ATP-binding cassette superfamily of transporters, MRP1 and MRP2 are homologous members of this superfamily [1] . In humans, it is encoded by the gene ABCC2 [2] . Structurally, MRP2 is a 190-kDa membrane protein consisting of 1545 amino acids. The predicted membrane topology consists of 17 transmembrane spanning domains (MSD0, MSD1, MSD2) which are linked together by conserved linker regions, and two highly conserved nucleotide-binding domains (NBD1 and NBD2) that serve as substrate–binding sites [3] . Whilst MRP1 localizes at the basolateral membrane of endothelial cells, MRP2 localizes at the apical membrane of polarized cells of hepatocytes, renal proximal tubular cells, enterocytes, and syncytiotrophoblasts of the placenta [4] . It is known to play critical roles in the export of conjugated bile salts in the liver as well as the transport of physiologically important substrates such as glutathione-S-conjugates, 17-beta-glucuronosyl estradiol, leukotriene C4 [5] . Overexpression of MRP2 is associated with multidrug resistance of tumor cells such as hepatocellular, ovarian, colorectal, lung, breast, and gastric carcinomas [6] , where it pumps drug conjugates and drug complexes across the plasma membrane into the extracellular space [5] . Thus, affecting the bioavailability and efficacy of anticancer drugs like cisplatin and methotrexate. Aside from cancer drugs, MRP2 also affects the efficacy of a broad spectrum of drug classes including HIV drugs (lopinavir), antibiotics (ampicillin, azithromycin), and antihypertensives (Olmesartan, Temocaprilate) [7] . With MDR being a major impediment to the chemotherapy regime and the overexpression of the MRP2 transporter being a major factor in this phenomenon, it is of great clinical interest to find ways of addressing this canker. Researchers have proposed two main approaches. One of which, is to completely block the efflux or pump activity of the transporter in these cells [8] . However, this approach would be destructive to the cells since it may also impede some important physiological activities of the transporter, thereby jeopardizing the overall well-being and physiological homeostasis of the cell or tissue [9] - [11] . Another possible approach that was proposed, was to modulate the activity of this transporter using biochemical modulation [9] [12] [13] . Using biochemical modulation, exogenously supplied metabolites can be used to selectively manipulate the activity of MRP2 in tumor cells to ensure the more selective response of cancer cells to the action of administered anticancer agents [14] . This would go a long way to improve the bioavailability and efficacy of anticancer drugs in tumor cells. Hence the identification of possible modulators of the MRP2 transporter is of great clinical importance [8] [13] . Moreover, the broad impact of the efflux activity of this transporter on the efficacy of a broad class of drugs makes it essential to investigate the possible interactions between various therapeutic drugs (both approved and those in clinical trials) and this transporter. Thus, in this study, a unique set of drugs from the FDA-approved drug library was screened using In-Cell ELISA to identify modulators of MRP2 protein expression.

Test compounds that modulated the protein expression levels of MRP2 in MDCKII/MRP2 cells above 50% (Hit compounds) in this study cut across a broad spectrum of drug classes and exhibit great diversity in their structure, molecular targets, and mode of action. This included anticancer drugs, antibiotics, antivirals, and anti-inflammatory drugs. This finding reaffirms the promiscuous nature of the MRP2 transporter, and how important it is to investigate the interaction between both old and newly developed drugs with MRP2. Although several studies have aimed at investigating the impact of various therapeutic agents on MRP2 efflux activity, it is necessary that researchers also pay critical attention to how these drugs may affect the protein expression levels of this transporter. From our screening, about 10.48% of the hit compounds increased the expression of MRP2 protein levels, this included drugs like vincristine (anticancer drug), oxaliplatin (antineoplastic medication), and irinotecan (anticancer drug). MRP2 has been reported to be one of the major ABC transporters that affect the bioavailability and therapeutic potency of anticancer drugs in both polarized and unpolarized cells [9] [15] . The anticancer drugs: Vincristine, Oxaliplatin, and Irinotecan have earlier been reported in other studies as substrates of MRP2 [7] [16] [17] . This indicates they are actively transported by MRP2/ABCC2 transporter thus the increase in MRP2 protein expression as observed in this study provides the possible explanation that more MRP2 is expressed in these cells to catalyze and ensure successful transport or efflux of these drugs across the plasma membrane.

Glucocorticoids like dexamethasone and prednisone also upregulated MRP2 protein expression in our screening. The ability of dexamethasone to increase the expression levels of MRP2 protein as observed from the screening also reaffirms the observation reported by Narang and his colleagues [18] who reported that dexamethasone increased the expression and activity of multidrug resistance transporters at the rat blood-brain barrier. Prednisone, on the other hand, has also been reported to induce the activity of the MRP2 promoter [19] , thus providing a possible reason for the increase in MRP2 protein levels observed in this study. Methylprednisolone, another glucocorticoid with anti-inflammatory and immunomodulating properties, also upregulated MRP2 protein levels in this screening. However, to the best of our knowledge, the interaction between MRP2 and methylprednisolone is yet to be reported, and it would be enlightening for further studies to be carried out to investigate how these drugs may affect MRP2 activity. Erythromycin (an antibiotic) and Lovastatin (a hypolipidemic agent and an HMG-CoA reductase inhibitor) which has been reported by other studies to be substrates of MRP2 [20] [21] also elevated the protein levels of MRP2 in this present study. Hence, this finding provides useful information on the modulatory effect of these drugs that can be further explored.

Furthermore, findings from this study also suggest that anticancer drugs like Pralatrexate, Afatinib, and Cladribine (an immunosuppressant) may decrease MRP2 protein expression levels in MDCKII MRP2-overexpressing cells. Pralatrexate has been reported in earlier studies to act as both a substrate and an inhibitor of MRP2 [22] , interestingly, results from our present study demonstrate that pralatrexate may downregulate the expression levels of MRP2. Further investigation can be conducted to provide more insight into the effect of pralatrexate on gene expression and other effectors that regulate MRP2 protein expression. Moreover, the effect of pralatrexate on MRP2 protein expression levels in other MRP2-overexpressing cell lines can be explored to confirm this initial finding. Afatinib is a known moderate inhibitor of Permeability glycoprotein (P-gp) [23] [24] , and a substrate/inhibitor of Breast Cancer Resistance Protein (BCRP) [24] . Results of this current study show that Afatinib may reduce MRP2 protein levels. A thorough search of current literature revealed that little is known about the impact of Afatinib on MRP2 activity and protein expression. Thus, it would be enlightening to conduct further investigation to confirm and ascertain how Afatinib affects the efflux activity of this transporter in other MRP2 overexpressing cells. Cladribine is an FDA-approved drug used in the treatment of multiple sclerosis and hairy cell leukemia. It is a known substrate of BCRP [25] [26] but proved otherwise on MRP2 when its impact on MRP2 membrane vesicles was explored [27] . Nonetheless, cladribine downregulated the protein expression levels of MRP2 in MDCKII MRP2 overexpressing cells in our present study. Thus, it would be illuminating to investigate the impact of cladribine on other MRP2-overexpressing cells since the specific interactions between Cladribine and MRP2 remain uncertain. Further probing using cell lines overexpressing this transporter would be a step in the right direction. Adrucil (Fluorouracil), a DNA and RNA synthesis inhibitor that irreversibly inhibits thymidylate synthase, and Asenapine, an antipsychotic medication belonging to the dibenzooxepino pyrrole class [28] downregulated the expression levels of MRP2 in our present study. To the best of our knowledge, the impact of Adrucil and Asenapine on MRP2 activity and expression has not been reported in literature.

Although HIV Protease Inhibitors (HPIs) have been reported to be substrates of MRP1 and MRP2, the majority of tested HPIs are transported by MRP2. As such the overexpression of MRP2 has great pharmacological implications on administered HPIs [29] [30] . Darunavir Ethanolate is the ethanolate form of darunavir and an antiretroviral drug that inhibits the human immunodeficiency virus type-1 (HIV-1) protease [31] - [33] . In this present study, Darunavir Ethanolate increased MRP2 protein expression in MDCKII overexpressing MRP2 cells. Although to the best of our knowledge, Darunavir Ethanolate has not been reported as a substrate of MRP2, its ability to increase MRP2 protein levels suggests that this antiretroviral drug may also be a prey of MRP2 efflux activity. Interestingly, Darunavir the parent compound of Darunavir Ethanolate has been reported in other studies to induce P-gp mRNA activity and expression in vitro as well as induce MRP1 protein expression in CD4 (+) T cells from healthy human volunteers [34] [35] . On the Contrary, Adefovir dipivoxil, a diester prodrug of adefovir and an antiviral medication used in the treatment of chronic Hepatitis B infection in adults, also lessened the protein levels of MRP2 in this screening. This is not surprising since adefovir is reported to be a known inhibitor of MRP2 [36] . However, no information has been reported on the interaction between this diester derivative of adefovir and MRP2. Investigating the modulatory effect of Darunavir Ethanolate and Adefovir dipivoxil on MRP2 activity and expression in other MRP2 overexpressing cells would be enlightening.

Our screening also identified other novel drugs whose effect on MRP2 activity or expression levels is yet to be reported or explored to the best of our knowledge. This included glyburide (medication for diabetes), Rasagiline mesylate (medication for Parkinson’s disease), dronedarone HCl (antiarrhythmic drug), and conivaptan HCl (vasopressin antagonist, endocrine-metabolic agent). These novel drugs downregulated the protein expression levels of MRP2 in MDCKII MRP2-overexpressing cells in this study. Other non-reported drugs that upregulated the expression levels of MRP2 in our present study included Streptozotocin, Megestrol acetate, Gestodene, Trilostane, and Ranolazine dihydrochloride among others. This initial data on these novel drugs would provide foreknowledge that can further be explored. Like most proteins, MRP2 can be regulated at the transcriptional and post-transcriptional levels. Studies have revealed that alterations in the intracellular concentrations of bile acids and of some lipophilic compounds that are ligands for nuclear hormone receptors can regulate MRP2/ABCC2 transcription levels [6] . Nuclear hormone receptors for hydrophobic molecules such as steroid hormones (estrogens, glucocorticoids, progesterone, mineralocorticoids, androgens, vitamin D3, ecdysone, oxysterols, and bile acids), retinoic acids (all-trans and 9-cis isoforms), thyroid hormones, fatty acids, leukotrienes and prostaglandins [37] [38] . Research has shown that the hormone response element in rat MRP2/Abcc2 promoter (ER-8) is bound by heterodimers of the retinoid receptor [39] with the ligand-activated transcription factors, pregnane X receptor (PXR), farnesoid X receptor (FXR) or constitutive androstane receptor (CAR). Thus, various xenobiotics that regulate bile acid concentration can activate these receptors which in turn upregulates the promoter region of the ABCC2 transporter [40] - [44] . This finding provides a possible explanation for the increase in MRP2 protein expression observed in this study after MDCKII/MRP2 cells were treated with Megestrol acetate (androgen receptor), Gestodene (estrogen/progestogen receptor), Methylprednisolone and Prednisone (Glucocorticoid receptor), and Evista (estrogen/progestogen receptor). Data from this study provides important information on otherwise potent drugs that can regulate the protein expression of MRP2. The early detection of drugs that upregulate MRP2 expression levels provides vital information on the performance of these drugs which is essential for clinical trials. Drugs that showed the ability to decrease protein expression could be used together with drugs that are known to be efficacious in treating cancer and other diseases but are unfortunately substrates of the MRP2 transporter. As such, in the presence of a drug that decreases protein expression of the MRP2 transporter, the more potent drug would have higher bioavailability to exert its effect thereby enhancing combinatorial drug therapy for the treatment of diseases.

This work was supported by the Competitive Research Grant (Award #3S9322) to Surtaj H. Iram, sponsored by the South Dakota Board of Regents and South Dakota’s Governor’s Office of Economic Development.