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Abstract
Multidrug resistance protein 1 (MRP1) can efflux a wide variety of molecules including toxic chemicals, drugs, and their derivatives out of cells. Substrates of MRP1 include anti-cancer agents, antibiotics, anti-virals, anti-human immunodeficiency virus (HIV), and many other drugs. To identify novel substrates and modulators of MRP1 by exploiting intramolecular fluorescence resonance energy transfer (FRET), we genetically engineered six different two-color MRP1 proteins by changing green fluorescent protein (GFP) insertion sites, while keeping the red fluorescent protein (RFP) at the C-terminal of MRP1. Four of six recombinant proteins showed normal expression, localization, and transport activity. We quantified intramolecular FRET using ensemble fluorescence spectroscopy in response to binding of known substrate or ATP alone, substrate/ATP, and trapping of the transporter in closed conformation by vanadate. Recombinant MRP1 proteins GR-881, GR-888, and GR-905 exhibited reproducible and higher FRET changes under all tested conditions and are very promising for use as MRP1 biosensors. Furthermore, we used GR-881 to screen 40 novel anti-cancer drugs and identified 10 hits that potentially directly interact with MRP1 and could be substrates or modulators. Profiling of drug libraries for interaction with MRP1 can provide very useful information to improve the efficacy and reduce the toxicity of various therapies.
Highlights
ATP-binding cassette (ABC) membrane proteins are a large superfamily of proteins consisting of seven subfamilies (A to G), which mediate the ATP-dependent transport of diverse solutes including lipids, peptides, heavy metals, ions, and a wide variety of endogenous and exogenous compounds and their metabolites across biological membranes [1,2,3,4,5,6,7]
To test if altering the position of the green fluorescent protein (GFP) insertion resulted in enhanced fluorescence resonance energy transfer (FRET) sensitivity, we generated six additional two-color Multidrug resistance protein 1 (MRP1) constructs by inserting the GFP tag at different sites within the MRP1 coding sequence, while keeping the red fluorescent protein (RFP) at the end of MRP1 (Figure 1B)
Our results indicate that the GR-881 MRP1 biosensor is a very powerful tool for identifying drugs and compounds that interact with MRP1
Summary
ATP-binding cassette (ABC) membrane proteins are a large superfamily of proteins consisting of seven subfamilies (A to G), which mediate the ATP-dependent transport of diverse solutes including lipids, peptides, heavy metals, ions, and a wide variety of endogenous and exogenous compounds and their metabolites across biological membranes [1,2,3,4,5,6,7]. ABC proteins such as P-glycoprotein (P-gp/ABCB1), multidrug drug resistance protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are implicated in poor patient response to chemotherapy. These ABC transporters function in an ATP-dependent manner and efflux their substrate drugs out of cells, negatively impacting the efficacy of those drugs. The prototypical functional ABC transporter is composed of four domains, two membrane spanning domains (MSDs), each containing six transmembrane (TM) α-helices, and two nucleotide-binding domains (NBDs) that are cytosolic [12,13,14]. The energy required to translocate the substrates is generated by the binding and hydrolysis of ATP [19,20,21]
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