Abstract 5217: Mechanism of polyspecificity of P-glycoprotein: Substitution of fifteen residues in the drug-binding pocket with tyrosine reveals a negative correlation between substrate size and transport
Authors: Vahedi Shahrooz , Chufan Eduardo , Ambudkar Suresh V. 
Published: July 2017

P-glycoprotein (P-gp) belongs to the superfamily of ATP-binding cassette (ABC) transporters. This energy-dependent cell surface transporter can pump various chemically dissimilar amphipathic or hydrophobic compounds out of cells. In cancer cells, the expression of P-gp is often linked to the development of multidrug resistance (MDR). Several studies have demonstrated that P-gp can recognize and transport various chemotherapeutic drugs and limit their bioavailability and effectiveness. The drug-substrate binding sites, translocation mechanism, and the signal transduction between drug-binding sites and ATP-hydrolysis at nucleotide-binding domains of this clinically important transporter are not yet well defined. Utilizing molecular modeling and mutagenesis, we previously identified key amino acid residues that interact with the most potent inhibitors of P-gp (zosuquidar, tariquidar, and elacridar). We found that replacing key interacting amino acids (Y307, Y953, and Q725) with alanine in the drug-binding pocket switches inhibition by these modulators of ATPase activity to stimulation. The data demonstrated that hydrogen bond interactions are critical for inhibition of ATP hydrolysis of P-gp by modulators. As tyrosine and glutamine residues can function as both hydrogen bond donors and acceptors, we hypothesized that increasing the potential for hydrogen bond interactions by introducing more tyrosine residues into the drug-binding pocket would increase the affinity of substrates for P-gp. To investigate this, we first identified residues in the transmembrane domains that interact with a number of substrates by employing a molecular modeling technique. Then we substituted fifteen conserved residues (11 Phe, 2 Leu, 2 Ilu and 1 Met) with tyrosine, creating a mutant we named 15Y. Compared to WT, for the majority of tested substrates, we observed normal transport of thirteen fluorescent substrates by the 15Y P-gp mutant demonstrating that fifteen mutations in the transmembrane domains is well tolerated in P-gp. Additionally, these changes have no effect on the cell surface expression or total level of the mutant protein and normal conformational changes occur during transport, further confirming flexible nature of transmembrane domains of P-gp. Interestingly, transport of three large-size substrates was significantly decreased. Physiochemical characterization of the substrates revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant, possibly due to increased hydrogen bond interactions. These data also demonstrate that hydrogen bond interactions are substrate- or modulator-specific.

Citation Format: Shahrooz Vahedi, Eduardo Chufan, Suresh V. Ambudkar. Mechanism of polyspecificity of P-glycoprotein: Substitution of fifteen residues in the drug-binding pocket with tyrosine reveals a negative correlation between substrate size and transport [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2017-5217