Abstract LB-224: Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping
Authors: Shukla Suneet , Chufan Eduardo E. , Singh Satyakam , Skoumbourdis Amanda P. , Kapoor Khyati , Boxer Matthew B. , Duveau Damien Y. , Thomas Craig J. , Talele Tanaji T. , Ambudkar Suresh V. 
Published: May 2015

Nilotinib (Tasigna®) is a novel tyrosine kinase inhibitor (TKI) that has been used to treat chronic phase chronic myeloid leukemia patients who develop resistance due to the emergence of point mutations within the BCR-ABL kinase domain. It is known to be transported by the ATP binding-cassette (ABC) drug efflux transporters P-glycoprotein (P-gp) and ABCG2, which not only alters the efficacy of nilotinib but might also result in lower bioavailability. Identification of a nilotinib binding conformation in the drug-binding pocket of these transporters is important to understand its interactions and can be used to design the next generation of TKIs that do not interact with ABC drug transporters. A structure-activity relationship (SAR)-based approach for nilotinib was used in this study to identify an initial pharmacophore interacting with P-gp. [125I]-Iodoarylazidoprazosin (IAAP) photolabeling and FACS-based transport of fluorescent substrates were carried out with 26 structural derivatives of nilotinib to evaluate their potency for inhibiting the binding of substrate and the transport function of P-gp. IC50 values from these assays were used for 3D pharmacophore modeling and quantitative structure-activity relationships (QSARs) to propose the spatial arrangement of chemical features that are essential for inhibitory activity of nilotinib towards P-gp. Molecular docking of nilotinib in a homology model of human P-gp was performed to identify interactions with the drug-binding pocket. This docked model was evaluated by mutational mapping studies replacing the Y307, M949 and A985 residues with Cysteine in the primary-binding site, which resulted in the loss of nilotinib's activity to inhibit substrate binding to P-gp. The orientation of nilotinib in this binding pocket was further substantiated by assessing the interaction of nilotinib's structural derivatives with P-gp using photoaffinity labeling, ATP hydrolysis and transport assays. These results demonstrate that the pyridine and pyrimidine rings in nilotinib play a key role in its interaction with the drug-binding pocket, while orientation of the imidazole ring determines the affinity of nilotinib for P-gp. These results for the first time identify the primary binding site of nilotinib in the drug-binding pocket of P-gp and can be exploited to design novel tyrosine kinase inhibitors that would not be recognized by ABC drug transporters.

Citation Format: Suneet Shukla, Eduardo E. Chufan, Satyakam Singh, Amanda P. Skoumbourdis, Khyati Kapoor, Matthew B. Boxer, Damien Y. Duveau, Craig J. Thomas, Tanaji T. Talele, Suresh V. Ambudkar. Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-224. doi:10.1158/1538-7445.AM2014-LB-224