Distinct Binding Mode Of Lenvatinib To Vegfr2 Revealed By Biochemical Characterization

Background: Lenvatinib mesilate (lenvatinib) selectively inhibits vascular endothelial growth factor receptors (VEGFR1-3), and other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib is currently evaluated in several clinical trials, including two phase 3 clinical trials in patients with thyroid cancers and hepatocellular carcinoma. Recently, lenvatinib achieved the primary endpoint in a phase 3 clinical trial in patients with differentiated thyroid cancer. To gain insight into the origin of the potency of lenvatinib in VEGFR2 inhibition, crystal structure analysis of VEGFR2 complexed with lenvatinib and kinetics interaction analysis of lenvatinib against VEGFR2 were conducted in comparison with other VEGFR2 inhibitors. Experimental procedures: Recombinant human VEGFR2 was crystalized with lenvatinib or sorafenib, respectively. The resulting crystals of kinase-inhibitor complexes were subjected to x-ray structural analysis and the three dimensional structures were solved. The reporter displacement assay was conducted by using recombinant human VEGFR2 and a synthetic reporter probe. Reporter displacement was measured after addition of compounds and the kinetic parameters were determined. Results: In crystal structure analysis, both lenvatinib and sorafenib bind to the ATP-binding site and the neighboring allosteric region. However there was a notable difference in the conformation of the DFG motif in the activation loop. While DFG motif assumed a DFG-out conformation in the complex with sorafenib as reported, it was maintained in a DFG-in conformation in the complex with lenvatinib. In the reporter displacement assay, lenvatinib showed intermediate binding kinetics of those of sorafenib and sunitinib. Its association rate constant (k on ) and dissociation rate constant (k off ) values were 4.8 × 10 5 s −1 × M −1 (61 times of sorafenib, a representative type 2 inhibitor) and 9.9 × 10 −4 s −1 (3.8 times), respectively. Sunitinib, a representative type 1 VEGFR2 inhibitor had a much faster binding kinetics and we could not determine kinetics parameters (k on u003e1.9 × 10 5 , k off u003e57 × 10 −4 ). Conclusions: Lenvatinib was suggested to have interaction with not only ATP region but also the neighboring allosteric region of VEGFR2, which is a typical feature of type 2 inhibitors. However, VEGFR2 adopted DFG-in conformation in complex with lenvatinib, which is a characteristic of type 1 inhibitors. Kinetics analysis revealed that lenvatinib had intermediate characteristics among these three types of VEGFR2 inhibitors. These results suggest lenvatinib is very distinct in its ability in binding with VEGFR2. Citation Format: Kiyoshi Okamoto, Megumi Ikemori Kawada, Anja Jestel, Konstanze von Konig, Yasuhiro Funahashi, Tomohiro Matsushima, Akihiko Tsuruoka, Atsushi Inoue, Junji Matsui. Distinct binding mode of lenvatinib to VEGFR2 revealed by biochemical characterization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1362. doi:10.1158/1538-7445.AM2015-1362