Small molecule inhibitors block Gas6-inducible TAM activation and tumorigenicity

Abstract

TAM receptors (Tyro-3, Axl, and Mertk) are a family of three homologous type I receptor tyrosine kinases that are implicated in several human malignancies. Overexpression of TAMs and their major ligand Growth arrest-specific factor 6 (Gas6) is associated with more aggressive staging of cancers, poorer predicted patient survival, acquired drug resistance and metastasis. Here we describe small molecule inhibitors (RU-301 and RU-302) that target the extracellular domain of Axl at the interface of the Ig-1 ectodomain of Axl and the Lg-1 of Gas6. These inhibitors effectively block Gas6-inducible Axl receptor activation with low micromolar IC50s in cell-based reporter assays, inhibit Gas6-inducible motility in Axl-expressing cell lines, and suppress H1299 lung cancer tumor growth in a mouse xenograft NOD-SCIDγ model. Furthermore, using homology models and biochemical verifications, we show that RU301 and 302 also inhibit Gas6 inducible activation of Mertk and Tyro3 suggesting they can act as pan-TAM inhibitors that block the interface between the TAM Ig1 ectodomain and the Gas6 Lg domain. Together, these observations establish that small molecules that bind to the interface between TAM Ig1 domain and Gas6 Lg1 domain can inhibit TAM activation, and support the further development of small molecule Gas6-TAM interaction inhibitors as a novel class of cancer therapeutics.

Tyro3 carboxyl terminal region confers stability and contains the autophosphorylation sites

“…It should be noted that we did not demonstrate intra-molecular autophosphorylation per se, but rather tyrosyl-phosphorylation that occurs upon forced overexpression. That this was dependent on the Tyro3 kinase activity was demonstrated by elimination upon replacement of the acceptor lysine at position K540 with either arginine or methionine, and the inhibition by the TAM-selective inhibitor R428 [23]. It has been suggested that Tyro3 can be cross-phosphorylated by the related family member Axl, which is also expressed at high levels in NR6WT (Figure 1A).…”

Rational Drug Design of Axl Tyrosine Kinase Type I Inhibitors as Promising Candidates Against Cancer

“…TAM activation can, thus, be efficiently inhibited by the smallmolecule inhibitors for this interaction. For the TAM activation blocking compounds RU-301 and RU-302, Kimani et al (2017) suggested and experimentally observed that these may act as pan-TAM inhibitors as they suppress the H1299 lung cancer tumor growth in the mouse xenograft NOND-SCIDγ model tested. Individual studies are, of course, limited to only either modeling or experiments in vitro or in cell culture.…”

The Dual Role of TAM Receptors in Autoimmune Diseases and Cancer: An Overview

“…The development of small molecules demonstrated strong inhibitory activity, however, most are not specific to the TAM receptors [ 165 ]. The Axl inhibitor BGB324 has been shown to inhibit both Mer and Tyro3 at high concentrations [ 166 ], while the Mer inhibitors UNC569 and UNC1666 also have inhibitory effects on Axl and Tyro3 [ 167 , 168 ]. These inhibitors have also been described as possessing off-target effects in other proteins such as vascular endothelial growth factor receptor (VEGFR), Abelson tyrosine-protein kinase (Abl), tunica internal endothelial cell kinase 2 (Tie-2), MET, Fms-like tyrosine kinase (Flt3), and rearranged during transfection (RET) protein [ 165 , 167 , 168 ].…”