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Kinetic and Mechanistic Characterization of a Potent and Selective Inhibitor for Human AXL Receptor Tyrosine Kinase
Author(s) -
Chen Yu,
Miles Dillon H.,
Udyavar Akshata,
Cho Sean,
Becker Annette,
Foley Corinne,
Leleti Manmohan R.,
Jin Lixia,
Young Stephen W.,
Powers Jay P.,
Walters Matthew J.,
Zhao Xiaoning
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.09160
Subject(s) - autophosphorylation , gas6 , receptor tyrosine kinase , axl receptor tyrosine kinase , tyrosine kinase , cancer research , signal transduction , chemistry , microbiology and biotechnology , mertk , kinase , biology , biochemistry , protein kinase a , jak stat signaling pathway
The AXL receptor tyrosine kinase (AXL) is a member of the TAM RTK family comprising TYRO3, AXL and MER. AXL signaling is activated by binding of the growth arrest‐specific protein 6 (Gas6) or vitamin K‐dependent protein S (PROS1), which subsequently induces homodimerization and autophosphorylation, thereby initiating a conventional RTK signaling pathway. Maximal stimulation requires an extracellular lipid, phosphatidylserine (PtdSer). Activation of AXL regulates an array of signal transduction pathways, including those mediating cell survival, proliferation and migration, as well as promoting an immunosuppressive phenotype within the tumor microenvironment 1, 2. AXL has been shown to be highly expressed in sarcoma, renal cell carcinoma, pancreatic adenocarcinoma and other types of tumors, correlative with poor prognosis in cancer patients. Furthermore, AXL has been shown to facilitate both intrinsic and acquired resistance to multiple anti‐cancer drugs 3, suggesting therapeutic targeting of AXL may be an effective anti‐cancer strategy. We have developed a potent, reversible and competitive small molecule AXL inhibitor. This inhibitor (Compound 1) inhibits the activity of recombinant human AXL kinase domain with nanomolar potency, as determined by detection of phosphorylated substrate using homogeneous time‐resolved fluorescence (HTRF). Compound 1 is also highly selective against TYRO3, MERTK and MET kinase domains. The compound binds to the intracellular AXL kinase domain with a nanomolar dissociation rate constant measured by monitoring displacement of a competitive fluorescent tracer using bioluminescence resonance energy transfer (BRET). In order to assess the inhibitory effect of the compound on downstream signal transduction, AXL autophosphorylation‐induced SH2 translocation was determined using an AXL PathHunter ® assay and phosphorylation of AKT Ser473 induced by AXL activation was also assessed by AlphaLISA. Support or Funding InformationReferences 1 Kurowska-Stolarska , Mariola et al. “ MicroRNA-34a dependent regulation of AXL controls the activation of dendritic cells in inflammatory arthritis .” Nature communications vol. 8 15877 . 22 Jun. 2017 . 2 Paolino , Magdalena et al. “ The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells .” Nature vol. 507 ,7493 ( 2014 ): 508 – 12 . 3 Hugo , Willy et al. “ Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma .” Cell vol. 165 , 1 ( 2016 ): 35 – 44 .