Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.
Submitter: Marie Ary
Submission Date: June 28, 2017, 12:44 p.m.
|Number of data points||90|
|Proteins||Tyrosine-protein kinase receptor Axl Ig1|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: kon to Gas6 ; Experimental Assay: koff to Gas6 ; Experimental Assay: Kd to Gas6 ; Derived Quantity: ΔΔGbind ; Computational Protocol: ΔGbind|
|Libraries||Calculated free energy of binding and changes in free energy of binding of Axl Ig1 and variants (Table S3) ; Kd, kon, koff to Gas6 for WT Axl Ig1, MYD1 and Axl-nb (Fig. 1d) ; Kd to Gas6 for all permutations of 4 mutations in MYD1 (Fig. 1e) ; Tm of Axl Ig1 variants (Table S4)|
|Structure ID||Release Date||Resolution||Structure Title|
|5VXZ||2017-06-21||2.3||High-affinity AXL decoy receptor|
|5U6B||2017-07-26||2.84||Structure of the Axl kinase domain in complex with a macrocyclic inhibitor|
|4RA0||2014-09-24||3.07||An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis|
|2C5D||2005-12-19||3.3||Structure of a minimal Gas6-Axl complex|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||Tyrosine-protein kinase receptor Axl Ig1||P30530||UFO_HUMAN|