In vitro activity: LDC1267 moderately affects cell proliferation in 11 of 95 different cell lines with IC50 of>5μM. In NKG2D-activated NK cells, LDC1267 abolishes the inhibitory effects of Gas6 stimulation.

Kinase Assay: For optimization of Axl/TAM receptor inhibitors, an Axl binding assay is established (HTRF method; Kinase tracer 236). This assay is based on the binding and displacement of the Alexa Fluor 647-labelled Kinase tracer 236 to each glutathione S-transferase (GST)-tagged kinase used in the binding assay. Binding of the tracer to the kinase was detected by using europium (Eu)-labelled anti-GST antibodies. Simultaneous binding of both the fluorescent tracer and the Eu-labelled antibodies to the GST-tagged kinase generates a fluorescence resonance energy transfer (FRET) signal. Binding of inhibitor to the kinase competes for binding with the tracer, resulting in a loss of the FRET signal. For the assay, the compound is diluted in 20 mM HEPES, pH 8.0, 1 mM DTT, 10 mM MgCl2 and 0.01% Brij35. Then, the kinase of interest (5 nM final concentration), fluorescent tracer (15 nM final concentration) and LanthaScreen Eu-anti-GST antibody (2 nM final concentration) are mixed with the respective compound dilutions (from 5 nM to 10 μM) and incubated for 1 h. The FRET signal is quantified using an EnVision Multilabellreader 2104.

Cell Assay: After incubation for 72 hours with LDC1267, CellTiterGlow reagent is used to determine the proliferation relative to the corresponding DMSO control. Cell lines: A panel of 93 cancer cell lines and two primary cells (x axis, IMR90 and human peripheral blood mononuclear cells).

Nature. 2014 Mar 27;507(7493):508-12.