Kinase binding assays

For optimization of Axl/TAM receptor inhibitors, an Axl binding assay was established (HTRF method; Kinase tracer 236). This assay was 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 generated a fluorescence resonance energy transfer (FRET) signal. Binding of inhibitor to the kinase competed for binding with the tracer, resulting in a loss of the FRET signal. For the assay, the compound was 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) were mixed with the respective compound dilutions (from 5 nM to 10 μM) and incubated for 1 hr. The FRET signal was quantified using an EnVision Multilabellreader 2104.

Cell lines

93 cancer cell lines and 2 primary cells (i.e. IMR90 and human peripheral blood mononuclear cells)

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

0 ~ 35 μM; 72 hrs

Applications

In 11 of 95 selected cell lines, LDC1267 moderately affected cell proliferation with the IC50 values of >5 μM. In NKG2D-activated NK cells, LDC1267 abolished the inhibitory effects on cell proliferation and IFN-γ production induced by Gas6 stimulation.

Animal models

Mice bearing B16F10 metastatic melanomas

Dosage form

20 mg/kg; i.p.

Applications

In mice bearing B16F10 metastatic melanomas, LDC1267 efficiently increased anti-metastatic NK cell activity, and inhibited tumor metastases without severe cytotoxicity.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

LDC1267 is a highly selective TAM kinase inhibitor with IC50 of < 5 nM, 8 nM, and 29 nM for Mer, Tyro3, and Axl, respectively.
LDC1267 preferentially inhibits Tyro3, Axl and Mer at low nano-molarity by tracer-based binding assays. Treatment of NKG2D- activated NK cells with LDC1267 indeed abolished the inhibitory effects of Gas6 stimulation; LDC1267 had no apparent additional effect in Cbl-b-deficient NK cells. Adoptive transfer of LDC1267-treated wild-type NK cells significantly increased the anti-tumour response to levels observed in mice transplanted with Cbl-b2/2 NK cells, but did not increase the anti-metastatic efficacy of Cbl-b-knockout NK cells, reinforcing the notion that Cbl-b acts downstream of TAM receptors for NK cell inhibition. [1]
In vivo, wild-type mice treated with LDC1267 showed enhanced cytotoxicity towards RMA cells overexpressing the NKG2D ligand Rae-1 (RMA-Rae1) to the same extent as C373AKI/KI mice, but had no effect on the already enhanced NK cytotoxicity in Cbl-b-mutant mice. LDC1267 markedly reduced metastatic spreading of melanomas; NK1.1 depletion abolished the therapeutic benefits of LDC1267. LDC1267 treatment significantly reduced the numbers and sizes of 4T1 micro-metastases in the liver, without any apparent effect on the primary vehicle LDC1267 mammary tumor. NK cell depletion using anti-asialoGM1 antibodies resulted in markedly enhanced 4T1 liver metastases and abolished the therapeutic benefits of LDC1267. Oral LDC1267 significantly reduced 4T1 liver micro-metastases. [1]
References:
[1]. Paolino M, Choidas A2, Wallner S et al. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature. 2014 Mar 27;507(7493):508-12. doi: 10.1038/nature12998. Epub 2014 Feb 19.