Bemcentinib (R428) belongs to small molecule inhibitors and is a highly selective oral Axl inhibitor (IC50 = 14 nM) with oral bioavailability and potent cell permeability. This compound effectively inhibits cancer cell migration and invasion, blocks tumor dissemination, and prolongs survival in various tumor models.

cells:14 nM (cell free)

A five-point R428 dose titration was performed in radiometric in vitro kinase assays on 133 kinases at the Km(ATP) for each kinase. Axl, Mer, and Tyro3 assays were also performed using a fluorescence polarization protocol. HER2 activity was determined by Z'-LYTE assay [1].

MDA-MB-231 or 4T1 cells (1 × 10^5) were allowed to migrate through Matrigel toward 20% FCS in an 8-μm pore 24-well Transwell plate at 37°C for 16 to 24 h. Noninvaded cells and Matrigel were removed by swabbing. Invaded cells were fixed in 4% formaldehyde, stained with 1% crystal violet, and quantified as for Axl cell-based assay. Cells were preincubated with R428 for 3 h. R428 was added to both upper and lower Transwell chambers [1].

Female BALB/c mice were inoculated in the mammary fat pad with 0.5 × 10^6 4T1 cells. Forty-eight hours after inoculation, mice were randomized into treatment groups (n = 10). Oral dosing with R428 (7–75 mg/kg twice daily) or vehicle continued until days 19 to 21. Cisplatin (1.2 or 4 mg/kg) was administered i.v. once weekly. Body weight and tumor size were measured thrice per week. Lungs were exposed postmortem. Total number and size of surface lung macrometastases were measured (small, <2 mm; medium, ≥2 mm and <3 mm; large, ≥3 mm). Half of each primary tumor was snap frozen in liquid nitrogen. The other half, and the livers were fixed in paraformaldehyde/lysine/periodate solution, paraffin embedded and sectioned (5 μm thick). Two H&E-stained liver sections per animal were examined microscopically for micrometastases in three view fields. Synergism was determined using Clark's synergy calculation [1].

R428, BGB324

H2O: < 1 mg/mL (insoluble or slightly soluble)

Ethanol: < 1 mg/mL (insoluble or slightly soluble)

DMSO: 23.4 mg/mL (46.19 mM), Sonication and heating are recommended.

10% DMSO+40% PEG300+5% Tween-80+45% Saline: 1 mg/mL (1.97 mM), Sonication is recommended.