In vitro activity: BGB-283 potently inhibits BRAFV600E-activated ERK phosphorylation and cell proliferation in vitro. It demonstrates selective cytotoxicity and preferentially inhibits proliferation of cancer cells harboring BRAFV600E and EGFR mutation/amplification. In BRAFV600E colorectal cancer cell lines, BGB-283 effectively inhibits the reactivation of EGFR and EGFR-mediated cell proliferation. It demonstrates selective cytotoxicity to cell lines harboring BRAFV600E or EGFR mutations. BGB-283 inhibits the EGF-induced EGFR autophosphorylation on Tyr1068 in A431 cells in a dose-dependent manner. In WiDr colorectal cancer cells, BGB-283 is shown to be able to inhibit the feedback activation of EGFR signaling and achieves sustained inhibition of pERK.

Kinase Assay: Lifirafenib (BGB-283) inhibits RAF kinases and EGFR activities in biochemical assays with IC50 values of 23, 29 and 495 nM for the recombinant BRAFV600E kinase domain, EGFR and EGFR T790M/L858R mutant. Compounds were tested for inhibition of RAF and WT EGFR kinase activity in assays based on time-resolved fluorescence-resonance energy transfer (TR-FRET) methodology. MEK1 (K97R) was used as a substrate for RAF kinases and a biotinylated peptide substrate was used for EGFR (61TK0BLC, CisBio Bioassys). The kinase was incubated with a serial dilution of compounds for 60 to 120 minutes at room temperature (RT), ATP (final concentration at 100 μmol/L) and kinase substrates were added to initiate the reaction. The reaction was stopped by an equal volume of stop/detection solution according to the manufacture's instruction (CisBio Bioassays). Plates were sealed and incubated at RT for 2 hours, and the TR-FRET signals (ratio of fluorescence emission at 665 nm over emission at 620 nm with excitation at 337 nm wavelength) were recorded on a PHERAstar FS plate reader (BMG Labtech). BGB-283 was screened for activity in a panel of 277 kinases at a fixed concentration of 10 μmol/L by Life Technologies using their standard assays at Km concentration of ATP for perspective kinases. The IC50 was then determined for kinases showing>80% inhibition at 10 μmol/L BGB-283.

Cell Assay: Cellular phospho-ERK and phospho-EGFR were measured using a TR-FRET–based method. Cells were seeded at 3 × 104 per well of a 96-well plate and left to attach for 16 hours. Growth medium was then replaced with 100 μL of DMEM containing no serum. Cells were then treated with a 10-point titration of compound. After 1 hour of compound treatment, 50 μL of lysis buffer (Cisbio) was added to each well. Plates were then incubated at room temperature with shaking for 30 minutes. A total of 16 μL of cell lysate from each well of a 96-well plate was transferred to a 384-well small volume white plate. Lysate from each well was incubated with 2 μL of Eu3+- or Tb3+- cryptate (donor) labeled anti-ERK or anti-EGFR antibody (Cisbio) and 2 μL of D2 (acceptor) labeled anti-phospho-ERK or anti-phospho-EGFR antibody (Cisbio) for 2 hours at room temperature. FRET signals were measured using a PHERAstar FS reader (BMG Labtech).