Identification and Characterization of Small-Molecule Inhibitors to Selectively Target the DFG-in over the DFG-out Conformation of the B-Raf Kinase V600E Mutant in Colorectal Cancer

V600E is the most common mutation in the B-Raf kinase domain and the B-Raf^V600E mutant has been recognized as an attractive target of colorectal Cancer. Here, the structural dynamics of V600E-induced conformational conversion in the B-Raf activation loop (A-loop) was characterized in detail using a computational simulation strategy. The simulations revealed that the V600E mutation would induce A-loop flipping from DFG-out to DFG-in, and the approved B-Raf inhibitor vemurafenib exhibits strong selectivity for the mutant over the wild-type kinase. The selectivity is closely associated with the kinase conformation, which can be influenced directly by the V600E mutation. The molecular structure of vemurafenib was applied to a chemical similarity search against a large library of drug/lead-like compounds, from which three hits with high structural similarity were identified, and their inhibitory activities against both the wild-type and mutant kinases were measured by in vitro kinase assay, from which two compounds were determined to possess higher selectivity for the B-Raf^V600E mutant than for the wild type (5.2- and 3.1-fold, respectively). They can potently inhibit the kinase mutant with IC₅₀ = 54 and 76 nM, respectively. Structural analysis suggested that specific noncovalent interactions play a crucial role in the selectivity of B-Raf inhibitors.

Activation loop; B-Raf kinase V600E mutation; Colorectal cancer; DFG-in and DFG-out; Inhibitor drug.