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-RafV600Emutant 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-RafV600Emutant 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.
Department of Gastroenterology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, P. R. China.
Huixiang
H
Yao
Department of Gastroenterology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, P. R. China. sunqun_1967@sina.com.
Qun
Q
Sun
Department of Gastroenterology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, P. R. China.
Jinshui
J
Zhu
D001665:Binding Sites; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000235:genetics*; D003198:Computer Simulation; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology; D000077484:Vemurafenib
D007211:Indoles; D050505:Mutant Proteins; D054852:Small Molecule Libraries; D013449:Sulfonamides; D000077484:Vemurafenib; D048493:Proto-Oncogene Proteins B-raf
Activation loop; B-Raf kinase V600E mutation; Colorectal cancer; DFG-in and DFG-out; Inhibitor drug