Kinase activity inhibition profiles of BRAFV600E and BRAFWT upon Ponatinib titration using SelectScreen assay.
PHI2 is an analogue of PHI1 with distinct binding specificity for BRAFV600E monomers and dimers compared to PHI1.
(a) The molecular structure of PHI2. (b) BRAF kinase inhibition profiles (SelectScreen, Invitrogen) of PHI2.
Ponatinib induces BRAF dimers with alfaC-CENTER conformation. Crystal structures of BRAF with inhibitors typically are determined in dimeric conformation and each protomer’s бChelix can adopt different conformations between the IN (active) and OUT (inactive) position.
The BRAF–Ponatinib structure corroborates the dimeric conformation of the kinase induced by alfaC-IN inhibitors, with both protomers occupied by Ponatinib [Inhibitors of BRAF dimers using an allosteric site]
PHI1 is selective for oncogenic BRAF dimers.
results demonstrate that PHI1 more effectively targets p61BRAFV600E dimers than BRAFV600E
monomers. [Inhibitors of BRAF dimers using an allosteric site]
Inhibition of kinase activity of BRAFV600E, BRAFWT, and other tyrosine kinases targets, identified by KinomeEDGE®, using SelectScreen (Invitrogen) in the presence of 100 μM ATP. Half-maximal inhibition values (IC50 ± SD) of two technical replicates from n = 2 independent experiments are tabulated. Source data are provided as a Source Data file. [Inhibitors of BRAF dimers using an allosteric site]
CETSA showed that Ponatinib stabilizes BRAFV600E monomers in cells (ΔTm = +6.3 °C) more effectively compared to PHI1 (ΔTm=+3.4 °C)
In contrast, PHI1 and Ponatinib display similar stabilization effect with p61BRAFV600E dimers
(ΔTm=+3.2 °C)
exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors.