Materials and methods Patient selection and clinical data collection With approval

Results Computer simulated binding of ponatinib to the native or mutated PDGFR kinase in DFG out state To gain insights into the structural basis for ponatinib to bind to the ATP binding site of T674I PDGFR, we per formed computer simulations of molecular docking be tween ponatinib and T674I PDGFR, ponatinib bound to native or mutated PDGFR pan JAK 阻害剤 with the same orientation in the DFG out state. These findings were similar to those observed in the complex between ponatinib and T315I Abl. The T674I gatekeeper mutation does not perturb the overall protein structure of PDGFR, except that the large ali phatic side chain causes a steric hindrance that prevents the binding of imatinib but not ponatinib. The imidazo pyridazine scaffold of pona tinib docks in the adenine binding pocket of T674I PDGFR and forms one hydrogen bond with the backbone N atom of C677 in the hinge region.

The LDE225 分子量 ethynylene linker makes VDW interactions with the side chain of I674 residue, and the methylphenyl group occupies the hydrophobic pocket behind the gatekeeper residue. The extended amide linker contacts T674I PDGFR by 2 hydrogen bonds, one with the backbone N atom of D836 in the DFG motif and the other with the side chain carboxyl of E644 in the C helix. Consequently, the trifluoromethylphenyl group binds to the pocket in the DFG out conformation, with the terminal methyl piperazion group oriented to a solvent exposed region. Ponatinib and WT PDGFR have an interaction profile similar to but slightly differ ent from that in Additional file 1, Figure S1B.

Specifically, the whole WT PDGFR molecule turns more closely to the DFG motif, with the corresponding H bond distance shortened to 2. 92. The in silico structural comparisons revealed the importance of the DFG out state and the ethynylene linker in ponatinib in avoiding a steric clash imposed by the mutated gatekeeper residue I674. Ponatinib inhibits PDGFR supplier LY2157299 phosphorylation To examine whether ponatinib is active against T674I FIP1L1 PDGFR, we exposed BaF3 T674I FIP1L1 PDGFR cells to ponatinib, sorafenib and imatinib, levels of phosphorylated and total PDGFR were mea sured by immunoblotting. The phosphorylation of T674I FIP1L1 PDGFR was altered by sorafenib but not ima tinib, which is consistent with a previous re port. In contrast to imatinib, 300 nM ponatinib inhibited phosphorylation of T674I FIP1L1 PDGFR to a similar degree as 1000 nM sorafenib.

It also inhibited the phosphorylation of FIP1L1 PDGFR in EOL 1, BaF3 WT FIP1L1 PDGFR and BaF3 T674I FIP1L1 PDGFR cells in concentration and time dependent manners. Ponatinib inhibits downstream signaling of PDGFR We next examined signal transduction downstream of PDGFR after ponatinib treatment. The phosphorylation of Stat3, Stat5, Akt and Erk1 2 were measured by immunoblot ting with phospho specific antibodies. Treatment for 24 h with ponatinib reduced the phosphorylation of Stat3, Stat5, Akt and Erk1 2 in cells expressing WT or T674I PDGFR at 0. 3 30 nM and over time. Ponatinib inhibits growth of imatinib resistant and sensitive CEL cells bearing PDGFR We examined the effect of TKIs on cell viability.