Chemical structure of FF-10101, and potent and irreversible inhibition of FLT3 by FF-10101 through binding interaction with cysteine residue at 695 (C695). (A) Chemical structure of FF-10101. (B) X-ray analysis of the co-crystal structure of the FLT3 protein bound to FF-10101 revealed covalent binding of FF-10101 to C695 of FLT3 molecule. (C) FF-10101 has a double bond to form a covalent bond to the C695 of the FLT3 molecule. FLA-9430 has the same chemical structure as FF-10101, with the exception of having a single bond instead of a double bond. Marked differences in inhibitory activities against the wild-type FLT3 enzyme between FF-10101 and FLA-9430 were shown, with IC₅₀ values of 0.14 nM and 2.72 nM, respectively. (D) FLT3-ITD–expressing or FLT3-ITD-C695S–expressing HEK293T cells were incubated with various concentrations of FF-10101 or FLA-9430 at 37°C for 1 hour. Phosphorylation status of FLT3 was assessed as described in “Methods.” Percent inhibition of FLT3 phosphorylation at each concentration was plotted to calculate IC₅₀ values using XLfit software. (E) FLT3 immobilized in a 96-well plate was preincubated with an excess concentration of FLT3 inhibitor (5 μM) for 1 hour followed by 5-time wash with TBST. After washing out theFLT3 inhibitors, ATP and substrate peptide labeled with FAM were added. Phosphorylated substrate was detected at each time point by a mobility shift assay.