Imatinib, a type II kinase inhibitor, has been very successful at treating chronic myelogenous leukemia (CML) caused by a fusion of Abelson (Abl) kinase with the breakpoint cluster region (BCR) that renders Abl constitutively active. However, over time mutations can arise that diminish or eliminate the effect of imatinib on the cancer cells.
One such mutation involves the replacement of the small gatekeeper residue with a bulky residue (T315I), thereby sterically blocking imatinib and the majority of second-generation inhibitors from the active site. This active-site mutation has been particularly hard to overcome and indicates the importance of continued research to find the next generation of CML drugs. Wild-type Abl is regulated by its Src homology domains (SH2 and SH3) and an N-terminal myristoylated region. This regulation is lost upon the fusion with BCR, which leads to a change in the conformation of an α-helix thereby blocking the SH2 domain from inhibiting Abl.
It was discovered previously that inhibitors can bind to the distal myristoyl site causing a 90° bend in the nearby α-helix, which once again allows the SH2 domain to inhibit the kinase domain of Abl. The initial discovery of a compound that acted on this distal site was by chance, and the goal here was to develop an assay that would specifically select for inhibitors of this distal site.
The authors used an assay format called FLiK (fluorescent labels in kinases), which had been previously applied to the activation and “glycine-rich” loops of kinases, to probe the distal myristoyl site. FLiK involves covalent modification of the protein with an environmentally sensitive fluorophore (acrylodan) at a site that will undergo a large conformational change upon compound binding (see Figure 1).
Prior to the covalent modification, mutagenesis of the protein was required. A cysteine residue was added to which the fluorophore was later linked (V338C), and other surface accessible cysteine residues were mutated (C305V and C330S) so that they would not be covalently modified. The assay involved monitoring the decrease in fluorescence upon compound addition due to the change in the environment of the fluorophore (see Figure 2).