Figure 1.
Inhibition of Abl proteins with compounds alone or in combination.
A. Enzymatic inhibition (in vitro) of recombinant wild-type and T315I Abl by dasatinib, GNF-5 and combination treatments. Percent inhibition of wt Abl or T315I Abl by dasatinib and GNF-5 or the combination. The combination curve (red) contains twice the total drug concentration of the single agent curves due to both drugs being present. B. Synergistic inhibition of Bcr-Abl T315I transformed Ba/F3 cells. Dose and effect curve of GNF-5, dasatinib and the combination of GNF-5 and dasatinib (1∶1 ratio) on Bcr-Abl T315I transformed cells. The combination curve (red) contains twice the total drug concentration of the single agent curves due to both drugs being present. C. CI values for fractional growth inhibitions of 0.50, 0.75, and 0.90 in Bcr-Abl T315I cells. Antagonism CI >1.00; additivity CI = 1.00; synergy CI <1.00.
Figure 2.
Deuterium incorporation curves for wild-type and T315I Abl in the presence of dasatinib, GNF-5 or both dasatinib and GNF-5.
Both dasatinib and GNF-5 were present at a molar ratio of 1∶7, protein:inhibitor. Only data for the peptides that showed changes in deuterium uptake in the presence of inhibitors are shown; all other regions indicated no changes in hydrogen exchange in the presence of the inhibitors in the time frame the experiment was performed (4 hours). Data for free or bound forms were acquired under identical conditions, in duplicate. The error of each data point is ±0.20 Da. Note that we have used Abl 1b numbering [45] throughout this work; for example, Abl position T315 is actually T334 in Abl 1b, but we are using the numbering designation of T315I according to the established clinical conventions [10].
Figure 3.
Summary of the hydrogen exchange results for all binding experiments in this study.
In each panel, the ribbon diagram of Abl (PDB 2F4J, [46]) is shown in the left and the space filling model is shown on the right. The differences in deuterium levels are colored on each peptide where changes were observed, according to the color code shown. The location of each specific peptide is labeled in Supplemental Fig. S5. Obvious changes (colored hot pink) were defined as a difference between deuterium exchange-in curves of 1.0 Da or more, subtle changes (colored light yellow) were defined as a difference of 0.4–1.0 Da and no changes were defined as differences of 0.0–0.4 Da. The ATP binding site is shown by rendering the drug VX-6, already present in the 2F4J crystal structure. A close-up of the myristic acid binding pocket is shown in Supplemental Fig. S6. Abl structure 2F4J was chosen to interpret the hydrogen exchange data as it has an extended αI helix thought to be present in the active form of Abl. Although the NCap SH3 domain, SH2 domain and the SH2-kinase linker are present in the constructs studied, no changes in hydrogen were detected in those regions in the presence of these inhibitors.