Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

Abstract

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.

Figure 1

AGT and Kinase Constructs. (A). A crystal structure of AGT denoting the relative location and distances of the N- and C-terminus from the active site cysteine (PDP entry 1EH8). (B). The four PP motif AGT fusions that were used in this study. (C). Structures of full length SRC and ABL. The domains contained in the SRC-3D and ABL-3D constructs used in this study are indicated. The location of two regulatory phosphorylation sites (Y461, and Y527) are shown on the linear diagram of SRC: [M] = membrane binding region, [U] = unique region, [L] = linker, [NTS] = nuclear-transport signal, [DB] = DNA binding domain, [AB] = actin-binding domain.

Figure 2

IC₅₀ values of bivalent inhibitors with variable tether lengths between the ATP-competitive inhibitor and AGT. (A). Chemical structures of BG-linked quinazoline inhibitors with variable linker lengths. Each inhibitor contains a benzyl guanine (BG) moiety linked to a chloromethoxyaniline quinazoline inhibitor through a flexible tether. (B). In vitro activities of unconjugated inhibitors 1, 2, and 3 and bivalent conjugates AGT(WT)-1, AGT(WT)-2, AGT(WT)-3, AGT(PP1)-1, AGT(PP1)-2, AGT(PP1)-3 against SRC-3D. IC₅₀ values of unconjugated 1, 2, and 3 and bivalent conjugates AGT(WT)-1, AGT(WT)-2, AGT(WT)-3, AGT(PP4)-1, AGT(PP4)-2, AGT(PP4)-3 against ABL-3D. All protein-small molecule conjugates were prepared in two independent labeling reactions, and values shown are the average of four assays ± SEM.

Figure 3

IC₅₀ values of various ATP-competitive inhibitors conjugated to AGT(PP1). (A). Chemical structures of BG-linked, ATP-competitive kinase inhibitors 4–6. (B). In vitro activities of unconjugated inhibitors 4, 5, and 6 and bivalent conjugates AGT(PP1)-4, AGT(PP1)-5, AGT(PP1)-6 against SRC-3D. In vitro activities of unconjugated 4, 5, 6 and bivalent conjugates AGT(WT)-4, AGT(WT)-6, AGT(PP4)-4, AGT(PP4)-5, AGT(PP4)-6 against ABL-3D. All protein-small molecule conjugates were prepared in two independent labeling reactions, and values shown are the average of four assays ± SEM.

Figure 4

Effect of PP motif affinity on bivalent inhibitor potency and selectivity. (A). The fluorescence polarization competition assay that was used to determine the affinities of AGT(WT), AGT(PP1), AGT(PP2), and AGT(PP3) for the SH3 domains of SRC and HCK. The SH3 domains of SRC and HCK were expressed as GST-fusion proteins (GST-SH3(SRC) and GST-SH3(HCK)) and used in a fluorescence polarization competition assay with the peptide FAM-AAVSLARRPLPPLP-NH₂. In each assay, an AGT fusion protein was titrated against GST-SH3(SRC) or GST-SH3(HCK) in the presence of FAM-AAVSLARRPLPPLP-NH₂ (100 nM when assaying GST-SH3(SRC), 300 nM when assaying GST-SH3(HCK)). (B). Affinities of AGT(WT), AGT(PP1), AGT(PP2), and AGT(PP3) for the SH3 domains of SRC and HCK. (C). Representation of the genetic relationship between the tyrosine kinases SRC, HCK, LCK and ABL. In vitro activities of unconjugated inhibitor 1 and protein-small molecule conjugates AGT(PP1)-1, AGT(PP2)-1, AGT(PP3)-1 against SRC-3D, HCK-3D, LCK-3D and ABL-3D.

Figure 5

Activity of bivalent inhibitors against specific phospho-isoforms of SRC. (A). SRC was auto-phosphorylated at tyrosine 416 by incubating SRC-3D (100 nM) with ATP (1 mM) and Na₃VO₄ (2.5 mM) in activation buffer for 30 minutes. Phosphorylation was monitored by immuno-blotting with antibodies specific for the pY416 and non-pY416 forms of SRC. (B). IC₅₀ values of BG-linked analogue 1 and protein-small molecule conjugates AGT(PP1)-1, AGT(PP2)-1, AGT(PP3)-1 for pY416 and non-pY416 forms of SRC. All protein-small molecule conjugates were prepared in two independent labeling reactions, and values shown are the average of four assays ± SEM.