Divergent Modulation of Src-Family Kinase Regulatory Interactions with ATP-Competitive Inhibitors

Abstract

Multidomain protein kinases, central controllers of signal transduction, use regulatory domains to modulate catalytic activity in a complex cellular environment. Additionally, these domains regulate noncatalytic functions, including cellular localization and protein–protein interactions. Src-family kinases (SFKs) are promising therapeutic targets for a number of diseases and are an excellent model for studying the regulation of multidomain kinases. Here, we demonstrate that the regulatory domains of the SFKs Src and Hck are divergently affected by ligands that stabilize two distinct inactive ATP-binding site conformations. Conformation-selective, ATP-competitive inhibitors differentially modulate the ability of the SH3 and SH2 domains of Src and Hck to engage in intermolecular interactions and the ability of the kinase–inhibitor complex to undergo post-translational modification by effector enzymes. This surprising divergence in regulatory domain behavior by two classes of inhibitors that each stabilize inactive ATP-binding site conformations is found to occur through perturbation or stabilization of the αC helix. These studies provide insight into how conformation-selective, ATP-competitive inhibitors can be designed to modulate domain interactions and post-translational modifications distal to the ATP-binding site of kinases.

Targeting transcription is no longer a quixotic quest

“…Thus by intercepting a particular conformer, a small molecule could have either a positive or a negative influence on the binary and ternary transcription factor–coactivator assembly process. Certainly the field can be guided by the success seen in targeting particular conformational states of kinases or of the protein folding machinery for enhanced selectivity and context-specific effects on downstream processes 8,18,19 .…”

SH2-Catalytic Domain Linker Heterogeneity Influences Allosteric Coupling across the SFK Family

“… 9 , 10 In fact, our lab has shown that ATP-competitive ligands that stabilize Src and Hck in an inactive αC helix-out conformation strengthen intramolecular SH3/SH2 domain engagement upon binding. 12 , 18 In contrast, inhibitors that stabilize helix αC in an active conformation weaken interactions between the SH2/SH3 domains and their respective intramolecular ligands. While the basic allosteric regulatory network between the catalytic and regulatory domains of SFKs is well-characterized, less is known about how SH2-CD linker heterogeneity affects the magnitude of this bidirectional relationship.…”

How ATP-Competitive Inhibitors Allosterically Modulate Tyrosine Kinases That Contain a Src-like Regulatory Architecture

“…Inhibitor 1 contains an extended 4-phenoxyphenyl substituent at the C-3 position on the pyrazolopyrimidine scaffold that occupies a hydrophobic pocket created by the outward movement of helix aC (Figure 2A, left). 14,[18][19]31 Inhibitor 2 stabilizes the DFG-out inactive conformation of tyrosine kinases (Figure 2A, right). Inhibitor 2 contains a 3-trifluoromethylbenzamide substituent that occupies the hydrophobic pocket formed by the displacement of the DFG-motif.…”

“…While inhibitors that stabilizes the DFG-out inactive conformation promote an open global conformation of tyrosine kinases that contain an SH3-SH2-CD architecture, 22,23 we have found that other classes of ATPcompetitive inhibitors are also capable of promoting allosteric disengagement of regulatory SH2 and SH3 domains. 18 To better understand the common features of inhibitors that promote an open global conformation of the SH3-SH2-CD regulatory module, we assembled a panel of 13 inhibitors that contain structurally diversified substituents that make varied ATP-binding site contacts and tested them in biochemical assays for the global conformational state of Src ( Figure 3A). Inhibitors 1, 3, and 4 contain pharmacophores that should stabilize the helix aC-out inactive conformation of Src, 14, 17 while 2 and 5 contain substituents that stabilize the DFG-out form.…”

“…7,16 We and others have shown that ATP-competitive inhibitors that stabilize different ATP-binding site conformations can exploit these allosteric networks to modulate the regulatory interactions of the SH3-SH2-CD module of SFKs. 14,[17][18][19][20][21][22][23] Inhibitors that stabilize the helix aC-out inactive conformation of the ATP-binding site, which involves outward movement of the catalytically important helix aC ( Figure 1B), promote an autoinhibited, closed global conformation of the SH3-SH2-CD module of SFKs. The presence of an extended substituent that occupies the hydrophobic pocket formed by the displacement of helix aC is necessary for inhibitors to stabilize this inactive conformation.…”