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

Abstract

<p>Small molecule kinase inhibitors that stabilize distinct ATP-binding site conformations can differentially modulate the glob-al conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP-binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects are general to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP-binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src’s regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix aC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src’s helix aC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Taken together, our study highlights that an ATP-competitive inhibitor’s interactions with helix aC can have a major influence on the global conformation of some tyrosine kinases in vitro and in cells.</p>

Differential impact of BTK active site inhibitors on the conformational state of full-length BTK

“…For the SRC family, the αC-in configuration has been correlated with release of the regulatory domains from the autoinhibited state ( Leonard et al, 2014 ; Tsutsui et al, 2016 ). Other studies suggest that the αC-helix conformation alone can be used to predict the regulatory domain conformation in the SRC, ABL, and TEC kinases; inhibitors that stabilize the αC-in conformation disrupt the regulatory domains from their autoinhibitory conformation, whereas αC-out stabilizing inhibitors do not ( Fang, 2020 ). However, ABL focused studies show that Dasatinib stabilizes αC-in but does not disrupt the regulatory domains from their compact, autoinhibitory state ( Sonti et al, 2018 ).…”

Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to catalytic domain dynamics

“…We next used the rate of proteolysis of the flexible linker that connects Src’s SH2 domain to its CD (SH2-CD linker) by the metalloprotease thermolysin to characterize how β1/β2 resistance cluster mutations affect intramolecular regulatory domain engagement. Previous studies have demonstrated an inverse correlation between the rate of thermolysin cleavage, measured as intact protein half-life (t1/2), of the SH2-CD linker and intramolecular SH2 and SH3 regulatory domain engagement levels ( Figure 4H ) (Agius et al, 2019; Fang et al, 2020). Concordant with the SH3 domain pull-down results, the SH2-CD linkers of E283M and W285T Src 3D were proteolyzed ∼3 times more rapidly than WT Src 3D ( Figure 4I , S4F ).…”

“…Inhibitors 1 , 2 , and 3 was synthesized as previously described in (Ahler et al, 2019; Fang et al, 2020).…”

“…Src mutational resistance to different modes of ATP-competitive inhibition. We further explored the intersection between the regulation of Src's phosphotransferase activity and drug resistance by determining how resistance arises in response to different modes of ATP-competitive inhibition (Figure S3A) (Fang et al, 2020;Ranjitkar et al, 2010). We profiled our Src variant library in the presence of each of a matched set of pyrazolopyrimidine-based ATP-competitive inhibitors that stabilize structurally distinct conformations of Src's ATP-binding site (Figure 3A).…”

Tyrosine kinases: complex molecular systems challenging computational methodologies

“…Furthermore, umbrella sampling computations indicate that the configuration of the SH2 and SH3 regulatory domains, greatly affects the ability of the catalytic domain to adopt the active or inactive conformation [87]. Conversely, individual inhibitors binding to the catalytic domain can have differing allosteric effects on whether the regulatory domains adopt a closed or open conformation [120]. Computational methods based on simplified CG models have played a useful role to interpret SAXS data on Hck kinase in solution in terms of the configuration of the multi-domain kinase [92,101].…”