A fluorescent reporter of ATP binding-competent receptor kinases

Abstract

ERBB receptor kinases play a crucial role in normal development and cancer malignancies. A broad range of modifications creates receptor subpopulations with distinct functional properties in live cells. Their apparent activation state, typically assayed by tyrosine phosphorylation of substrates, reflects a complex equilibrium of competing reactions. With the aim of developing optical tools to investigate ERBB populations and their state of activation, we have synthesized a fluorescent 'turn-on' probe, DMAQ, targeting the ERBB ATP binding pocket. Upon binding, probe emission increases due to the hydrophobic environment and restricted geometry of the ERBB2 kinase domain, facilitating the analysis of receptor states at low occupancy and without the removal of unbound probes. Cellular ERBB2 autophosphorylation is inhibited with saturation kinetics that correlate with the increase in probe fluorescence. Thus, DMAQ is an example of a new generation of 'turn-on' probes with potential applications in querying receptor kinase populations both in vitro and in live cells.

Figure 1.

Design strategy for a ‘turn-on’ fluorescent ligand: incorporation of a push–pull chromophore similar to DCS and an ERBB-targeting pharmacophore resembling 1, yields DMAQ, which is comparable in overall size to gefitinib.

Figure 2.

(A) UV–vis and emission spectra of DMAQ in PEG and PBS. Emission is enhanced 23-fold in PEG relative to PBS. The lowest energy electronic transition correlates well with the predicted energy (DF B3LYP 6–31G*). (B) The frontier orbital distribution and energies of DMAQ compare are remarkably similar to DCS, a well-studied stilbene that functions as a ‘turn-on’ fluorophore, suggesting similar photophysical behavior for DMAQ.

Figure 3.

(A) Saturating titration of DMAQ binding to ERBB2 (λ_ex = 375 nm, λ_em = 450 nm) compared with the relative inhibition of ERBB2 autophosphorylation in BT474 cells. B Representative Western blot of ERBB2 inhibition by DMAQ and Canertinib (CI) and constitutively active AKT after treatment with DMAQ or the dual PI3K/mTOR inhibitor: BEZ-235 (BEZ). (B) The impact on SRC activity was tested in vitro using biotinylated HNRNPK, a known SRC client; PP1 was used as positive control. (C) Titration of DMAQ dependent fluorescence in whole cell lysate containing a fusion protein of soluble ERBB2 kinase domain and mCherry with and without Canertinib pretreatment (CI) prior to lysis. Soluble kinase domain fusions in both samples were standardized by mCherry fluorescence.