Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4‑Kinase IIIβ (PI4KB)

The lipid kinase
phosphatidylinositol 4-kinase IIIβ (PI4KB)
is an essential host factor for many positive-sense single-stranded
RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV),
Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses.
Inhibitors of PI4KB are considered to be potential broad-spectrum
virostatics, and it is therefore critical to develop a biochemical
understanding of the kinase. Here, we present highly potent and selective
fluorescent inhibitors that we show to be useful chemical biology
tools especially in determination of dissociation constants. Moreover,
we show that the coumarin-labeled inhibitor can be used to image PI4KB
in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.