Fluorescence Linked Enzyme Chemoproteomic Strategy for Discovery of a Potent and Selective DAPK1 and ZIPK Inhibitor

DAPK1 and ZIPK (also called DAPK3)
are closely related serine/threonine
protein kinases that regulate programmed cell death and phosphorylation
of non-muscle and smooth muscle myosin. We have developed a fluorescence
linked enzyme chemoproteomic strategy (FLECS) for the rapid identification
of inhibitors for any element of the purinome and identified a selective
pyrazolo­[3,4-d]­pyrimidinone (HS38) that inhibits
DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations.
In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated
in mouse aorta, rabbit ileum, and calyculin A stimulated arterial
muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor
also promoted relaxation in Ca2+-sensitized vessels. A
close structural analogue (HS43) with 5-fold lower affinity for ZIPK
produced no effect on cells or tissues. These findings are consistent
with a mechanism of action wherein HS38 specifically targets ZIPK
in smooth muscle. The discovery of HS38 provides a lead scaffold for
the development of therapeutic agents for smooth muscle related disorders
and a chemical means to probe the function of DAPK1 and ZIPK across
species.