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 Ca(2+)-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.
Department of Pharmacology and Cancer Biology, Duke University Medical Center , Durham, North Carolina 27710, United States.
David A
DA
Carlson
Aaron S
AS
Franke
Douglas H
DH
Weitzel
Brittany L
BL
Speer
Philip F
PF
Hughes
Laura
L
Hagerty
Christopher N
CN
Fortner
James M
JM
Veal
Thomas E
TE
Barta
Bartosz J
BJ
Zieba
Avril V
AV
Somlyo
Cindy
C
Sutherland
Jing Ti
JT
Deng
Michael P
MP
Walsh
Justin A
JA
MacDonald
Timothy A J
TA
Haystead
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D064550:Death-Associated Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007082:Ileum / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010766:Phosphorylation; D061251:Primary Cell Culture; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D040901:Proteomics; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D011744:Pyrimidinones / Q000737:chemistry / Q000494:pharmacology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
C000590333:(2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo(3,4-d)pyrimidin-6-yl)thio)propanamide; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011744:Pyrimidinones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; C579096:DAPK1 protein, human; C579100:Dapk3 protein, mouse; D064550:Death-Associated Protein Kinases; D009219:Myosin-Light-Chain Kinase; D043263:Myosin-Light-Chain Phosphatase; C510645:Ppp1r12a protein, mouse; D002118:Calcium