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

Abstract

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 and 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.

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle

“…HS38 appeared to attenuate the velocity of the contractile responses, whereas the steady-state level of force induced by CLa was unaffected by ZIPK inhibition (Fig. 1, C and D), a result that was previously demonstrated (Carlson et al, 2013). As shown in Table 1, the mean steady-state force induced by CLa was ∼160% of that induced by KCl (87 mM) and was unaffected by pretreatment with HS38.…”

“…The pyrazolo [3,4-d]pyrimidinone derivative HS38 was previously characterized as a potent inhibitor of ZIPK (Carlson et al, 2013). HS38 acts as a competitive inhibitor with respect to ATP and was reported to be most potent toward ZIPK (K d 5 280 nM), DAPK1 (K d 5 300 nM), and proviral integrations of Moloney virus 3 (PIM3) (K d 5 810 nM), 10-fold less potent against IRAK4 and PIM1, 100-fold less potent against PIM2 and MLCK, and inactive against ROCK2.…”

“…HS38 and 1-(3-chlorophenyl)-6-((2-hydroxyethyl)thio)-1,5-dihydro-4H-pyrazolo [3,4-d]pyrimidin-4-one (HS43) were synthesized in the Haystead Laboratory at Duke University as previously described (Carlson et al, 2013). Only trace impurities were identified (,1% by liquid chromatography coupled to mass spectrometry).…”

“…In contrast to MLCK and the other DAPKs, ZIPK does not have a calmodulin-binding domain, and its activity is regulated independently of Ca 21 . ZIPK appears to be a key regulator of VSM contractility, and the kinase is implicated in diphosphorylation of the myosin 20-kDa regulatory light chain (LC 20 ) subunits of myosin II at Ser19 and Thr18 (Murata-Hori et al, 1999;Niiro and Ikebe, 2001;Borman et al, 2002;Moffat et al, 2011;Carlson et al, 2013); phosphorylation of the myosin phosphatase-targeting subunit 1 (MYPT1) of myosin light chain phosphatase (MLCP) at the inhibitory sites, Thr697 and Thr855 (Endo et al, 2004;MacDonald et al, 2001a); and phosphorylation of the protein kinase C (PKC)-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17) at Thr38 (MacDonald et al, 2001b). Ultimately, these targets are thought to be the primary mediators of ZIPK effects on VSM contraction; however, several novel ZIPK regulators have been revealed in recent years.…”

“…Four independent groups have recently reported the development of novel ZIPK inhibitory compounds: 1) Velentza et al (2003) reported the first small molecule DAPK inhibitor, an alkylated 3-amino-6-phenylpyridazine; 2) Okamoto et al (2009Okamoto et al ( , 2010 used structure-based, virtual screening to identify DAPK1 and ZIPK inhibitors with a 2-phenyl-4-(3-pyridinylmethylene)-5(4H)-oxazolone core; 3) Huber et al (2012) identified inhibitory oxo-b-carboline compounds that do not rely on canonical ATP competition; and 4) Carlson et al (2013) used a chemoproteomic fluorescence linked enzyme chemoproteomic strategy screening to identify an aryl-thiopyrazolo [3,4-d]pyrimidinone-i.e., 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38)-that competitively inhibits ZIPK. HS38 displays similar or greater potency for ZIPK and has fewer off-target liabilities than other published ZIPK inhibitors (Carlson et al, 2013). The availability of a cell-permeable, potent, and selective inhibitor provides a novel opportunity to examine aspects of ZIPK signaling in VSM that were previously not attainable, and we have used HS38 to study the mechanistic role of ZIPK in Ca 21 sensitization of VSM.…”