br Materials and methods br Results

Materials and methods
Results
Discussion Although no increase in DAPK activity was detected at early time points associated with neuronal apoptosis, it is possible that the spread of apoptosis through the KH CB19 following HI is not synchronized enough to yield a large, consistent fluctuation in DAPK activity that would be quantifiable by our assay. However, small changes in kinase activity at an early point in a signal transduction cascade can be amplified by orders of magnitude [44], so inhibition of small increases in an early step in signal transduction pathways that are true biochemical cascades can result in profound biological effects. Furthermore, the mechanism of cell death in HI occurs along a spectrum from apoptosis to necrosis, varying with the severity of the ischemic insult [50]. The severity of the hypoxic-ischemic insult delivered to the animals in our experiments may have resulted in predominant necrotic cell death in the early phase of the cellular response. In this case, the contribution of DAPK catalytic activity to the process of apoptotic cell death would be overwhelmed by necrosis, and hence be less readily detectable using our methodology. Therefore, we cannot make any conclusions about the presence or absence of a transient increase in DAPK activity in the early time window following hypoxic-ischemic injury. Indeed, a report [14] of DAPK activity regulation in PC12 cells in response to neuronal apoptosis induced by treatment with ceramide demonstrates a rapid increase in DAPK catalytic activity that quickly returns to basal levels. In addition, there is precedent for the presence of temporal waves of protein kinase activity following cerebral ischemia. For example, activity of the pro-survival kinase Akt has been shown to increase in the cerebral cortex during reperfusion in the hours following transient focal ischemia, as well as in a 4- to 7-day period after injury [51]. Although cellular responses in the injured brain are probably more synchronized by the 7-day time point where we detected an increase in DAPK specific activity, the observed increase in activity may be a minimal estimate due to the inherent complexity of the in vivo injury model and the potential presence of rapid, temporal variations in kinase activity. Regardless, the phenomenon of increased DAPK activity was confirmed in a more controlled cell model of neuronal anabolic responses to a physiological stimulus, NGF treatment of PC12 cells. The set of protein substrates involved in this function of DAPK will most likely be distinct from those involved in other proposed roles for DAPK, and the results with MLC phosphorylation are consistent with this possibility. Determining the role of DAPK in animal models of disease, such as the rat model of perinatal hypoxic-ischemic injury, can best be facilitated by the use of bioavailable small molecule inhibitors of DAPK. For example, a potential DAPK activity responsible for mediating initial cell death following HI might be blocked by pretreatment with small-molecule inhibitors of DAPK, possibly resulting in decreased tissue loss. Such feasibility data would be consistent with the presence of a DAPK-mediated cell death pathway following injury, and would be useful in the validation of DAPK as a potential therapeutic target for stroke [2], [52]. Our results indicate that development of bisubstrate site targeted inhibitors might be a viable approach to the discovery of small molecule, bioavailable inhibitors of DAPK. The ATP site of protein kinases is a validated drug discovery target [53], [54], and the novel features of the ATP site of DAPK are attractive. However, the smaller volume of this site in DAPK may preclude full exploitation of this site as has been done with other kinases. The potential for exploiting the triphosphate tunnel that connects the ATP and the peptide substrate recognition sites is one that has not been actively pursued in kinase inhibitor development, but the docking results raise this as a possible site. The docking results also suggest targeting of the tunnel region in conjunction with the peptide recognition region. The ability to exploit the peptide recognition site of protein kinases, which has inherently more potential selectivity, using structure assisted approaches is limited by the molecular topology and the demand by most docking programs for more well-defined ligand binding sites. Our docking results identify two ligand binding sites, the ATP and triphosphate tunnel sites, that are preferred by design algorithms and make use of the novel 3-aminopyridazine template discovered with other CaMKs [32], [46]. Taken together, the prior proof-of-principle, in which chemical genomics hits were refined into first generation compounds by use of in-parallel syntheses and enzyme assay screens, and the docking results indicate the feasibility for discovery of selective DAPK inhibitors for use in future investigations of HI induced brain injury.