Structure–activity relationship of novel DAPK inhibitors identified by structure-based virtual screening
Graphical abstract
An in silico approach lead to the efficient discovery of novel potent and selective DAPK inhibitors.
Introduction
DAP-kinase (DAPK), a Ca2+/calmodulin activated Ser/Thr kinase, was originally identified by Adi Kimchi and co-workers at the Weizmann Institute while screening for genes critical for IFN-γ-induced cell death.1 DAPK belongs to a family of related death kinases, all of which share significant sequence and functional homology. This family includes two closely related homologues of DAPK (DAPK1): ZIPK [ZIP kinase, also known as Dlk (DAP-like kinase) or DAPK3] and DRP-1 (DAPK-related protein 1, also known as DAPK2). There are two other protein kinases which display homology to DAPK: DRAK-1 and DRAK-2 (DAPK-related apoptosis-inducing protein kinase-1 and -2), although these are more distantly related2 and have been less well characterized.
DAPK is necessary for the regulation or execution of cell death in response to various stimuli, including death receptor activation, cytokines, matrix detachment, ceramide,3 and others. DAPK is linked to both type I apoptotic and type II autophagic cell death in both caspase-dependent and caspase-independent manners.2
Shamloo et al. showed that DAPK plays an important role in ischemic rat brain injuries. They characterized the mechanisms of DAPK activation during ischemia and showed that DAPK is dephosphorylated and activated following ischemia in the brain. Based on their findings, Shamloo et al. suggested that DAPK could be a good therapeutic target for treating acute ischemic stroke.4
Inhibition of DAPK would likely intercept cell death and prevent further damage of ischemic regions in cerebral infarction and other ischemic diseases. Interestingly, Velentza et al. reported that a single intraperitoneal injection of a DAPK non-selective inhibitor, an alkylated 3-amino-6-phenylpyridazine, reduced in vivo brain injury in an animal model when administered 6 h after the insult, which is the minimal desired therapeutic time window.5 Although there have been only a few detailed studies of DAPK as a drug target, it would be useful to have more potent and selective inhibitors against DAPK in order to determine their possible application to unmet needs.
We have previously reported DAPK inhibitors discovered through our structure-based virtual screening (SBVS) research program.6 In this paper, we describe the general protocol of our in silico approach, and the strategy used to develop hit compounds. In addition, the studies conducted on the structure–activity relationship (SAR) of DAPK inhibitors are discussed in detail.
Section snippets
Structure-based virtual screening
The general protocol of our in silico approach using structure-based virtual screening is shown in Figure 1. First, for more efficient virtual screening, we typically use several hundreds of thousands of compounds that have been refined using drug-likeness filtering and clustering from commercially available compound databases.7 Second, we generate protein–ligand complex models using MultiCopyMD,6, 8 an in-house molecular dynamics simulation program. Third, using the model protein structure, we
Conclusion
After obtaining the first series of hit compounds, a similarity search and a substructure search were used to obtain more potent compounds. The binding mode of the active compounds was predicted using CONSENSUS-DOCK, an in-house docking calculation program, and the structure–activity relationship was analyzed using solvated interaction energy calculations at the ATP binding site. The strategy described in this paper lead to the efficient identification of novel potent DAPK inhibitors.
Compounds
The database of commercially available compounds was produced by Namiki Shoji Co., Ltd (http://www.namiki-s.co.jp/) and Summit Pharmaceuticals International (SPI; http://www.summitpharma.co.jp/). The databases include compounds produced by several suppliers. Tested compounds in this paper were purchased from several suppliers (Enamine, ChemDiv, etc.). Their characterizations and purity were confirmed using 1H NMR and HPLC.
Similarity search
We calculated a fingerprint based on the descriptors of the BIT_MACCS:
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2016, GeneCitation Excerpt :DAPK3 (also referred to as Zipper-interacting protein kinase (ZIPK)), a Ca2 +/Calmodulin activated Ser/Thr kinase, belongs to the family of DAPK (death-associated protein kinases) and shares significant sequence and functional homology with other members of the family (Van Eldik, 2002; Okamoto et al., 2010; Bialik and Kimchi, 2006). DAPK family is majorly involved in the regulation of cell death in response to various stimuli, including activation of death associated receptors, cytokines and cell detachment from the extracellular matrix (Okamoto et al., 2010; Bialik and Kimchi, 2006). They have been associated with apoptosis (type I) and autophagy (type II), programmed cell death in either a caspase-dependent or caspase-independent manner (Van Eldik, 2002; Okamoto et al., 2010).
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2015, International Journal of Biochemistry and Cell BiologyCitation Excerpt :Second, orthologue-specific DAPK inhibitors should be developed. Currently, DAPK inhibitors were described by two independent research groups (Huang et al., 2014; Okamoto et al., 2009, 2010; Velentza et al., 2003), which seem to target the different DAPK family members at similar IC50s (Geering et al., 2014). Third, animals with a genetic ablation of DAPK2 must be challenged with disease models associated with the hematopoietic lineage (cancer and inflammation) in order to gain insight into the range of diseases worth targeting in a DAPK2-specific manner.