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  • We have previously reported DAPK inhibitors discovered throu

    2020-05-22

    We have previously reported DAPK inhibitors discovered through our structure-based virtual screening (SBVS) research program. 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.
    Results and discussion
    Conclusion
    Experimental
    Introduction An epigenetic pathway to colorectal neoplasia is CpG island methylation of genes regulating the balance between cell growth and death. Hypermethylation of CpG islands in promotor regions were found to be a major mechanism of inactivation of tumor suppressor genes in human cancer [1], [2], [3]. Death-associated protein kinase (DAPK) was discovered as a novel 160-kD calmodulin-dependent serine/threonine kinase operating as a positive mediator of apoptosis [4]. DAPK is localized to the Melittin synthesis and carries a kinase domain with 11 subdomains, eight ankyrin repeats, two P-loop motifs, a microfilament-binding domain and a death domain near the C-terminal end [5], [6]. It participates in TNF-α-, Fas- and IFN-γ-induced apoptosis [5], [7]. Lung carcinoma clones (in murine models) with high metastatic behavior did not express DAPK, whereas their low-metastatic counterparts expressed the protein [8]. Therefore, DAPK may Melittin synthesis function as a suppressor of metastatic activity. Overexpression of DAPK induces apoptosis, whereas loss of its function leads to protection against programmed cell death [5]. A repressed expression of DAPK by hypermethylation in the promotor CpG region of gene has been shown for various human cancers [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Previous studies have already investigated the methylation status of DAPK in primary colorectal cancer [19], [20], [21].
    Materials and methods
    Results
    Discussion The frequency of DAPK promotor methylation (81.8%) proven in macrodissected tissue samples of carcinomas was found to be higher than reported previously. The frequency given in other studies of colorectal tumors varied between 19 and 55% [19], [20]. The higher detection rate in our study might be due to the macrodissection preparation method of formalin-fixed, paraffin-embedded tissue blocks. There were only two cases in which aberrant methylation was present in both the tumor and the adjacent colon mucosa from the same patient. Given the sensitivity of MSP, it is possible that normal-appearing specimens contained a rare cancer cell population that was undetectable by histomorphology. The fact that, in one case, methylation was found only in the tumor-adjacent mucosa and not in the corresponding tumor-distant one is consistent with the hypothesis that the cancer in this individual originated from a methylated clonal precursor. In support of this hypothesis, a recent study documented p16 promotor methylation in pathologically normal-appearing esophageal specimens obtained from a patient who later developed Barrett\'s metaplasia [23]. Also, in other epithelial tumors, DAPK promotor methylation was observed rather occasionally in non-malignant cells [24], [25], indicating the aberrant methylation of the DAPK promotor as a tumor-dependent phenomenon. It is noteworthy that there were no tumors showing exclusively the methylated PCR-product. There may be two explanations for this phenomenon: (i) the specimen contained heterogeneous tissue of both methylated and unmethylated promotors and (ii) only one allele of the DAPK gene was hypermethylated. At this point, we cannot differentiate between both hypotheses. If there is one wild-type allele still available, it should be able to produce also a gene product at a low level as described in the Knudson theory of tumor suppressor gene inactivation. For the p16 tumor suppressor gene, it has been reported that methylation, as one of the two genetic hits, cooperates with mutations or loss of heterozygosity and completely inactivates the p16 protein function [26], [27]. It still needs to be clarified whether there are mutations in the DAPK gene that act as primary genetic hits, although deletions of the DAPK promotor region have been described in pituitary tumors [15]. Possibly, loss or reduced amounts of DAPK protein caused by promotor methylation may be one important step in abrogating apoptosis and may be the precondition for further accumulation of other additional genetic aberrations. Also for invasive pituitary tumors, co-operating aberrations other than DAPK promotor methylation are necessary to complete the final transition into a metastatic tumor [15]. Otherwise, the high frequency of DAPK methylation in T1-carcinomas demonstrates that methylation of the DAPK gene occurs independently of the metastasis. This is in contrast to studies of head and neck cancer, where the presence of DAPK promotor methylation was correlated with lymph node involvement and advanced stage of disease [28]. A more detailed study on a colon cancer group with known clinical follow-up would shed light on this matter.