DNA
methyltransferases (DNMTs) are important enzymes involved in
epigenetic control of gene expression and represent valuable targets
in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi)
have been studied in cancer, including in cancer stem cells, and two
of them (azacytidine and decitabine) have been approved for treatment
of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi
have been identified so far, and even fewer have been validated in
cancer. Through a process of hit-to-lead optimization, we report here
the discovery of compound 5 as a potent non-nucleoside
DNMTi that is also selective toward other AdoMet-dependent protein
methyltransferases. Compound 5 was potent at single-digit
micromolar concentrations against a panel of cancer cells and was
less toxic in peripheral blood mononuclear cells than two other compounds
tested. In mouse medulloblastoma stem cells, 5 inhibited
cell growth, whereas related compound 2 showed high cell
differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem
cell line.
Image-based profiling has emerged as a powerful technology for various steps in basic biological and pharmaceutical discovery, but the community has lacked a large, public reference set of data from chemical and genetic perturbations. Here we present data generated by the Joint Undertaking for Morphological Profiling (JUMP)-Cell Painting Consortium, a collaboration between 10 pharmaceutical companies, six supporting technology companies, and two non-profit partners. When completed, the dataset will contain images and profiles from the Cell Painting assay for over 116,750 unique compounds, over-expression of 12,602 genes, and knockout of 7,975 genes using CRISPR-Cas9, all in human osteosarcoma cells (U2OS). The dataset is estimated to be 115 TB in size and capturing 1.6 billion cells and their single-cell profiles. File quality control and upload is underway and will be completed over the coming months at the Cell Painting Gallery: https://registry.opendata.aws/cellpainting-gallery. A portal to visualize a subset of the data is available at https://phenaid.ardigen.com/jumpcpexplorer/.
DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.
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