RESUMEN
BACKGROUND: G9a is a histone H3K9 methyltransferase enzyme found highly upregulated in many cancers. H3 binds to the rigid I-SET domain and the cofactor, S-adenosyl methionine, binds to the flexible post-SET domain of G9a. Inhibition of G9a is known to inhibit the growth of cancer cell-lines. METHODS: Recombinant G9a and H3 were used to develop radioisotope-based inhibitor screening assay. The identified inhibitor was evaluated for isoform selectivity. The mode of enzymatic inhibition was studied by enzymatic assays and bioinformatics. Anti-proliferative activity of the inhibitor was studied in cancer cell lines by utilizing MTT assay. The mechanism of cell death was studied by western blotting and microscopy. RESULTS: We developed a robust G9a inhibitor screening assay that led to the discovery of SDS-347 as a potent G9a inhibitor with IC50 of 3.06 µM. It was shown to reduce the levels of H3K9me2 in cell-based assay. The inhibitor was found to be peptide competitive and highly specific as it did not show any significant inhibition of other histone methyltransferases and DNA methyltransferase. Docking studies showed that SDS-347 could form direct bonding interaction with Asp1088 of the peptide-binding site. SDS-347 showed anti-proliferative effect against various cancer cell lines especially the K562 cells. Our data suggested that SDS-347 mediated antiproliferative action via ROS generation, induction of autophagy and apoptosis. CONCLUSION: Overall, the findings of the current study include development of a new G9a inhibitor screening assay and identification of SDS-347, as a novel, peptide competitive and highly specific G9a inhibitor with promising anticancer potential.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Neoplasias , Humanos , N-Metiltransferasa de Histona-Lisina/metabolismo , Histona Metiltransferasas , Péptidos , Línea CelularRESUMEN
Euchromatic histone lysine methyltransferase-2, also known as G9a, is a ubiquitously expressed SET domain-containing histone lysine methyltransferase linked with both facultative and constitutive heterochromatin formation and transcriptional repression. It is an essential developmental gene and reported to play role in embryonic development, establishment of proviral silencing in ES cells, tumor cell growth, metastasis, T-cell immune response, cocaine induced neural plasticity and cognition and adaptive behavior. It is mainly responsible for carrying out mono, di and tri methylation of histone H3K9 in euchromatin. G9a levels are elevated in many cancers and its selective inhibition is known to reduce the cell growth and induce autophagy, apoptosis and senescence. We carried out a thorough search of online literature databases including Pubmed, Scopus, Journal websites, Clinical trials etc to gather the maximum possible information related to the G9a. The main messages from the cited papers are presented in a systematic manner. Chemical structures were drawn by Chemdraw software. In this review, we shed light on current understanding of structure and biological activity of G9a, the molecular events directing its targeting to genomic regions and its post-translational modification. Finally, we discuss the current strategies to target G9a in different cancers and evaluate the available compounds and agents used to inhibit G9a functions. The review provides the present status and future directions of research in targeting G9a and provides the basis to persuade the development of novel strategies to target G9a -related effects in cancer cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Epigénesis Genética/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Animales , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Terapia Molecular Dirigida , Neoplasias/enzimología , Neoplasias/patologíaRESUMEN
Sildenafil, a phosphodiesterase-5 inhibitor is FDA approved drug against erectile dysfunction. It is currently undergoing many clinical trials, alone or in combinations against different diseases. Treatment of neural progenitor cells with sildenafil is known to regulate their basal cGMP levels and enhance neurogenesis and differentiation. cGMP as well as cAMP are known to play a central role in the maintenance, repair and remodelling of the nervous system. In the present study, we report the neurodifferentiation property of sildenafil in neuroblastoma cancer cell line IMR-32. Sildenafil was found to induce the formation of neurite outgrowths that were found expressing neuronal markers, such as NeuN, NF-H and ßIII tubulin. IS00384, a recently discovered PDE5 inhibitor by our laboratory, was also found to induce neurodifferentiation of IMR-32 cells. The effect of IS00384 on differentiation was even more profound than sildenafil. Both the compounds were found to elevate and activate the Guanine nucleotide exchange factor C3G, which is a regulator of differentiation in IMR-32 cells. They were also found to elevate the levels of cGMP and activate the AMPK-ACC and PI3K-Akt signalling pathways. These pathways are known to play important role in cytoskeletal rearrangements necessary for differentiation. This study highlights the role of phosphodiesterases-5 in neurodifferentiation and use of sildenafil and IS00384 as small molecule tools to study the process of cellular differentiation.