Identification Of Novel Cell Active SIRT1 Inhibitors,And Activity Evaluation Of JARID1B In Vitro

As an important branch in epigenetics, histone modification including methylation, acetylation, phosphorylation and so forth, can modulate gene expression and repression.Histone deacetylase(HDAC) and histone acetyltransferase(HAT), acting as two families of the histone modification regulators, work in cooperation on keeping global histone acetylation patterns. Histone deacetylase(HDAC) has been characterized three families as HDAC I, HDAC II and Sirtuins(HDACIII). Sirtuins are distinct different with HDAC I and II, because they can deacetylate substrates in the NAD-dependent manner. To the date, 7 homologues of Sirtuin family have been discovered in mammals, displaying various targets, cell functions and sub-cellular localizations. Silent mating type information regulation 2 homolog 1(SIRT1), the best characterized Sirtuin member in human, shuttles between nucleus and cytoplasm. Different location allows SIRT1 deacetylating not only histone substrates such as H1K26, H3K9 and H4K16, but also a large spectrum of transcription factors and cofactors, containing IR、 p53、 FOXO、 IRS、 PPARγ and so on. The interaction between SIRT1 and tumor suppressor p53 implicates SIRT1’s function in regulating tumorgenesis. The SIRT1 inhibitors are explored in human breast cancer, colon cancer, prostate cancer, chronic myelogenous leukemia, lung cancer and so on. These results provide evidence that SIRT1 can function as an oncogene in the tumor formation and chemotherapy drug resistance.In addition to histone acetylation, the status of histone lysine methylation plays a vital role in the gene expression, too. The first reported histone lysine-specific demethylase(KDM) was KDM1 A, which was published in the journal of Cell by Prof. Yang Shi and his team. So far lysine-specific demethylases ever reported have been divided into two distinct families. The one family is amine oxidase, such as KDM1 A, which removes the methyl group through a FAD-dependent amine oxidation reaction. And the other one is oxygenase, represented by JARID1 B, use Fe(II) and α-keto-glutarate as cofactors to demethylate substrates. JARID1 family has characteristic JmjC, Jmj N, PHD and ARID domain; they can demethylate H3K4me2 and H3K4me3 in vitro, and the demethylation of H3K4me1 is only available in vivo, which indicates additional factors. There are four members in JARID1 family for mammals, known as JARID1 A, JARID1 B, JARID1 C and JAIRD1 D. JARID1 B protein interacts with a large number of oncogene. It is shown to be over-expressed in breast cancer, melanoma cancer, gastric cancer, prostate cancer and bladder cancer as oncogene.In general, a group of novel 1,2,3-triazole–dithiocarbamate hybrids were obtained in our previous experiment, and they were identified as weak anticancer agents by our group. With molecular docking and biochemical assay, most of them were characterized to inhibit SITR1 potently in recombinant level. In the cellular level, the most potent compound 3a increased the expressing level of p53 K382 Ac amount by targeting SIRT1, and induced H4K16 Ac accumulation. Furthermore, compound 3a inhibited SIRT2 in the cellular level, which led to the increasing amount of acetylation at alpha-tubulin(K40). These findings indicate that compound 3a is a non-selective SIRT1/2 inhibitor. Meanwhile, we find that the compound 3a can upregulate the amount of p53 by accumulating the K382 acetylation of p53, which lead to the stabilization of p53 in human gastric cancer cell line MGC-803 cells. Refer to the vital roles of SIRT1 and p53 in tumorgenesis, further investigation is needed as an anticancer agent or a biological tool.And we have constructed a prokaryotic expression vector of JARID1 B with C-terminal truncated, the fusion recombinant protein JARID1 B has a molecular weight about 97 kDa. After the construction of plasmid pET30a-JARID1 B, we transform it to the Rosetta(DE3) strain, which is a kind of E coli that can efficiency express exogenous fusion protein by genetic engineering. The JARID1 B direct assay protocol in vitro was established through optimized fluorescent assay coupled with FDH(formaldehyde dehydrogenase). The assay platform is applied with the compound library of our lab, from which several potent compounds have been discovered in the screening of other therapeutic drug targets. The screening of new, potent, specific modulators of JARID1 B is underway.