Design Of SIRT6 Allosteric Activator And Study Of Its Anti-HCC Activity

Sirtuin is a family of mammalian proteins that homologue to Sir2(Silent information regulator 2)in budding yeast S.cerevisiae,belong to class?histone deacetylases(HDACs),which is a cluster of nicotinamide adenosine dinucleotide(NAD~+)dependent deacetylases.Mammalian sirtuins has seven members,SIRT1-SIRT7.SIRT6 is a multifunctional enzyme of sirtuin family that has been reported to efficiently catalyze the defatty-acylation and ADP-ribosyltransferase activities on substrates besides its NAD~+dependent deacetylation activity.SIRT6 is an important factor involved in a variety of physiological and pathological processes,such as Aging and longevity,glycolipid metabolism,DNA damage repair and tumor suppression.NAD~+dependent deacetylation activity,defatty-acylation and ADP-ribosyltransferase activities of SIRT6 have its unique substrates.The classic substrates of deacetylation activity of SIRT6 are N~?-acetyllysines 9 and 56 of histone H3(H3K9Ac and H3K56Ac).SIRT6 binding to transcription factors such as NF-?B?HIF-1??c-Myc regulates their transcriptional activities through remove N~?-acetyllysines 9 and 56 of histone H3 in promoter regions of these transcription factors'target genes.The reported long chan fatty acylation substrates of SIRT6 including TNF?and R-Ras2;the ADP-ribosyltransferase substrates of SIRT6 including PARP1 and SIRT6 itself.There has been a lot of evidence that SIRT6 exists as a tumor suppressor in most solid tumors.SIRT6 expression was down-regulated in liver cirrhosis and hepatocellular carcinoma specimens,SIRT6 dependent genetic and epigenetic alterations are associated with poor clinical outcome in hepatocellular carcinoma patients.Therefore,activate the deacetylase activity of SIRT6 in liver cancer cells may have potential therapeutic value.Herein,we using the computational tool in ASD,a allosteric database developed by our laboratory,identify potential allosteric regulatory site of SIRT6 based on the protein structure of SIRT6.A small-molecule activator MDL-800 was obtained combining virtual and experimental screening at this site afterwards medicinal chemistry structure optimization.MDL-800 directly binding and activated SIRT6 deacetylation but no effect on its defatty-acylation and ADP-ribosyltransferase activities.Mechanically MDL-800increasing the binding affinities of acetylated substrates and cofactor as well as elevating the catalytic efficiency of SIRT6.MDL-800 was selective against numerous other histone deacetylase(HDAC)enzymes.Structural and mutagenesis data show the direct binding of MDL-800 to a surface allosteric site of SIRT6.MDL-800 decreased both H3K9Ac and H3K56Ac levels in liver cancer cells,inhibited the growth inhibition of human hepatocellular carcinoma cells via cell cycle arrest.MDL-800 has SIRT6-dependent antitumor activity that inhibits tumor growth in mouse model of subcutaneous transplantation of hepatocellular carcinoma cells.Collectively,we designed MDL-800 is first cellular active deacetylation small molecule activator of SIRT6,MDL-800 binding to allosteric site of SIRT6 and activate its deacetylation specifically.MDL-800 has good selectivity in vitro,and in vivo.It can inhibit the cell growth of hepatocellular carcinoma cells by cell cycle arrest,may advance our understanding of SIRT6 deacetylation in more physiological and pathological processes.