Troelsen Kathrin S, Bæk Michael, Nielsen Alexander L, Madsen Andreas S, Rajabi Nima, Olsen Christian A
Center for Biopharmaceuticals & Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Denmark
RSC Chem Biol. 2021 Feb 1;2(2):627-635. doi: 10.1039/d0cb00216j. eCollection 2021 Apr 1.
Sirtuin 3 (SIRT3) is the major protein lysine deacetylase in the mitochondria. This hydrolase regulates a wide range of metabolically involved enzymes and has been considered as a potential drug target in certain cancers. Investigation of pharmacological intervention has been challenging due to a lack of potent and selective inhibitors of SIRT3. Here, we developed a strategy for selective inhibition of SIRT3 in cells, over its structurally similar isozymes that localize primarily to the nucleus (SIRT1) and the cytosol (SIRT2). This was achieved by directing the inhibitors to the mitochondria through incorporation of mitochondria-targeting peptide sequences into the inhibitor structures. Our inhibitors exhibited excellent mitochondrial localization in HeLa cells as indicated by fluorophore-conjugated versions, and target engagement was demonstrated by a cellular thermal shift assay of SIRT3 using western blotting. The acetylation state of documented SIRT3 target MnSOD was shown to be increased in cells with little effect on known targets of SIRT1 and SIRT2, showing that our lead compound exhibits selectivity for SIRT3 in cells. We expect that the developed inhibitor will now enable a more detailed investigation of SIRT3 as a potential drug target and help shed further light on the diverse biology regulated by this enzyme.
沉默调节蛋白3（SIRT3）是线粒体中主要的蛋白质赖氨酸脱乙酰酶。这种水解酶调节多种参与代谢的酶，并被认为是某些癌症中的潜在药物靶点。由于缺乏强效且选择性的SIRT3抑制剂，药物干预的研究一直具有挑战性。在此，我们开发了一种在细胞中选择性抑制SIRT3的策略，相对于其结构相似且主要定位于细胞核的同工酶SIRT1和定位于细胞质的同工酶SIRT2。这是通过将线粒体靶向肽序列掺入抑制剂结构中，从而将抑制剂导向线粒体来实现的。如荧光团偶联形式所示，我们的抑制剂在HeLa细胞中表现出出色的线粒体定位，并且通过使用蛋白质印迹法对SIRT3进行细胞热迁移分析证明了靶点结合。已记录的SIRT3靶点锰超氧化物歧化酶（MnSOD）的乙酰化状态在细胞中显示增加，而对SIRT1和SIRT2的已知靶点影响很小，这表明我们的先导化合物在细胞中对SIRT3具有选择性。我们预计，所开发的抑制剂现在将能够更详细地研究SIRT3作为潜在药物靶点，并有助于进一步阐明该酶所调节的多种生物学过程。
