Stabilizing HDAC11 with SAHA to assay slow-binding benzamide inhibitors |
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Authors: | Yinping Tian Wenhui Lv Xuewei Li Congying Wang Dayuan Wang Peng G Wang Jin Jin Jie Shen |
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Institution: | 1. College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic of China;2. State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People’s Republic of China |
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Abstract: | Among 18 human histone deacetylases (HDAC), HDAC11 is least studied. MS275, a benzamide HDAC inhibitor (HDACi), was stereotypically considered to selectively target Class I HDACs. We verified this slow-binding inhibitor also targeted HDAC11. In a traditional enzyme based assay, MS275 at low concentrations surprisingly behaved as an agonist. This was attributed to the poor stability of HDAC11 which lost 40% activity in 3 h at 37 °C. By adding 0.2 μM SAHA, HDAC11 activity was stabilized during the 3-h assay period. Since 0.2 μM SAHA inhibited 50% HDAC11 activity, the apparent IC50′ of MS275 was adjusted to the true IC50 = 0.65 μM. Finally, the new method demonstrated its superiority in one-dose-screening assays by decreasing false negative results. This work highlighted an optimized strategy to assay slow-binding inhibitors of unstable proteins with known fast-binding inhibitors. It should be especially useful in a hit-discovery stage to find moderate potent compounds. |
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Keywords: | Inhibitor screening Slow-binding Unstable protein HDAC11 Chaperone molecule |
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