Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery, Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
Chemical Genomics Facility, Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA.
Int J Mol Sci. 2021 Jan 8;22(2):594. doi: 10.3390/ijms22020594.
-terminal acetylation catalyzed by -terminal acetyltransferases (NATs) has various biological functions in protein regulation. -terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z'-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.
-末端乙酰化由 -末端乙酰基转移酶（NATs）催化，在蛋白质调节中具有多种生物学功能。NAT 中的 NatD 是最具特异性的一种，仅将组蛋白 H4 和 H2A 蛋白作为已知的底物。NatD 的失调与结直肠癌和肺癌的进展有关，这表明它在癌症治疗方面具有潜力。然而，目前还没有报道 NatD 的抑制剂。为了促进小分子 NatD 抑制剂的发现，我们通过监测辅酶 A 的形成，报告了在 384 孔高通量筛选（HTS）格式中基于荧光的乙酰基转移酶测定法的开发。荧光信号是在辅酶 A 与荧光探针 ThioGlo4 反应中形成的加合物产生的。该测定法的 Z'-因子为 0.77，变异系数为 6%，表明它是一种用于 HTS 的稳健测定法。对 1280 种具有药理活性的化合物进行的初步筛选和后续验证确定了两个命中物，证实了该荧光测定法在 HTS 中的应用。
