Abstract：
Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing\'s sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug\'s mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.
摘要：
赖氨酸特异性脱甲基酶-1（LSD1）作为转录调节因子，代表抗癌治疗的有希望的表观遗传靶标。LSD1抑制剂正在临床试验中，用于治疗尤因肉瘤（EWS），急性髓系白血病,和小细胞肺癌，强大的抑制剂的开发需要准确的方法来探测去甲基化，效力，效力和选择性。这里,检查了对H3K4me2肽和核小体底物的抑制动力学，比较可逆[CC-90011(PD)和SP-2577(SD)]和不可逆[ORY-1001(ID)和反式环丙胺(TCP)]抑制剂存在下的去甲基化率。抑制剂还在三种人细胞系中进行了活力研究，并进行了蛋白质印迹分析，以监测EWS（TC-32）细胞中的H3K4me2核小体水平。实现药物效力的相关性，体外抑制，和基于细胞的研究。例如,SP-2577，一种用于EWS临床试验的药物，使用间接偶联测定法抑制小肽底物（Ki=60±20nM）的活性，但使用直接Western印迹方法不抑制H3K4me2肽或核小体的去甲基化。此外,该药物对TC-32细胞中的H3K4me2水平没有影响。这些数据表明，SP-2577不是LSD1酶抑制剂，尽管由于其在TC-32细胞中的细胞毒性选择性，该药物可能不依赖于去甲基化。一起来看,这项工作突出了使用耦合分析来赋予药物的作用模式的陷阱，强调在表观遗传药物靶向策略中使用生理相关底物，并提供了对LSD1底物选择性抑制剂的开发的见解。
