CX5461 is an rRNA synthesis inhibitor with oral activity that inhibits Pol I-driven rRNA transcription. CX5461 activates the DNA damage response and has antitumor activity in tumors such as ovarian cancer.

Pol I-driven transcription of rRNA:142 nM

方法：HCT-116、A375、MIA PaCa-2 和 BJ hTERT 细胞用 CX5461 (0-10 µM) 处理 96 h，通过 CyQUANT assay 检测细胞活力。
结果：HCT-116、A375 和 MIA PaCa-2 细胞系的抗增殖剂量反应评估分别得出了 167、58 和 74 nmol/L 的 EC50。BJ-hERT 正常细胞系的 EC50 约为 5000 nmol/L。[1]
方法：CaSki 细胞用 CX5461 (1-5 µM) 处理 24-72 h，通过 Western Blot 检测靶点蛋白表达水平。
结果：CX5461 处理后，LC3I 完全转化为 LC3II，同时 p62 表达减少。LC3I 和 LC3II 条带的密度测定分析显示，CX5461 治疗 72 h 后，LC3II:LC3I 比值显著增加。[2]

方法：为检测体内抗肿瘤活性，将 CX5461 (50 mg/kg，50 mmol/L NaH2PO4 pH 4.5) 口服给药给携带 MIA PaCa-2 或 A375 异种移植物的小鼠，每天一次或每三天一次，持续 32 天。
结果：CX5461 在第 31 天显示出明显的 MIA PaCa-2 TGI，TGI 等于 69%。同样，CX5461 显示出明显的 A375 TGI，第 32 天的 TGI 等于 79%。[1]

Pol I and Pol II Transcription Assay: Two short-lived RNA transcripts (half-lives ~20-30 minutes), one produced by Pol I and another by Pol II, are quantitated by qRT-PCR as a measure of CX-5461-related effects on transcription. The 45S pre-rRNA served as the Pol I transcript and the mRNA for the protooncogene c-myc served as the comparator Pol II transcript. Both Pol I and Pol II transcription are known to be affected by general cellular stress. To minimize the potential effects of such stress, cells are exposed to test agents for only a short period of time (2 hours). This is sufficient time for these transcripts to be reduced by greater than 90% if CX-5461 affects their synthesis.

Cells are plated on 96-well plates and treated the next day with dose response of CX-5461 for 96 hours. Cell viability is determined using Alamar Blue and CyQUANT assays(Only for Reference)