AG14361 is an effective inhibitor of PARP1 (Ki<5 nM).

PARP1:<5 nM(Ki)

在无毒剂量内,AG14361可提高伊立替康、x射线照射及替莫唑胺诱导的LoVo移植瘤生长延迟,提高2到3倍.在SW620移植瘤中, AG14361（10 mg/kg,i.p.）处理4小时以上可抑制75%以上的PARP-1活性.在携带LoVo移植瘤的小鼠体内,先以AG14361处理再进行统计学辐射,看显著提高对放射疗法的敏感性.在移植瘤中,AG14361可显著提高血流,故可能促进药物输送到移植瘤.

AG14361（0.4 μM）对癌细胞基因表达或生长无影响,但可提高替莫唑胺和拓扑替康抗增殖活性,且抑制LoVo细胞从潜在的致命γ辐射损伤中恢复,抑制达73％。AG14361（0.4 μM）对基因表达无大幅影响。在A549细胞中, AG14361（0.4 μM）在17小时后对6800种基因表达均无影响。因此,尽管0.4 μM AG14361抑制85%以上细胞PARP-1 活性,但其不影响基因表达和细胞增殖。AG14361在更高浓度会影响基因表达,但是这种影响与PARP-1受抑制无关,因为在PARP-/-和PARP-1+/+细胞中,细胞增殖一样受影响。AG14361可增加喜树碱诱导DNA单链断裂的持久性。AG14361的效果比苯甲酰胺至少高1×103倍。AG14361对透性化SW620细胞（IC50：29 nM）和完整SW620细胞（IC50：14 nM）有抑制作用。AG14361对细胞生长的抑制作用与PARP-1抑制无关,因为在比GI50浓度低很多时(≤1 μM),对PARP-1也有最大抑制效果。

PARP-1 Activity Assays: The activity of full-length recombinant human PARP-1 is measured in a reaction mixture containing 20 nM PARP-1, 500 μM NAD+ plus [32P]NAD+ (0.1–0.3 μCi per reaction mixture), and activated calf thymus DNA (10 μg/mL) at 25oC; the reaction is terminated after 4 minutes by adding ice-cold 10% (wt/vol) trichloroacetic acid. The reaction product [32P]ADP-ribose incorporated into acid-insoluble material is deposited onto Whatman GF/C glass fiber filters with a Bio-Dot microfiltration apparatus and quantified with a PhosphorImager. Inhibition of PARP-1 activity by AG14361 at 0–600 nM is measured, and the Ki for AG14361 is calculated by nonlinear regression analysis.

LoVo and SW620 colorectal cancer cells and A549 non–small-cell lung carcinoma cells are maintained in RPMI-1640 medium containing 10% fetal calf serum. Cell growth inhibition is estimated in exponentially growing LoVo, A549, and SW620 cells in 96-well plates. Cells are exposed to AG14361 (0–20 μM) alone or in the presence of 400 μM temozolomide. After 5 days of culture, these cells are fixed with 10% trichloroacetic acid and stained with sulforhodamine B. The concentration of temozolomide, topotecan, and AG14361 alone or in combination that inhibits growth by 50% (GI50) is calculated from computer-generated curves. Recovery from potentially lethal damage is measured in confluent LoVo cell cultures arrested in G1 phase to mimic the radiation-resistant quiescent cell population in tumors. Such cells are exposed to 8 Gy of γ-irradiation and then harvested and plated for colony formation assay immediately or maintained as growth-arrested confluent cultures for a 4-hour or 24-hour recovery period before harvesting and plating for the colony formation assay. Where indicated, 0.4 μM AG14361 is added 30 minutes before irradiation and is present in the recovery incubation. (Only for Reference)