β-Lapachone (ARQ-501) is a specific DNA topoisomerase I inhibitor, and no inhibitory activities against DNA topoisomerase II or ligase.

IDO1:0.44 μM

Beta-Lapachone通过剂量依赖性方式抑制由DNA拓扑异构酶I引起的DNA松弛。[1] 对Beta-Lapachone（100 nM或更高浓度）的处理导致与DMSO对照相比，Topo I DNA解旋活性的抑制率超过95%。Beta-Lapachone（1-5 μM）通过锁定Topo I到DNA上并阻止复制叉移动，导致HL-60和三种人类前列腺癌细胞（DU-145, PC-3, 和LNCaP）的细胞周期在G0/G1阶段停滞并诱导细胞凋亡。[2] Beta-Lapachone促进小鼠3T3成纤维细胞和人类内皮EAhy926细胞通过不同的MAPK信号途径迁移，从而在体外加速刮擦伤口的愈合。[3] 此外，Beta-Lapachone通过不竞争性抑制作用抑制纯化的重组IDO1活性，IC50为0.44 μM；且Beta-Lapachone还展示出具有IC50为1.0 μM的高效细胞内IDO1抑制活性，该活性部分依赖于NQO1的生物转化。[4] Beta-Lapachone通过NQO1依赖性的活性氧（ROS）生成和PARP1过度激活诱导NQO1+癌细胞的程序性坏死。[5]

Beta-lapachone (50 mg/kg) 的处理在人类卵巢癌异种移植小鼠模型中显著抑制体内肿瘤生长，且与taxol联合使用可协同诱导凋亡。[6] 在正常及糖尿病(db/db)小鼠中，beta-lapachone的治疗相比于仅用载体的治疗可加速愈合过程。[3]

Topoisomerase I Catalytic Actioity Assay [1]: Topoisomerase I Catalytic Actioity Assay: The enzymatic activity is analyzed by the DNA unwinding assay. DNA topoisomerase I, from TopoGEN (1 unit, which is defined as the amount of enzyme that converts 0.5 μg of superhelical DNA to the relaxed state in 30 minutes at 37 °C), is incubated with 0.5 μg of 6x174 RF DNA, in the presence or absence of Beta-Lapachone, in 20 μL of relaxation buffer (50 mM Tris (pH 7.5). 50 mM KCI, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30 μg/mL bovine serum albumin) for 30 minutes at 37 °C. Reactions are stopped by adding 1% SDS and proteinase K (50 μg/mL). After an additional 1-hour incubation at 37 °C, the products are separated by electrophoresis in 1% agarose gel in TAE buffer (0.04 M tris acetate, 0.001 M EDTA). The gel is stained with ethidium bromide after electrophoresis. The photographic negative is scanned with an NIH image analysis system.

IC50 calculations for each cell line are determined by DNA amount (IS) and anchorage-dependent colony formation (CF) assays. For the CF assay, cells are seeded at 500 viable cells/well in 6-well plates and incubated overnight, then treated with equal volumes of media containing beta-lapachone at final concentrations ranging from 0.005 to 50 μM in half-log increments (controls were treated with 0.25% DMSO, equivalent to the highest dose of beta-lapachonc used) for 4 hour or for continuous 12-hour exposures. Plates (3 wells/condition) are stained with crystal violet, and colonies of >50 normal-appearing cells are enumerated. IC50 values for various cells are calculated using drug doses with numbers of colonies surrounding 50% of control. For DNA assays, plates are harvested for IC50 determinations 8 days after treatment using a CytoFluor 2350 fluorescence measurement system. Six-well samplings are included in the calculation of DNA fluor units for each dose. A graph of beta-lapachone dose versus percentage control DNA in fluor units is used to calculate each IC50. All experiments are repeated at least twice, each in duplicate. (Only for Reference)