A-96649 is a new-type and effective inhibitor. The Ki of A-966492 for PARP1 and PARP2 is 1 nM and 1.5 nM, respectively.

PARP2:1.5 nM(Ki), PARP1:1 nM(ki)

A-966492是一种高效的PARP抑制剂，对PARP-1酶显示出极高的效力，其Kiof为1 nM，全细胞测定中的EC50也为1 nM。A-966492能显著以剂量依赖性增强TMZ的疗效。此外，A-966492具有通过口服在多种物种中具有生物利用度，能穿过血脑屏障，并且似乎能分布到肿瘤组织中。A-966492作为一个前景广阔的结构多样的苯并咪唑类似物，正在进行进一步的临床前特征研究。[1]

A-966492在与替莫唑胺联合应用时，在B16F10亚皮下小鼠黑色素瘤模型中展现出良好的体内功效，并在MX-1乳腺癌异种移植模型中作为单一化合物以及与卡铂联合应用时均显示出效果。此外，A-966492在与TMZ和卡铂联合使用时，在临床前小鼠肿瘤模型中展示了体内功效，同时作为单一化合物在BRCA1缺陷的MX-1肿瘤模型中也显示活性。进一步的研究表明，在斯普拉格-道利大鼠、比格犬和食蟹猴中，A-966492展现出34-72%的口服生物利用度和1.7-1.9小时的半衰期。在体内研究中，A-966492显著增强了在小鼠B16F10同源性黑色素瘤模型中TMZ的疗效，与A-966492联合治疗组表现出更优的疗效。[1]

PARP Enzyme Assay: The enzyme assay is conducted in buffer containing 50 mM Tris, pH 8.0, 1 mM dithiothreitol(DTT), and 4 mM MgCl2. PARP reactions contains 1.5 μM [3H]-NAD+ (1.6 μCi/mmol), 200 nM biotinylated histone H1, 200 nM slDNA, and 1 nM PARP-1 or 4 nM PARP-2 enzyme. Autoreactions utilizing SPA bead-based detection are carried out in 100 μL volumes in white 96-well plates. Reactions are initiated by adding 50 μL of 2X NAD+ substrate mixture to 50 μL of 2× enzyme mixture containing PARP and DNA. These reactions are terminated by the addition of 150 μL of 1.5 mM benzamide (～1 × 103-fold over its IC50). A 170 μL amount of the stopped reaction mixtures is transferred to streptavidin-coated Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter. Ki data are determined from inhibition curves at various substrate concentrations.

C41 cells are treated with A-966492 for 30 minutes in a 96-well plate. PARP are activated by damaging DNA with 1 mM Water2 for 10 minutes. Cells are washed with ice-cold phosphate-buffered saline (PBS) once and fixed with prechilled methanol/acetone (7:3) at -20 °C for 10 minutes. After they are air-dried, plates are rehydrated with PBS and blocked using 5% nonfat dry milk in PBS-Tween(0.05%) (blocking solution) for 30 minutes at room temperature. Cells are incubated with anti-PAR antibody 10H (1:50) in blocking solution at room temperature for 60 minutes followed by washing with PBS-Tween20 five times, and incubation with goat antimouse fluorescein 5(6)-isothiocyanate (FITC)-coupled antibody (1:50) and 1 μg/mL 40,6-diamidino-2-phenylindole (DAPI) in blocking solution at room temperature for 60 minutes. After washing with PBS-Tween20 5 times, analysis is performed using an fmax Fluorescence Microplate Reader set at the excitation and emission wavelength for FITC or the excitation and emission wavelength for DAPI. PARP activity (FITC signal) is normalized with cell numbers (DAPI).(Only for Reference)