AZ6102 is a potent TNKS1/2 inhibitor that has 100-fold selectivity against other PARP family enzymes and shows IC50 of 5 nM for Wnt pathway inhibition in DLD-1 cells.

TNKS2:5 nM, TNKS1:5 nM

AZ6102在酶促实验和TCF4报告基因实验中（<5 nM）抑制TNKS1和TNKS2。在Colo320DM细胞中，AZ6102能抑制增殖（GI50约40 nM），但在含有β-catenin突变的HCT-116细胞系和BRCA突变的MDA-MB-436细胞系中不显示抗增殖活性。在Colo320DM中，AZ6102稳定了axin2蛋白并以剂量和时间依赖的方式在体内外调控Wnt目标基因[1]。

裸鼠每千克体重被给予25mg的AZ-6102。该化合物半衰期为4小时，清除率(Clearance, CL)为24 mL/min.kg。进一步在小鼠和大鼠的分析显示，AZ-6102的生物利用度分别为12%和18%，表现出中等水平。通过对DLD-1细胞处理后的Western blot 分析发现，AZ-6102在较低浓度(at 24, 48 和 72h)时，相比XAV-939对TNKS1、TNSK2 和Axin2的稳定作用更强且持续时间更长。在临床前物种中，AZ-6102表现出良好的药代动力学特性，具有低Caco2外排作用（以避免可能的肿瘤抗性机制）。此外，这种化合物可以使用SBECD作为辅料，在pH4时配制成每毫升20mg的临床相关静脉注射溶液。利用AZ-6102作为静脉(i.v.)探针化合物探索其对肿瘤异种移植物和正常组织中TNKS1及TNSK2抑制作用的体内效果的结果即将公布[2]。

Sphingosine Kinase Assays: The IC50 values for ABC294640 and DMS are determined by a newly developed HPLC-based SK activity assay. In brief, the test compounds are incubated with recombinant SK1 or SK2 and NBD-Sph in the isozyme-selective assay buffers detailed below with 1 mg/ml fatty acid-free bovine serum albumin, 100 μM ATP, and 400 μM MgCl2. The product, i.e., NBD-S1P, is separated from NBD-Sph by HPLC as follows: Waters 2795 HPLC system with a Waters 2495 fluorescence detector, C8 Chromolith RP-8e column (100 × 4.6 mm), 1 ml/min mobile phase (acetonitrile/20 mM sodium phosphate buffer, pH2.5, at 45:55). Fluorescence is monitored with excitation at 465 nm and emission at 531 nm. The ratio of NBD-S1P/(NBD-Sph + NBD-S1P) is used as a measure of SK activity. SK-isozyme selective assay buffers each contained 20 mM Tris, pH7.4, 5 mM EDTA, 5 mM EGTA, 3 mM β-mercaptoethanol, 5% glycerol, 1× protease inhibitors and 1× phosphatase inhibitors. For the SK1 assay buffer, 0.25% (final) Triton X-100 is added; and for the SK2 buffer, 1 M (final) KCl is added. Assays are run for 2 h at room temperature, and then a 1.5 volume of methanol is added to terminate the kinase reaction. After 10 min, the samples are centrifuged at 20,000 g to pellet the precipitated protein, and the supernatants are analyzed by HPLC. In experiments to determine the Ki for inhibition of SK2 by ABC294640, the ADP Quest assay system is used to measure kinase activity in the presence of varying concentrations of sphingosine and ABC294640. To determine the effects of ABC294640 on cellular SK activity, near-confluent MDA-MB-231 cells are serum-starved overnight, and then treated with varying concentrations of ABC294640. The cells are then incubated with [3H]sphingosine at a final concentration of 1 μM. The cells take up the exogenous sphingosine, which is converted to S1P via SK activity, and [3H]S1P is separated from [3H]sphingosine by extraction and quantified by scintillation counting.