Biocenter Oulu, Department of Biochemistry, University of Oulu, Oulu, Finland.
Eur J Pharm Sci. 2013 May 13;49(2):148-56. doi: 10.1016/j.ejps.2013.02.012. Epub 2013 Feb 26.
Poly(ADP-ribose) polymerases (PARPs) or diphtheria toxin like ADP-ribosyl transferases (ARTDs) are enzymes that catalyze the covalent modification of proteins by attachment of ADP-ribose units to the target amino acid residues or to the growing chain of ADP-ribose. A subclass of the ARTD superfamily consists of mono-ADP-ribosyl transferases that are thought to modify themselves and other substrate proteins by covalently adding only a single ADP-ribose moiety to the target. Many of the ARTD enzymes are either established or potential drug targets and a functional activity assay for them will be a valuable tool to identify selective inhibitors for each enzyme. Existing assays are not directly applicable for screening of inhibitors due to the different nature of the reaction and different target molecules. We modified and applied a fluorescence-based assay previously described for PARP1/ARTD1 and tankyrase/ARTD5 for screening of PARP10/ARTD10 and PARP15/ARTD7 inhibitors. The assay measures the amount of NAD(+) present after chemically converting it to a fluorescent analog. We demonstrate that by using an excess of a recombinant acceptor protein the performance of the activity-based assay is excellent for screening of compound libraries. The assay is homogenous and cost effective, making it possible to test relatively large compound libraries. This method can be used to screen inhibitors of mono-ARTDs and profile inhibitors of the enzyme class. The assay was optimized for ARTD10 and ARTD7, but it can be directly applied to other mono-ARTDs of the ARTD superfamily. Profiling of known ARTD inhibitors against ARTD10 and ARTD7 in a validatory screening identified the best inhibitors with submicromolar potencies. Only few of the tested ARTD inhibitors were potent, implicating that there is a need to screen new compound scaffolds. This is needed to create small molecules that could serve as biological probes and potential starting points for drug discovery projects against mono-ARTDs.
聚（ADP-核糖）聚合酶（PARPs）或白喉毒素样 ADP-核糖基转移酶（ARTDs）是一类通过将 ADP-核糖单元共价连接到靶氨基酸残基或 ADP-核糖链上，从而催化蛋白质共价修饰的酶。ARTD 超家族的一个亚类由单 ADP-核糖基转移酶组成，这些酶被认为通过仅将单个 ADP-核糖部分共价添加到靶标上，来修饰自身和其他底物蛋白。许多 ARTD 酶要么是已确立的，要么是潜在的药物靶点，因此针对它们的功能性活性测定将是识别每种酶的选择性抑制剂的有价值工具。由于反应性质和不同的靶分子不同，现有的测定方法不能直接用于抑制剂的筛选。我们修改并应用了以前针对 PARP1/ARTD1 和 tankyrase/ARTD5 描述的荧光测定法，用于筛选 PARP10/ARTD10 和 PARP15/ARTD7 抑制剂。该测定法测量化学转化 NAD（+）为荧光类似物后剩余的 NAD（+）量。我们证明，通过使用过量的重组受体蛋白，该活性测定法的性能非常适合化合物文库的筛选。该测定法是均相的，且具有成本效益，因此可以测试相对较大的化合物文库。这种方法可用于筛选单 ADP-核糖基转移酶抑制剂，并对酶类抑制剂进行分析。我们对 ARTD10 和 ARTD7 进行了优化，但该方法可直接应用于 ARTD 超家族的其他单 ADP-核糖基转移酶。在验证性筛选中，用已知的 ARTD 抑制剂对 ARTD10 和 ARTD7 进行分析，确定了具有亚微摩尔效力的最佳抑制剂。测试的 ARTD 抑制剂中只有少数是有效的，这表明需要筛选新的化合物骨架。这对于筛选可以作为单 ADP-核糖基转移酶的生物学探针和潜在药物发现项目起点的小分子是必要的。
