Clausse Victor, Fang Yuhong, Tao Dingyin, Tagad Harichandra D, Sun Hongmao, Wang Yuhong, Karavadhi Surendra, Lane Kelly, Shi Zhen-Dan, Vasalatiy Olga, LeClair Christopher A, Eells Rebecca, Shen Min, Patnaik Samarjit, Appella Ettore, Coussens Nathan P, Hall Matthew D, Appella Daniel H
Synthetic Bioactive Molecules Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, United States.
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850, United States.
ACS Pharmacol Transl Sci. 2022 Sep 28;5(10):993-1006. doi: 10.1021/acsptsci.2c00147. eCollection 2022 Oct 14.
Wild-type P53-induced phosphatase 1 (WIP1), also known as or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator.
野生型P53诱导磷酸酶1（WIP1），也称为PP2Cδ，是一种在基因毒性应激后由P53诱导产生的丝氨酸/苏氨酸蛋白磷酸酶。WIP1抑制已被提议作为治疗WIP1过表达的P53野生型癌症的一种治疗策略，但这种方法在P53阴性癌症中无效。此外，在一些P53突变的癌症中，WIP1起着肿瘤抑制作用。因此，根据P53状态，激活WIP1磷酸酶也可能是一种治疗策略。迄今为止，尚未报道具有适当药代动力学性质的特异性、强效WIP1抑制剂，也没有WIP1特异性激活剂。在此，我们报告了通过高通量筛选（HTS）发现新的WIP1调节剂，该筛选使用了先前描述的适用于鉴定抑制剂和激活剂的正交生化测定法。首次高通量筛选是使用快速液相色谱-质谱联用分析，针对102277种化合物的文库在单一浓度下进行的。通过快速液相色谱-质谱联用分析和基于正交荧光的测定法，在11种浓度范围内对筛选出的阳性结果进行了进一步评估。对已确认的阳性结果进行的进一步生物物理、生化和基于细胞的研究揭示了一种WIP1激活剂和两种抑制剂，一种是竞争性抑制剂，一种是非竞争性抑制剂。这些新的骨架是优化的主要候选对象，这可能会产生具有改善药代动力学的抑制剂和首个WIP1激活剂。
