Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4-MK2 interactions.
Development of cell-based high throughput luminescence assay for drug discovery in inhibiting OCT4/DNA-PKcs and OCT4-MK2 interactions.
复制标题
DOI：
10.1002/bit.27712
复制
发表时间：
2021-05
影响因子：
3.8
通讯作者：
Kang MH
中科院分区：
文献类型：
--
作者：
Mohiuddin IS;Wei SJ;Yang IH;Martinez GM;Yang S;Cho EJ;Dalby KN;Kang MH
扩增非依赖性c-MYC过表达在多种癌症中被提出。靶向c-MYC活性具有治疗潜力，但迄今为止的努力大多不成功。为了找到一个药物靶点来调节癌症中的c-MYC活性，我们鉴定了两种激酶，MAPKAPK 2（MK2）和DNA依赖性蛋白激酶催化亚基（DNA-PKcs），它们分别磷酸化OCT 4的Ser 111和Ser 93残基，以转录激活c-MYC。利用这些观察结果，我们在这里提出了一种新的基于细胞的发光测定，以确定抑制这些激酶和OCT 4之间的相互作用的化合物。在筛选了大约80，000种化合物后，我们鉴定了56种抑制DNA-PKcs和OCT 4之间的发光反应的化合物（“命中”），以及65种抑制MK 2-OCT 4相互作用的命中。使用对pOCT 4S 93和pOCT 4S 111具有特异性的定制抗体，验证“命中”对OCT 4磷酸化和活化的影响。使用两步法进行验证，我们从DNA-PKcs试验中鉴定了两种候选化合物，从MK2试验中鉴定了三种。所有五种化合物都表现出在纳摩尔范围内杀死癌细胞的显著能力。总之，我们开发了一种基于细胞的发光测定来鉴定靶向c-MYC转录激活的新型抑制剂，并发现了五种化合物，它们可能作为进一步开发的先导化合物。
Amplification-independent c-MYC overexpression is suggested in multiple cancers. Targeting c-MYC activity has therapeutic potential, but efforts thus far have been mostly unsuccessful. To find a druggable target to modulate c-MYC activity in cancer, we identified two kinases, MAPKAPK2 (MK2) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylate the Ser111 and the Ser93 residues of OCT4, respectively, to transcriptionally activate c-MYC. Using these observations, we present here a novel cell-based luminescence assay to identify compounds that inhibit the interaction between these kinases and OCT4. After screening approximately 80,000 compounds, we identified 56 compounds (“hits”) that inhibited the luminescence reaction between DNA-PKcs and OCT4, and 65 hits inhibiting the MK2–OCT4 interaction. Using custom antibodies specific for pOCT4S93 and pOCT4S111, the “hits” were validated for their effect on OCT4 phosphorylation and activation. Using a two-step method for validation, we identified two candidate compounds from the DNA-PKcs assay and three from the MK2 assay. All five compounds demonstrate a significant ability to kill cancer cells in the nanomolar range. In conclusion, we developed a cell-based luminescence assay to identify novel inhibitors targeting c-MYC transcriptional activation, and have found five compounds that may function as lead compounds for further development.
登录
查看更多内容
影响因子：
7.3
作者：
Fiore M;Forli S;Manetti F
通讯作者：
Manetti F
影响因子：
3.1
作者：
Cho, Eun Jeong;Devkota, Ashwini K.;Dalby, Kevin N.
通讯作者：
Dalby, Kevin N.
影响因子：
--
作者：
Newbatt, Y;Burns, S;Aherne, W
通讯作者：
Aherne, W
DOI：
10.1038/nrc.2017.36
发表时间：
2017-08
期刊：
Nature reviews. Cancer
影响因子：
--
作者：
Dang CV;Reddy EP;Shokat KM;Soucek L
通讯作者：
Soucek L
影响因子：
8
作者：
Kim, YH;Girard, L;Pollack, JR
通讯作者：
Pollack, JR