Nano-Luciferase Based High-throughput Screening System For YAP1 Inhibitors

As a most critical downstream transcriptional regulator for the hippo signaling pathway,YAP1 is an attractive target for cancer therapy and regenerative medicine.Although some inhibitors targeting the hippo signaling pathway have been investigated,their application remains challenging.High-throughput screening(HTS)provides a technical basis for the discovery of lead compounds.NanoBiT assay system is an ideal detection system for HTS as it can quickly and accurately quantify intracellular HiBiTtagged proteins.Delivery of CRISPR/Cas9 in the form of RNP enables rapid gene editing and significantly improves efficiency and specificity.Therefore,we hope to deliver CRISPR/Cas9 RNP and knock-in HiBiT at the C terminal of YAP 1 to establish an HTS system based on NanoBiT for YAP1 inhibitors.First,we purified EGFP,Cas9,and T7 RNA polymerase.Then we established the RNP electroporation method using homemade EGFP.Together we constructed HiBiT knock-in cell lines using an AAV or ssDNA donor.In comparison,we found that HiBiT knock-in efficiency using an AAV donor is higher.Subsequently,we experimentally confirmed that the NanoBiT detection system could quickly detect the protein expression of YAP 1-HiBiT in cells.Using this system,we identified four compounds that inhibited YAP1 expression from a small set of compounds,which can be used as positive controls for HTS.Then,we identified twenty-four compounds with potential as YAP1 inhibitors by HTS from 1,500 natural products.We will further clarify the candidate compounds’ mechanism and explore the potential of these compounds for clinical applications at the cellular and animal levels.