Discovery and evaluation for PCSK9 post-transcriptional inhibitors
Objective To discover small molecule compounds with the inhibitory activity of PCSK9 post-transcriptional expression by establishing high-throughput screening assay for PCSK9 post-transcriptional inhibitor,and to discover PCSK9 inhibitors with potential lipid-lowering activity.Methods The high-throughput screening assay for PCSK9 post-transcriptional inhibitors was developed by inserting the 3'UTR regulatory sequence of PCSK9 gene mRNA to downstream of luciferase gene to construct psi-PCSK9-3'UTR and stably transfected into HepG2 cells.After optimization and evaluation,the assay was applied in large-scale screening.RT-qPCR and Western blot were applied to measure the expression level of PCSK9 and LDLR,and flow cytometry to assess the LDL-C uptake by HepG2 cells.Results Six positive compounds were selected by the screening assay,and the dose-effect relationship was analyzed.It was shown that these compounds down-regulated the luciferase activity dose-dependently.Then HepG2 cells were used to evaluate the effect of IMB-57 to PCSK9 expression.The results showed that IMB-57 significantly lowered PCSK9 mRNA and protein level and increased the protein level of LDLR and cholesterol uptake function of HepG2 cells.It was further confirmed that the compound inhibited PCSK9 expression by decreasing the stability of PCSK9 mRNA by preliminary molecular mechanism analysis.Conclusion As a PCSK9 post-transcriptional inhibitor,IMB-57 plays an important regulatory role on cellular cholesterol uptake,implying that the compound has the potential to be further developed into a novel lipid-lowering drug candidate.It is also confirmed that the constructed HTS assay in this work is reliable and can be used for the discovery of PCSK9 inhibitors with novel mechanisms.
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