A luciferase-based assay identifies niclosamide derivatives antagonizing Mcl-1 through post-translational down-regulation

Abstract

The up-regulation of Mcl-1 expression is a major mechanism of cancer cell survival and therapy resistance. However, the underlying molecular mechanism remains incompletely understood, limiting the number of druggable approaches to selectively inhibit Mcl-1 function. In this study, we designed and employed a novel mechanistic high-throughput screening system to selectively uncover post-translational modulators of Mcl-1. We generated a cell-based high-throughput screening assay in which myeloid leukemia K562 cells constitutively express Mcl-1 or Bcl-xL fused with luciferase (Luc-Mcl-1 or Luc-Bcl-xL, respectively) under a viral promoter. 1,650 bioactive compounds were screened for their ability to selectively induce Mcl-1 down-regulation in a 2-hour assay. A family of niclosamide derivatives were eventually identified for their remarkable ability to decrease Mcl-1 protein stability, exemplified by N007. These salicylate derivatives did not alter Mcl-1 mRNA levels, but selectively induced proteasome-dependent Mcl-1 down-regulation independent of Noxa, Mule, or GSK3{beta}. We also demonstrate that N007 potently induced cell death in leukemia cell lines, including those resistant to Bcl-2 inhibitors. Our work highlights the versatility of the mechanistic high-throughput screening approach as a valuable tool in identifying novel agents with the ability to down-regulate proteins crucial to human diseases.