LC-363: Optimization and in vitro characterization of a novel series of small molecule inhibitors of the Wnt/β-catenin signaling pathway.

C222 Proteins within the Wnt/β-Catenin signaling pathway are directly involved in the initiation and progression of several types of cancers, most notably colon cancer. Mutations in this pathway are common and result in the stabilization of β-Catenin protein and subsequent activation of the Wnt/β-Catenin signaling pathway making it an attractive target for development of anti-cancer therapeutics. To this end, we have identified small molecule compound series (LC-363) inhibitors of the β-Catenin pathway from a gene expression biomarker-based high throughput screen. Specifically, we treated a colon cancer cell line with siRNA against β-Catenin and identified a set of gene expression biomarkers that were correlated with a decrease in pathway activity. The gene based biomarkers were reduced to a set of seven genes that specifically identified small molecule compounds known to disrupt β-Catenin signaling. A series of HIT compounds, LC-363, were identified which induced a G1 cell cycle arrest, decreased cytoplasmic levels of β-Catenin protein and inhibited T-cell factor (TCF) transcriptional activity (TOPFLASH). These compounds were also found to induce apoptosis, as detected by Annexin V labeling, in colon cancer cell lines (HT-29, SW-480, and DLD-1). Since the initial LC-363 HIT compounds were not potent inhibitors of the β-Catenin pathway gene set, chemical optimization of the series was instituted with the goal of creating compounds of sufficient potency and pharmacological properties necessary to initiate in vivo efficacy studies. Newly synthesized analogs were assayed for potency on cell growth inhibition (GI50) and the 7-gene β-Catenin profile thus linking the increasing potency of optimized analogs to on-target activity. Analysis of LC-363 compounds by full genome microarray verified the selectivity for the β-Catenin pathway and allowed for a comparison to our reference deck containing gene expression signatures from more than 300 small molecule compounds of known mechanism. This analysis validated the novelty of the compound series in that the gene expression signature for LC-363 compounds did not closely match (Pearson >0.90) any reference compound but there was a partial match (Pearson 0.60) to multiple compounds that affect the proteasome (Velcade, MG-132, and Curcumin). Based on these observations, we explored activity of LC-363 with regards to affects on the proteasome and detected an accumulation of ubiquitinated proteins, including β-Catenin, which is a hallmark of proteasome inhibition. In conclusion, we have developed a novel biomarker based method for chemical optimization of small molecule compounds resulting in the rapid development (9 months) of potent and selective inhibitors of the Wnt/β-Catenin signaling pathway.