Novel Tetrazolo-Pyridazine-Based MACC1 Transcriptional Inhibitor Reduces NFκb Signaling Pathway as Key Antimetastatic Mechanism of Action

Author(s): Paul Curtis Schöpe, Mathias Dahlmann, Dennis Kobelt, Bjoern-O Gohlke, Abdelrhman M Shoman, Wolfgang Walther, Robert Preissner, Marc Nazaré, Ulrike Stein

Targeted therapies for patients at high risk of metastasis are crucial to improving treatment outcomes and survival. Metastasis Associated in Colon Cancer 1 (MACC1) has emerged as a key molecule driving metastasis and serves as a prognostic and predictive biomarker for therapy response. Elevated MACC1 expression correlates with poor survival and resistance to standard chemotherapeutics. Recently, we identified a novel tetrazolo-pyridazine-based transcriptional inhibitor of the MACC1 gene through a high-throughput screen using the human MACC1 promoter linked to a luciferase reporter. While the initial phenotypic screening confirmed MACC1 inhibition, the molecular target and affected signaling pathways remained unknown, prompting further investigation. RNA sequencing revealed that treatment with Compound 22 attenuates TNF-α signaling via the NFκB pathway. Gene ontology analysis of differentially expressed genes indicated alterations in cell cycle regulation and intermediate filament-based processes, resulting in reduced proliferation and enhanced epithelial characteristics. Molecular analyses demonstrated decreased NFκB p65 and p50 activity, reduced nuclear translocation, and diminished activity on the MACC1 promoter. In silico predictions identified p105 as a potential protein target, and experimental validation showed that Compound 22 blocks TNF-α-induced phosphorylation of p105. Although additional studies are required to confirm direct binding to p105, our data suggest that this tetrazolo-pyridazine-based compound effectively suppresses NFκB signaling, leading to reduced MACC1 expression. Consequently, inhibition of this pathway and it subsequent targets such as MACC1 reduces the metastatic potential of cancer cells, highlighting a promising therapeutic strategy for metastasis prevention.