NSH76: a selective inhibitor of RRN3 and RNA polymerase I transcription with potential for cancer therapy

Background: Aberrant upregulation of RNA polymerase I (Pol I) transcription and its dedicated machinery plays a pivotal role in tumor progression and chemoresistance. RRN3, a Pol I-specific transcription initiation factor, is frequently overexpressed in malignancies contributing to oncogenic processes. Despite the therapeutic potential of Pol I transcription inhibition, existing inhibitors lack specificity and are associated with DNA damage, mutagenicity, and toxicity, limiting their clinical utility. To fully realize the potential of Pol I-targeted cancer therapies, selective Pol I transcription inhibitors with minimal off-target effects are essential.

Methods: Molecular docking and virtual screening were conducted to identify ligands targeting RRN3. Biochemical and spectroscopic analyses validated the direct ligand-RRN3 binding. The mechanism of action of the ligand was investigated through biochemical, cellular and molecular assays. Functional studies assessed the effects of the ligand on cancer cell viability, clonogenicity, cell cycle progression, and apoptosis, in comparison to non-cancerous cells. The ligand efficacy was further evaluated in chemoresistant cancer cell lines and 3D tumor spheroid models. Genotoxicity and mutagenicity were assessed using DNA damage and mutagenicity assays.

Results: We demonstrate that (N-(1-amidino-2-thiourea-alkyl-7-chloroquinoline-4-amine)) (NSH76) selectively inhibits Pol I transcription by disrupting the Pol I pre-initiation complex at the rDNA promoter through direct RRN3 binding. Notably, NSH76 does not affect cMyc expression, a Pol II-driven transcript, confirming its specificity. NSH76 preferentially inhibits Pol I transcription in cancer cells with high RRN3 expression, while sparing non-cancerous cells with low RRN3 levels. Functionally, NSH76 exhibits potent antiproliferative activity against cancer cells, with minimal impact on non-cancerous cells. NSH76 induces cell cycle arrest, suppresses clonogenicity, and significantly enhances apoptosis in cancer cells, including cisplatin- and doxorubicin-resistant cell lines. These effects are recapitulated in 3D tumor spheroid assays. Furthermore, NSH76 triggers nucleolar stress, leading to the activation of tumor suppressors p53 and p21. Notably, NSH76 does not induce DNA damage or mutagenicity.

Conclusion: These findings establish NSH76 as a potent and selective Pol I inhibitor with significant therapeutic potential in cancer and possible implications for overcoming chemoresistance.