Aleksandra Antonczyk ¹ Katarzyna Kluzek ¹ Natalia Herbich ¹ Bart Krist ¹ Dorota Wronka ² Anna Karlik ² Lukasz Przybyl ² Adam Plewinski ³ Joanna Wesoly ⁴ Hans AR Bluyssen ^1*

• ¹ 1. Human Molecular Genetics Research Unit, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland
• ² 2. Laboratory of Mammalian Model Organisms, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
• ³ 3. Animal Facility, Center for Advanced Technologies, Adam Mickiewicz University, Poznan, Poland
• ⁴ Laboratory of High Throughput Technologies, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland

Cardiovascular diseases (CVDs) include atherosclerosis, which is an inflammatory disease of large and medium vessels that leads to atherosclerotic plaque formation. Key factors contributing to onset and progression of atherosclerosis include the pro-inflammatory cytokines Interferon (IFN)α and IFNγ and the Pattern Recognition Receptor (PRR) Toll-like receptor 4 (TLR4). Together, they trigger activation of IFN Regulatory Factors (IRFs) as well as Signal Transducer and Activator of Transcription (STAT)s. Based on their promoting role in atherosclerosis, we hypothesized that inhibition of pro-inflammatory target gene expression through multi-IRF inhibitors may be a promising strategy to treat CVDs.Using comparative in silico docking of multiple IRF-DBD models on a multi-million natural compound library, we identified the novel multi-IRF inhibitor, ALEKSIN. This compound targets the DBD domain of IRF1, IRF2, and IRF8 with the same affinity and simultaneously inhibits expression of multiple IRF-target genes in HMECs in response to IIFNα and IFNγ. Under the same conditions, ALEKSIN was also shown to inhibit phosphorylation of STATs, potentially through low-affinity STAT-SH2 binding, however with lower potency as the known multi-STAT inhibitor STATTIC. This was in line with the common inhibition of ALEKSIN and STATTIC observed on genome-wide expression of pro-inflammatory IRF/STAT/NF-κB target genes, as well as on migration of HMECs. Finally, we identified a novel signature of 46 ALEKSIN and STATTIC commonly inhibited pro-atherogenic target genes, that was upregulated in atherosclerotic plaques in aorta's of high-fat diet fed ApoEKO mice and associated with inflammation, proliferation, adhesion, chemotaxis and response to lipid. Interestingly, the majority of these genes could be linked to macrophage subtypes present in aortic plaques in HFD fed LDLR-KO mice.Together, this suggests that ALEKSIN represents a novel class of multi-IRF inhibitors, which inhibit IRF-, STAT-and NF-κB-mediated transcription and could offer great promise for the treatment of CVDs. Also, the ALEKSIN and STATTIC commonly inhibited pro-inflammatory gene signature could serve to monitor plaque progression during experimental atherosclerosis.