Identification of ALEKSIN as a novel multi-IRF inhibitor of IRF- and STAT-mediated transcription in vascular inflammation and atherosclerosis [HMEC]

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)a and IFN? 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 combined inhibition of IRFs and STATs blocks pro-inflammatory target gene expression and 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 IFNa and IFNg. 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-?B 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 APOE-/- mice and associated with inflammation, proliferation, adhesion, chemotaxis and response to lipid. Together, this suggests that ALEKSIN represents a novel class of multi-IRF inhibitors, which inhibit IRF- , STAT- and NF-?B-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. Overall design: Gene expression profiling by high throughput sequencing in HMEC cell line after tretment with ALEKSIN and STATTIC, inhibitors of IRF- and STAT-mediated transcription