Covalent Modulators of Immune Regulatory Transcription Factors IRF8 and IRF5

Transcription factors are among the most challenging targets for drug discovery due to their lack of classical binding pockets and high degree of intrinsic disorder, despite the therapeutic importance of many of these proteins. IRF5 and IRF8 are key transcriptional regulators of innate immune signaling that orchestrate pro-inflammatory gene expression programs in response to stimuli such as toll-like receptor activation, making them central players in autoimmune and inflammatory diseases. Despite their therapeutic interest, direct targeting of IRF5 and IRF8 has remained challenging. Here, we screened a library of cysteine-reactive covalent ligands to identify hits that could degrade IRF5. We identified acrylamide EN1033 as the top hit, which not only led to IRF5 loss in a proteasome-dependent manner but also bound directly and covalently to the IRF5 protein, inhibiting IRF5-specific transcriptional activity in a macrophage cell line. Upon further analysis, however, we found that EN1033 not only engaged and degraded IRF5 but also more robustly and rapidly engaged and degraded a related inflammatory transcription factor, IRF8. We further demonstrated that EN1033 destabilized and degraded IRF5 and IRF8 by covalently targeting C28 and C223, respectively, as evidenced by the attenuation of their degradation through mutagenesis of these cysteines. We also found that IRF8 loss led to the downregulation and inhibition of IRF5 activity, suggesting a crosstalk between these two transcription factors, which are both targeted by EN1033. Overall, we identify an early-stage pathfinder molecule that covalently targets IRF8 and IRF5, thereby degrading these transcription factors and inhibiting their pro-inflammatory transcriptional activity.