Covalent Modulators of Immune Regulatory Transcription Factors IRF8 and IRF5

Abstract 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. Through a screen originally intended to identify cysteine-reactive electrophilic ligands that could directly and covalently engage and degrade IRF5, we identified an acrylamide hit, EN1033, that not only targeted and degraded IRF5 but also degraded a related inflammatory transcription factor, IRF8, more robustly and rapidly. 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 downregulation and inhibition of IRF5 activity, suggesting crosstalk between these two transcription factors, both of which are targeted by EN1033. Upon exploring structure-activity relationships, we identified an optimized compound, TH-B10, that more potently and selectively targeted and degraded IRF8 and, secondarily, downregulated IRF5 by targeting C223 on IRF8. Overall, we identify an early-stage pathfinder molecule, TH-B10, that directly covalently targets and degrades IRF8 and secondarily modulates IRF5 to shut down pro-inflammatory transcriptional activity.