Selective IRAK4 degradation, not kinase inhibition, blocks TLR-activated NF-Kb and p38 Signaling leading to broad cytokine inhibition

Abstract IL-1R/TLR activation plays a central role in the pathophysiology of multiple autoimmune and inflammatory diseases via the Myddosome that is dependent on both the kinase and scaffolding functions of Interleukin-1 receptor associated kinase 4 (IRAK4). Therefore, hetero-bifunctional molecules that selectively target IRAK4 for degradation and elimination by the ubiquitin proteasome pathway have the greatest potential to block IL-1R/TLR signaling, including NF-kb activation and cytokine production. To interrogate downstream signaling, phosphorylation events were monitored in monocytes and B cells following TLR7/8 or TLR9 activation, respectively. IRAK4 degradation, but not kinase inhibition, inhibited NF-kb p65 activation and phosphorylation of p38 pathway members by TLR agonists in both cell types. PBMCs pretreated with an IRAK4 degrader and then stimulated with the TLR7/8 agonist, R848, exhibited significantly broader inhibition of cytokines (IL-6, TNFa, IL-8 and IL-1b) compared to those pretreated with a selective IRAK4 kinase inhibitor. Compound washout experiments demonstrated a sustained effect of IRAK4 degrader on both target pharmacodynamics and cytokine inhibition that was differentiated from IRAK4 kinase inhibition. Overall, through removal of both scaffolding and kinase functions, IRAK4 degradation demonstrated greater activity compared to kinase inhibition across multiple TLR stimuli and cell types. These data highlight the potential for IRAK4 degraders to block multiple TLR signaling pathways across different immune cell types and thereby impact TLR/IL-1R-driven inflammatory and autoimmune diseases.