PROTAC-Mediated HDAC7 Protein Degradation Unveils Its Deacetylase-Independent Proinflammatory Function in Macrophages.

Class IIa histone deacetylases (Class IIa HDACs) play critical roles in regulating essential cellular metabolism and inflammatory pathways. However, dissecting the specific roles of each class IIa HDAC isoform is hindered by the pan-inhibitory effect of current inhibitors and a lack of tools to probe their functions beyond epigenetic regulation. In this study, a novel PROTAC-based compound B4 is developed, which selectively targets and degrades HDAC7, resulting in the effective attenuation of a specific set of proinflammatory cytokines in both lipopolysaccharide (LPS)-stimulated macrophages and a mouse model. By employing B4 as a molecular probe, evidence is found for a previously explored role of HDAC7 that surpasses its deacetylase function, suggesting broader implications in inflammatory processes. Mechanistic investigations reveal the critical involvement of HDAC7 in the Toll-like receptor 4 (TLR4) signaling pathway by directly interacting with the TNF receptor-associated factor 6 and TGFß-activated kinase 1 (TRAF6-TAK1) complex, thereby initiating the activation of the downstream mitogen-activated protein kinase/nuclear factor-κB (MAPK/NF-κB) signaling cascade and subsequent gene transcription. This study expands the insight into HDAC7's role within intricate inflammatory networks and highlights its therapeutic potential as a novel target for anti-inflammatory treatments.

A practical "preTACs-cytoblot" platform accelerates the streamlined development of PROTAC-based protein degraders.

With the growing importance of PROTAC-mediated protein degradation in drug discovery, robust synthetic methodologies and rapid screening assays are urgently needed. By harnessing the improved alkene hydroazidation reaction, we developed a novel strategy to introduce azido groups into the linker-E3 ligand conjugates and effectively created a range of prepacked terminal azide-labeled "preTACs" as PROTAC toolkit building blocks. Moreover, we demonstrated that preTACs are ready to conjugate to ligands targeting a protein of interest to generate libraries of chimeric degraders, which are subsequently screened for effective protein degradation directly from cultured cells with a cytoblot assay. Our study exemplifies that this practical "preTACs-cytoblot" platform allows efficient PROTAC assembly and rapid activity assessments. It may help industrial and academic investigators to accelerate their streamlined development of PROTAC-based protein degraders.