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Targeted protein degradation (TPD) of neo-substrates with proteolysis targeting chimeras (PROTACs) or molecular glues has emerged as a key modality in exploring new biology as well as designing new drug candidates where catalytic inhibition is neither efficacious nor an option. TPD is mediated through harnessing E3 ligases and redirecting them to ubiquitinatede novotarget proteins for subsequent proteasomal degradation. Until recently, E3 ligase chemical matter available for mediating TPD has been limited to a relatively low number of ligases, considering that over 600 E3 ligases are encoded by the human genome. In addition, the most utilized ligase for TPD approaches, CRBN, has been observed to be downregulated in settings of acquired resistance to immunomodulatory inhibitory drugs (IMiDs). IMiDs are molecular glues that target IKZF transcription factors to CRBN for degradation. Resistance is potentially accelerated by non-essentiality of CRBN for cell viability. Here we investigated if the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a potent, non-covalent DCAF1 binder. We show that this binder, selective for the CRL4DCAF1E3 ligase complex, can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments confirm specific degradation via the CRL4DCAF1E3 ligase. We further highlight the versatility of DCAF1 for TPD by developing a DCAF1-dasatininb PROTAC targeting multiple cytosolic and membrane bound tyrosine kinases. We expand these findings towards Bruton’s tyrosine kinase (BTK) selective PROTACs and through extensive optimization and characterization efforts share key observations that led to a potent and selective DCAF1-BTK PROTAC (DBt-10). Finally, with this PROTAC DBt-10, we show rescue of BTK degradation in a BTK-dependent, CRBN-degradation-resistant cell line and provide a rationale for E3 ligase swap to overcome CRBN mediated resistance.

Stimulator of interferon genes (STING) is a central component of the pathway sensing the presence of cytosolic nucleic acids, having a key role in type I interferon innate immune response. Localized at the endoplasmic reticulum (ER), STING becomes activated by cGAMP, which is generated by the intracellular DNA sensor cyclic GMP-AMP synthase (cGAS). Due to its critical role in physiological function and its ‘ involvement in a variety of diseases, STING has been a notable focus for drug discovery. Recent advances in drug discovery allow the targeting of proteins previously considered “un-druggable” by novel mechanism of actions. Molecular glue degraders are defined as the compounds leading targeted protein degradation (TPD) by creating novel ligase-substrate interactions. Here, we identified AK59 as a novel molecular glue degrader for STING. A genome-wide, CRISPR/Cas9 knockout screen showed that the compound-mediated degradation of STING by AK59 is compromised by the loss of HECT and RLD domain containing E3 ubiquitin protein ligase 4 (HERC4), ubiquitin-like modifier activating enzyme 5 (UBA5) and ubiquitin like modifier activating enzyme 6 (UBA6). While UBA5 and UBA6 could be the auxiliary factors for AK59 activity, our results indicate that HERC4 is the main E3 ligase for the observed degradation mechanism. Validation by individual CRISPR knockouts, co-immunoprecipitations, as well as proximity mediated reporter assays suggested that AK59 functions as a glue degrader by forming a novel interaction between STING and HERC4. Furthermore, our data reveals that AK59 was effective on the most common pathological STING mutations that cause STING-associated vasculopathy with onset in infancy (SAVI), suggesting a potential clinical application of this mechanism. Thus, these findings not only reveal a novel mechanism for compound-induced degradation of STING but also utilize HERC4 as potential E3 ligase that for TPD, enabling novel therapeutic applications.

Bile acids are critical metabolites in the gastrointestinal tract and contribute to maintaining intestinal immune homeostasis through cross-talk with the gut microbiota. The conversion of bile acids by the gut microbiome is now recognized as a factor affecting both host metabolism and immune responses, but its physiological roles remain unclear. We conducted a screen for microbiome metabolites that would function as inflammasome activators and herein report the identification of 12-oxo-lithocholic acid (BAA485), a potential microbiome-derived bile acid metabolite. We demonstrate that the more potent analogue 11-oxo-12S-hydroxylithocholic acid methyl ester (BAA473) can induce secretion of interleukin-18 (IL-18) through activation of the inflammasome in both myeloid and intestinal epithelial cells. Using a genome-wide CRISPR screen with compound induced pyroptosis in THP-1 cells, we identified that inflammasome activation by BAA473 is pyrin-dependent (MEFV). To our knowledge, the bile acid analogues BAA485 and BAA473 are the first small molecule activators of the pyrin inflammasome. We surmise that pyrin inflammasome activation through microbiota-modified bile acid metabolites such as BAA473 and BAA485 plays a role in gut microbiota regulated intestinal immune response. The discovery of these two bioactive compounds may help to further unveil the importance of pyrin in gut homeostasis and autoimmune diseases.

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.

Multiciliated cells (MCCs) function to promote directional fluid flow across epithelial tissues. Wang et al. show that TRRAP, a component of multiple histone acetyltransferase complexes, is required for airway MCC formation and regulates a network of genes involved in MCC differentiation and function., The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including Xenopus laevis epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.