Kymera Therapeutics

Mycobacterium tuberculosis (M. tb) is a leading cause of infectious disease mortality worldwide, and M. tb subverts host immunity to persist intracellularly. Type I interferon (IFN-I) modulates host defense during M. tb infection, but the mechanisms controlling their induction remain incompletely defined. In this research, a previously uncharacterized mycobacterial factor, M. tb Rv3248c, which regulates IFN-I response, was identified using pull-down and mass spectrometry. Overexpression of Rv3248c suppresses IFN-β expression in macrophages at both the transcriptional and protein levels. In addition, it also enhances intracellular bacterial load, whereas in bone marrow-derived macrophages (BMDMs) from Ifnar1-/- C57BL/6 mice, the difference of bacterial load was no longer statistically significant, suggesting that Rv3248c enhances mycobacterial survival in IFN-I-dependent manner. Furthermore, the study shows that M. tb Rv3248c inhibits IFN-I response via the cGAS-STING pathway. Mechanistically, Rv3248c binds TANK-binding kinase 1 (TBK1) and promotes its ubiquitination-mediated proteasomal degradation. The N-terminal region of Rv3248c (amino acids 1-134) is required for TBK1 interaction and IFN-I inhibition. These findings identify a previously uncharacterized mechanism by which M. tb dampens the IFN-I response by targeting TBK1, highlighting Rv3248c as a contributor to pathogenesis and a potential therapeutic target.

Kynurenine (Kyn), a tryptophan catabolite, is a key mediator of tumor-associated immunosuppression, and its production depends mainly on indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2). IDO1 is potently induced by interferon-γ (IFN-γ), and its transcriptional regulation is well characterized. In contrast, TDO2 is clinically relevant in cancer, but the mechanism controlling its expression is unclear. Here, we show that bromodomain and extra-terminal domain (BET) proteins directly regulate TDO2 transcription in cancer cells. In multiple TDO2-expressing cell lines, BET inhibitor JQ1 and BET degrader dBET6 suppressed TDO2 transcription and protein expression, with dBET6 showing broader and more potent activity. Chromatin immunoprecipitation followed by quantitative PCR showed BET protein occupancy at the TDO2 promoter, consistent with direct promoter binding. This BET-dependent regulation was observed across diverse cancer cell contexts, indicating that TDO2 is a conserved and pharmacologically tractable BET target. When IDO1 expression was induced by IFN-γ, dBET6 suppressed IDO1 and TDO2 expression and reduced Kyn production. Taken together, these findings reveal a BET-dependent mechanism controlling TDO2 expression and provide a rationale for evaluating BET inhibition or degradation as a strategy to suppress Kyn production in cancer.

Toll-like receptor 8 (TLR8) is an endosomal pattern recognition receptor highly expressed in myeloid cells that senses RNA degradation fragments. Monogenic gain-of-function (GOF) variants in TLR8 cause an inborn error of immunity (IEI) with a spectrum of immune-mediated pathology ranging from severe lymphoproliferation with neutropenia and bone marrow failure to anemia and autoimmunity. Half of identified patients have mosaic disease-causing variants, with variant allele fractions as low as 7% sufficient to drive systemic immune dysregulation. This, along with immunologic profiling of patients, demonstrates a dominant mechanism of disease, likely mediated by inflammatory cytokines. Disease-causing variants in TLR8 are enriched at or near agonist and antagonist binding sites in the encoded protein. As a spectrum of disease and genotypes emerges, identifying sensitivity to agonism and resistance to antagonism may provide insight into molecular mechanisms and therapeutic targets. Here, we review the genetics and clinical features of 19 reported cases of TLR8 GOF diseases, and future areas of investigation for this IEI.

Regnase-1 is an essential RNase that suppresses immune cell activation by targeting the mRNA of pro-inflammatory cytokines such as interleukin 6 (IL-6). Although genetic inhibition of Regnase-1 activity in CD8+ T cells has been shown to enhance antitumor immunity, no molecular inhibitors of Regnase-1 have been reported. By exploiting a library of nucleic acid analogs in conjunction with an RNA displacement assay, we identified multiple candidate competitors of Regnase-1 RNA substrates. Among these, suramin-an approved drug for treating African sleeping sickness-and several of its derivatives displaced Regnase-1 RNA targets with submicromolar affinity, competitively inhibited Regnase-1-mediated degradation of RNA in vitro, and enhanced Il6 mRNA levels and IL-6 production in mouse embryonic fibroblasts. A 2.62 Å crystal structure revealed that suramin bound to the Regnase-1 active site, coordinating with the catalytic Mg2+. Quantum chemical calculations further elucidated qualitative interactions between suramin and surrounding residues of Regnase-1. These findings establish suramin as a small-molecule inhibitor of Regnase-1-mediated RNA decay.

Triazole antifungals inhibit the lanosterol C14-demethylase Cyp51A/B and are the first-line treatment for invasive aspergillosis caused by Aspergillus fumigatus. We previously proposed that triazoles impose a secondary constraint on ergosterol biosynthesis through negative feedback regulation of HMG-CoA reductase (Hmg1) through its sterol sensing domain (SSD), and SSD mutations compromise this regulation to produce resistance. The molecular mechanism underpinning this feedback remained unknown. In this study, we show that voriconazole drives accelerated protein degradation of Hmg1 and that SSD mutation abolishes this regulated degradation. Triazole-induced Hmg1 degradation is proteasome-dependent and partially requires the ERAD E3 ubiquitin ligase, HrdA, and INSIG ortholog, InsA, both conserved regulators of HMG-CoA Reductase abundance. We provide genetic evidence that the initiating negative feedback signal is not triazole acting directly on Hmg1 but lanosterol accumulation resulting from Cyp51A/B inhibition. To test whether depleting Hmg1 protein could re-sensitize resistant strains, we utilized a GFP-targeted degradation system designed to target Hmg1 for degradation without engaging its SSD. Combining this system with voriconazole treatment reduced the MIC at least four-fold in both conidia and established hyphae in susceptible and resistant strains. Together, our findings support a model wherein triazole-induced lanosterol accumulation drives HrdA/InsA-dependent accelerated degradation of Hmg1 and establish that pharmacologically inducing this secondary mechanism can enhance triazole activity in both susceptible and resistant isolates.

The organization of signaling proteins into multi-component complexes is essential for innate immune responses and is often achieved through the oligomerization of death domains (DDs). The Myddosome, composed of MyD88 and IRAK family kinases, exemplifies this mechanism. However, how DDs assemble in an ordered and regulated manner to form Myddosomes to control signaling output remains unclear. Here, we show that IRAK4 kinase activity and autophosphorylation are required for IRAK4:IRAK1 heterotypic DD interactions. We show that IRAK1 is autoinhibited by its kinase domain, and IRAK4 phosphorylation relieves this inhibition, enabling IRAK1 DD incorporation into Myddosomes. This phosphorylation-dependent switch similarly controls IRAK2/3 incorporation. Thus, IRAK4 autophosphorylation acts as an energy-dependent molecular switch that integrates phosphorylation with protein oligomerization to drive Myddosome maturation and signal transduction. This mechanism demonstrates how enzymatic activity can impose temporal and spatial control over macromolecular assembly to ensure directional and context-dependent inflammatory signaling.