Kymera Therapeutics

Tubulointerstitial fibrosis (TIF) is a crucial pathological feature and inevitable consequence of progressive diabetic kidney disease (DKD). Transfer RNA-derived fragments (tRFs), a novel class of small non-coding RNA by cleavage of tRNAs, have recently attracted attention due to their functional significance in diverse biological processes. However, the role of tRFs in TIF is largely elusive. In the present study, we found that a tRF, tRF‑1:30‑Gln‑CTG‑4, was markedly decreased in proximal renal tubules and highly correlated with TIF in DKD mice. The overexpression of tRF‑1:30‑Gln‑CTG‑4 revealed a therapeutic effect against TIF. Further, RNA pull-down and RNA-immunoprecipitation assay were performed to identify that FBXO7, an adaptor protein in the SCF E3 ligase complex, was the targeted protein of tRF‑1:30‑Gln‑CTG‑4. Mass spectrometry and co-immunoprecipitation experiments revealed that PINK1, a master mitophagy-regulating protein, acted as the substrate of FBXO7 in tubular epithelial cells. Mechanistically, elevated FBXO7 promoted mitophagy defect through mediating PINK1 ubiquitylation and proteasomal degradation. The overexpression of tRF‑1:30‑Gln‑CTG‑4 alleviated defective mitophagy. Taken together, this study not only represents a novel insight into the pathogenesis of TIF but also provides a promising therapeutic targeting for the delaying the progression of DKD.

One of the alternative splicing variants of retinal G protein-coupled receptor (RGR), RGR-d, is recognized as a misfolded protein. With or without inhibition of the proteasome system, RGR-d has a cytotoxic effect on retinal pigment epithelium (RPE) cells. RGR-d mutant mice exhibit age-related macular degeneration-like pathologic changes. However, humans with RGR-d likely express both wild-type and splice variant forms. In this study, we investigate the protective influence of the co-expression of RGR and RGR-d protein in RPE cells. ARPE-19 cells overexpressing both RGR and RGR-d were treated with or without MG132. The protein-protein interaction, ubiquitination, endoplasmic reticulum (ER) stress, autophagy flux, and subcellular localization were explored. Aged RGR-d mice were subjected to ocular examination. We confirm that RGR binds RGR-d in RPE cells and decreases the polyubiquitin of RGR-d. Formation of the RGR/RGR-d complex could assist RGR-d in correctly folding on the endoplasmic reticulum (ER), and ameliorate ER stress and autophagy inhibition that were induced by misfolded RGR-d protein. The complexes highly colocalize with Golgi apparatus and maintain its typical morphology, avoiding degradation by lysosomes. We observed that RGR-d mice exhibited AMD-like subretinal hyper-reflective deposits on fundus examination. We have identified a fundamental mechanism in which RGR-opsin binds its splice variant, RGR-d, to form a protective complex that alleviates cytotoxicity to RPE. This discovery is significant as it reveals a previously unknown endogenous defense against proteotoxicity in the retina. Targeting the RGR-d may represent a novel and highly promising intervention for the protein-misfolding pathology of dry age-related macular degeneration.

The persistent threat of porcine epidemic diarrhea virus (PEDV) to the global swine industry is compounded by high neonatal piglet mortality and the absence of effective antiviral therapies. Host-directed strategies that reinforce immunity offer a promising avenue to counter viral immune evasion. Through screening of an FDA-approved compound library, we identify the small-molecule cyclocytidine hydrochloride (Cyclo-C) as a potent inhibitor of PEDV replication that acts by stabilizing the peroxisomal biogenesis factor PEX13, a previously unrecognized host restriction factor. The antiviral activity of Cyclo-C is strictly PEX13-dependent, as it is completely abrogated in PEX13 knockout cells. Mechanistically, Cyclo-C disrupts the interaction between PEX13 and the viral nonstructural protein 8 (NSP8), thereby preventing NSP8-mediated PEX13 degradation and the subsequent induction of PI3K/AKT/mTOR-driven pexophagy. Preservation of peroxisomal integrity stabilizes the peroxisome-localized pool of MAVS, leading to a robust enhancement of type III interferon (IFN-III) responses that suppress viral replication. Critically, this mechanism translates in vivo, where Cyclo-C treatment of PEDV-challenged piglets significantly reduces mortality, lowers viral loads, and protects intestinal villus architecture. Our findings establish Cyclo-C as a first-in-class host-directed therapeutic candidate and validate the concept that pharmacological preservation of peroxisome-mediated innate immunity represents an effective antiviral strategy against enteric coronaviruses. The high genetic variability of porcine epidemic diarrhea virus (PEDV) limits current vaccine efficacy, and no antiviral therapeutics exist. Host-directed therapies targeting cellular pathways that viruses exploit for immune evasion offer an alternative approach. Here, we identify the FDA-approved compound Cyclo-C as a potent inhibitor of PEDV replication. Cyclo-C acts by stabilizing PEX13, a host protein that the virus degrades to evade immunity. By blocking viral protein NSP8 from binding PEX13, Cyclo-C prevents virus-induced pexophagy, thereby preserving peroxisomal integrity. This preserves peroxisome-localized MAVS and enhances type III interferon responses. In infected neonatal piglets, Cyclo-C reduced mortality and viral loads while protecting intestinal integrity. This study provides proof of concept that targeting peroxisomal immune regulation is a viable antiviral strategy and identifies Cyclo-C as a promising candidate for treating PEDV infection.

Esophageal squamous cell carcinoma (ESCC) is a significantly fatal gastrointestinal malignancy worldwide, with complex proteomic remodeling during its onset and progression. Despite advancements in existing multimodal therapy regimens, the prognosis for advanced patients remains inadequate, necessitating the urgent identification of novel therapeutic targets. The Ubiquitin‑proteasome system is the principal regulatory mechanism for intracellular protein homeostasis, with deubiquitinating enzymes (DUBs) serving as crucial 'editors' that reverse ubiquitination modifications, significantly influencing the stability, localization and functional regulation of oncoproteins. This review aims to systematically delineate the complex regulatory network of DUBs in ESCC, comprehensively investigate their specific mechanisms within critical oncogenic signaling pathways, including TGF‑β, Wnt/β‑catenin, NF‑κB and Hippo, as well as their roles in epithelial‑mesenchymal transition, epigenetic remodeling and the regulation of the immune microenvironment. Furthermore, this review provides a novel cross‑cancer perspective by comparing the similarities and differences of DUBs in ESCC and uterine corpus endometrial carcinoma to determine the conserved and tissue‑specific functions of ubiquitin‑specific protease (USP)14, USP7 and BRCA1‑associated protein 1. Furthermore, the preclinical research progress of small molecule inhibitors and proteolysis‑targeting chimeras targeting DUBs was also assessed to identify the theoretical basis and translational pathway for the development of next‑generation precision oncology therapies.

Hypothyroidism is a prevalent endocrine disorder characterized by insufficient thyroid hormone (T3T4) production. Thyroglobulin (Tg) serves as the prohormone for T3 and T4 production, with many variants of uncertain clinical significance due to genetic diversity in the Tg gene. We leveraged the large-scale All of Us biobank to investigate the disease association of prevalent yet undercharacterized Tg variants. We related variant presence to thyroid-stimulating hormone levels and levothyroxine (LT4) usage as proxies for thyroid function. This identified R152H, Q870H, A993T, P1012L, and P1494L variants linked to increased LT4 usage and decreased thyroid function, while the R320C variant was associated with decreased thyroid function. Molecular characterization in Fisher rat thyroid cells revealed decreased secretion efficiency of R152H, Q870H, and R320C variants. Affinity purification-mass spectrometry demonstrated that secretion-deficient variants showed higher engagement with the protein homeostasis network, indicating protein quality control defects as the pathophysiology mechanism. In contrast, secretion-competent A993T and P1494L variants showed elevated interactions with degradation and antigen-presentation pathways, suggesting an alternative pathophysiology possibly linked to Hashimoto's disease, an autoimmune condition with overproduction of autoantibodies that target thyroid proteins. In support, participants carrying the A993T or P1494L variants had elevated anti-thyroidperoxidase (TPO) antibody levels. We estimate ∼115,000 US individuals currently taking levothyroxine could benefit from precision medicine targeting these variants, with ∼85,000 carrying Q870H. Our findings highlight the power of combining large public biobank data with molecular characterization to understand Tg genotype-to-phenotype relationships. Q870H represents a candidate for molecular therapies to restore secretion, offering precision medicine beyond LT4 replacement therapy.

Mesothelin is an attractive target for CAR T therapy on a number of cancer types; however, the efficacy of this therapy is diminished because the bulk of the cell surface-expressed mesothelin is shed through naturally occurring proteolysis leaving behind a short juxtamembrane peptide "stump". The two problems this creates are one, the bulk of the target protein is no longer on the tumor cell and two, soluble, shed mesothelin persists in the tumor microenvironment and circulates in blood and other body fluids, where it can bind mesothelin-targeted CAR T cells and act as a decoy that reduces engagement with tumor cell-surface mesothelin. These issues have contributed at least in part to the lack of desired efficacy in human clinical trials utilizing CAR T cells that target membrane distal regions of mesothelin (i.e., the shed domain) such as those utilizing the variable domains of anti-mesothelin monoclonal antibodies SS1 and M5. In addition, there have been safety concerns regarding the targeting of mesothelin on normal tissues. Here we describe CAR T cells that utilize novel phage display-derived antibodies specific for the mesothelin stump domain, thus being unaffected by the natural process of mesothelin shedding. Mesothelin "stump-specific" CAR T cells (CAR 422) had cytotoxicity and in vivo activity that were comparable to previously studied anti-mesothelin CAR T cells. Importantly, CAR 422 T cells were effective against tumor cells that were resistant to conventional anti-mesothelin CAR T cells and showed reduced on-target/off tumor toxicity in a human mesothelin knock-in mouse model. Thus, CAR 422 holds potential as a next-generation therapy for challenging solid tumors.

The membrane receptor MerTK is critical for the resolution of inflammation and thus is of pharmacological interest. MerTK function is inhibited by the proteolytic cleavage of its extracellular domain leading to the formation of soluble Mer (sMer). We describe here the NANOBODY molecule A0445046C08 and its half-life-extended version A044500050. Both bound selectively to MerTK and blocked lipopolysaccharide-induced MerTK cleavage in primary macrophages without influencing ligand binding or kinase activity of MerTK. A044500050 reduced Zymosan-induced sMer levels in the peritoneal lavage fluid of a mouse model with sterile peritonitis. The study demonstrates that NANOBODY molecules can be generated that selectively inhibit ectodomain shedding and outlines a novel pharmacological approach for targeting membrane proteins where aberrant cleavage plays a pathogenic role.