Rational design of RORγT small molecule modulators results in tunable LXRα pleiotropic effects in Th17 cells 2267479

NR1D1‐Mediated Suppression of JAK1/STAT3 Signaling Contributes to the Therapeutic Effects of Triptolide in Rheumatoid Arthritis

Background Triptolide demonstrates potent antiarthritic effects in rheumatoid arthritis (RA), although its complete mechanism is not fully elucidated. This study aimed to systematically identify its core targets and regulatory pathways by integrating network pharmacology, molecular docking, and experimental validation. Methods Potential targets of triptolide and RA‐associated genes were sourced from public databases. Network module analysis identified key targets, the binding affinity of which to triptolide was subsequently assessed by molecular docking, molecular dynamics (MD) simulations, and drug affinity responsive target stability (DARTS) assay. The functional effects of triptolide on RA fibroblast‐like synoviocytes (RA‐FLS) behavior, joint inflammation, and bone erosion were further investigated using in vitro cellular assays and a collagen‐induced arthritis (CIA) mouse model. Results Our analysis identified 73 candidate targets for triptolide against RA, which were primarily enriched in biological processes such as transcription factor activity and inflammatory response. Protein–protein interaction (PPI) network analysis highlighted a core module of five genes, among which STAT3 and its upstream regulator NR1D1 were selected for subsequent investigation. Molecular docking, MD simulation, and DARTS assay confirmed the high‐affinity and direct binding of triptolide to NR1D1. In vitro, triptolide effectively suppressed RA‐FLS migration and invasion, upregulated NR1D1, and inhibited JAK1/STAT3 signaling. In vivo, triptolide administration ameliorated arthritis severity and reduced bone erosion in CIA mice, as well as modulation of the NR1D1/STAT3 axis. Conclusion This study demonstrates that triptolide alleviates RA through NR1D1‐mediated inhibition of JAK1/STAT3 signaling. Our findings elucidate a novel molecular mechanism for triptolide’s antiarthritic effects, thereby revealing the NR1D1/JAK1/STAT3 axis as a potential therapeutic target for RA.

ES302, A Novel TL1A/IL-23p19 Bi-specific Antibody Demonstrates Robust Efficacy and Developability for Inflammatory Bowel Disease 2305836

The TL1A/DR3 and IL-23 pathways exhibit well-documented synergy in driving chronic intestinal inflammation, with TL1A enhancing IFN-γ and IL-17 production in a T cell-intrinsic manner and IL-23 stabilizing the Th17 lineage. We hypothesize that simultaneously co-targeting these two non-redundant axes with a single bispecific agent will deliver superior efficacy by fundamentally reshaping the dysregulated immune landscape in conditions like inflammatory bowel disease (IBD). We developed a fully human, symmetric 1 + 1 IgG-formatted BsAb. And the Fc portion was engineered to extend serum half-life. Binding affinity for both TL1A and IL-23p19 were determined by surface plasmon resonance (SPR). The dual functionality was assessed using cell-based reporter assays: Inhibition of TL1A-induced NF-κB activation and IL-23-induced STAT3 phosphorylation as well as IL17 secretion from PBMCs. The immune complex formation was assessed by SEC-MALS. In vivo efficacy was determined in human TL1A/IL-23 KI mice using TNBS-induced colitis model. Developability was assessed and PK profile was evaluated in FcRn transgenic mice. The ES302 demonstrated high-affinity binding to both targets. It potently neutralized both TL1A and IL-23 functionality from in vitro assays. In animal model, the ES302 showed significantly superior efficacy over monospecific therapies, markedly reducing disease activity, histopathological scoring, and pro-inflammatory cytokines. The molecule exhibited low immunogenicity risk, and excellent developability properties, including high Tm value, low viscosity, and superior stability under stress conditions, compatible with high-concentration formulation for subcutaneous administration. Furthermore, ES302 exhibited excellent PK profile (e.g. very long in vivo half-life) in humanized FcRn mice and NHP. ES302 is a highly differentiated antibody with strong potential for the treatment of inflammatory bowel diseases. n/a Therapeutic Approaches to Autoimmunity (THER)

Discovery of potent, selective and insurmountable MRGPRX2 antagonists for mast cell-driven disorders 2308475

Mas-related G protein—coupled receptor X2 (MRGPRX2) is an important mediator of non-IgE—dependent mast cell activation and has emerged as a key target in pseudo-allergic reactions and chronic inflammatory diseases. Yet, drugs targeting this mechanism remain limited. We report the discovery and characterization of novel series of potent, selective, and insurmountable MRGPRX2 oral small-molecule antagonists. Compounds were evaluated in vitro using CHO-K1 and HEK293 cells overexpressing human MRGPRX2, LAD2 human mast cells, and mast cells differentiated from mobilized CD34+ hematopoietic progenitor cells. Functional activity was assessed across multiple assay platforms – including calcium flux, β-arrestin recruitment, inositol monophosphate (IP1) accumulation and mast cell degranulation assays – using different MRGPRX2 agonists. Selectivity against the closest human homologs MRGPRX1 and MRGPRX4, dog and monkey MRGPRX2 orthologs, and a broad panel of G protein-coupled receptor proteins were tested to ensure high target specificity. Lead molecules were further evaluated in vivo in a humanized MRGPRX2 mouse model of cutaneous anaphylaxis for efficacy. Lead antagonists demonstrated potent and insurmountable inhibition of MRGPRX2-mediated responses across all cellular models and were active against all MRGPRX2 agonists tested. They exhibited minimal off-target activity, no cross-reactivity with dog and monkey orthologs, and no activity against MRGPRX1 and MRGPRX4, underscoring their high target selectivity. In vivo, lead compounds showed favorable pharmacokinetics and significantly reduced mast cell—dependent vascular leakage in a dose-dependent manner. These findings highlight the therapeutic promise of those insurmountable MRGPRX2 antagonists for the treatment of mast cell—driven disorders. Blueprint Medicines Corporation Translational and Interventional Immunology (TI)

Discovery of highly potent and selective VAV1 molecular glue degraders for the treatment of inflammatory disorders 2332299

VAV1 is a highly specific target for immune-mediated diseases. VAV1 functions as a guanine nucleotide exchange factor (GEF) in T- and B-cell receptor signaling and is essential for immune cell activation, proliferation, and inflammatory responses. Despite its therapeutic potential, VAV1 has historically been difficult to target with small molecules due to its multidomain architecture and dual roles as both a GEF and a scaffolding protein involved in GEF-independent signaling pathways. Here, we describe a novel protein degradation strategy using molecular glue degraders (MGDs) to overcome these challenges. Endogenous Vav1 protein levels were detected in Jurkat cells by HiBiT assay. In human and mouse whole blood assays, cells were profiled for VAV1 levels in CD3+ T cells with flow cytometry. Label-free mass spectrometry with data-independent acquisition (DIA) proteomics were used to detect protein abundance levels in human PBMC treated with compounds. In vivo pharmacokinetics and efficacies of the compounds upon oral treatment were evaluated in ICR mice and inflammatory disease models including collagen-induced arthritis (CIA), experimental autoimmune encephalomyelitis (EAE) and naïve CD4+ T cell transfer model of inflammatory bowel disease (IBD). The compounds demonstrated robust VAV1 degradation in both cellular and whole blood assays. The half-maximal degradation concentration (DC50) ranged from sub-nanomolar to low nanomolar, with Dmax values exceeding 95%. Proteomics analysis of human PBMCs confirmed consistent and selective VAV1 degradation. Oral administration of the compounds in animal models resulted in dose-dependent VAV1 degradation and corresponding improvements in disease scores. We have discovered potent and selective VAV1 molecular glue degraders with favorable in vivo pharmacokinetic and pharmacodynamic properties. Further investigations are needed to explore their potential in treating a range of inflammatory conditions, including neuroinflammation. n/a Therapeutic Approaches to Autoimmunity (THER)

Development of Potent G Protein Pathway-Biased GPR183 Agonists

Design and evaluation of engineered MyD88 fusion proteins for enhancing CAR-T therapy 2333869

MyD88 is a scaffold protein that plays a key role in propagating innate immune signals. Recent work has shown that a chemically induced dimerization (CID) platform comprising MyD88 fused to the intracellular domain of CD40 can enhance CAR-T cell fitness via multiple mechanisms. However, this system was associated with toxicity in the clinic due to excessive pro-inflammatory signaling. We sought to better understand the mechanistic levers by which engineered MyD88 assembles and propagates pro-inflammatory signals, with the goal of developing next-generation variants with improved safety profiles. Using structural analysis and bioinformatics, a series of engineered MyD88 fusion proteins were designed, cloned, and expressed in Jurkat cells and primary human T cells in combination with the Tmod™ NOT logic-gated CAR-T platform. Effects on NF-kB induction, cytokine production and cell expansion were evaluated. Consistent with previous reports, we observe that Myd88-based CID systems synergize with TCR signaling pathways in human T cells. We demonstrate that engineered MyD88 assembly can be modulated by either raising or lowering the stability of an initial dimeric “seed.” We then characterize how the integration of either natural or synthetic TRAF recruitment motif(s) impart distinct cytokine profiles, enabling the construction of variants with reduced pro-inflammatory signaling. Our results shed light on the mechanisms underlying inducible Myddosome assembly and offer insight into why MyD88 is particularly amenable to CID. We show that functional outputs can be modulated via altering assembly thresholds as well as tuning signaling motifs, two parameters which help to define design goals for next-generation immunotherapies integrating innate and adaptive signals. n/a Vaccines and Immunotherapy (VAC)