Gain-of-Function Variants in SMAD4 Compromise Respiratory Epithelial Function.

BACKGROUND: Myhre syndrome (MIM #139210) is an exceedingly rare yet increasingly diagnosed genetic disorder arising from germline variants in the SMAD4 gene. Its core manifestation is the progression of stiffness and fibrosis across multiple organs. Individuals with Myhre syndrome exhibit a propensity for upper respiratory remodeling and infections. The molecular and cellular mechanisms underlying this phenotype remain unclear. OBJECTIVE: We aim to investigate how SMAD4 pathogenic variants associated with Myhre syndrome impact SMAD4 protein levels, activation, and physiological functions in patient-derived nasal epithelial cells. METHODS: Clinical observations were conducted on a cohort of 47 patients recruited at MGH from 2016 to 2023. Nasal epithelial basal cells were isolated and cultured from inferior turbinate brushings of healthy subjects (n=8) and Myhre syndrome patients (n=3, SMAD4-Ile500Val, Arg496Cys, and Ile500Thr). Transcriptomic analysis and functional assays were employed to assess SMAD4 levels, transcriptional activity, and epithelial cell host defense functions, including cell proliferation, mucociliary differentiation, and bacterial elimination. RESULTS: Clinical observations revealed a prevalent history of otitis media and sinusitis among most individuals with Myhre syndrome. Analyses of nasal epithelial cells indicated that SMAD4 mutations do not alter SMAD4 protein stability or upstream regulatory SMAD phosphorylation but enhance signaling transcriptional activity, supporting a gain-of-function mechanism, likely attributable to increased protein-protein interaction of the SMAD complex. Consequently, Myhre syndrome nasal basal cells exhibit reduced potential in cell proliferation and mucociliary differentiation. Furthermore, Myhre syndrome nasal epithelia are impaired in bacterial killing. CONCLUSIONS: Compromised innate immunity originating from epithelial cells in Myhre syndrome may contribute to increased susceptibility to upper respiratory infections.

A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting the SMAD4-SMAD3-DNA complex.

The transforming growth factor-beta (TGFß) signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFß agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFß agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small-molecule inhibitors specifically targeting SMAD4, the downstream master regulator of the TGFß pathway, would offer an alternative approach with significant therapeutic potential for anti-TGF-ß signaling. In this study, we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) 1536-well plate format. This assay enables simultaneous monitoring of the protein‒protein interaction between SMAD4 and SMAD3, as well as the protein‒DNA interaction between SMADs and their consensus DNA-binding motif. The multiplexed TR-FRET assay exhibits high sensitivity, allowing the dynamic analysis of the SMAD4-SMAD3-DNA complex at single-amino acid resolution. Moreover, the multiplexed uHTS assay demonstrates robustness for screening small-molecule inhibitors. Through a pilot screening of an FDA-approved bioactive compound library, we identified gambogic acid and gambogenic acid as potential hit compounds. These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small-molecule inhibitors that target the SMAD4-SMAD3-DNA complex as novel anti-TGFß signaling agents.

A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting SMAD4-SMAD3-DNA complex.

The signaling pathway of transforming growth factor-beta (TGFß) plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFß agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFß agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small molecule inhibitors specifically targeting SMAD4, the downstream master regulator of TGFß pathway, would offer an alternative approach with significant therapeutic potential for anti-TGF-ß signaling. In this study, we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) 1536-well plate format. This assay enables simultaneous monitoring of the protein-protein interaction (PPI) between SMAD4 and SMAD3, as well as the protein-DNA interaction (PDI) between SMADs and their consensus DNA binding motif. The multiplexed TR-FRET assay exhibits high sensitivity, allowing the dynamic analysis of the SMAD4-SMAD3-DNA complex at single amino acid resolution. Moreover, the multiplexed uHTS assay demonstrates robustness for screening small molecule inhibitors. Through a pilot screening of an FDA-approved and bioactive compound library, we identified gambogic acid and gambogenic acid as potential hit compounds. These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small molecule inhibitors that target the SMAD4-SMAD3-DNA complex as novel anti-TGFß signaling agents.