TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS

SARS-CoV-2,the coronavirus that causes the disease COVID-19,has claimed millions of lives over the past 2 years.This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle.The interaction between host transmembrane serine protease 2(TMPRSS2)and viral SPIKE protein is an important initial step in SARS-CoV-2 infection,offering an opportunity for therapeutic development of viral entry inhibitors.Here,we report the development of a time-resolved fluorescence/Förster resonance energy transfer(TR-FRET)assay for monitoring the TMPRSS2–SPIKE interaction in lysate from cells co-expressing these proteins.The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance.To enable large-scale compound screening,we further miniaturized the assay into 1536-well ultrahigh-throughput screening(uHTS)format.A pilot screen demonstrated the utilization of the assay for uHTS.Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein–protein interaction.

Development of a miniaturized 3D organoid culture platform for ultra-high-throughput screening

The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery.However,organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening(HTS)-based drug discovery due to technical difficulties.Using genetically engineered human colon organoids as a model system,here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS.We first established organoid culturing in a 384-well plate format and validated its application in a cell viability HTS assay by screening a 2036-compound library.We further miniaturized the 3D organoid culturing in a 1536-well ultra-HTS format and demonstrated its robust performance for large-scale primary compound screening.Our miniaturized organoid culturing method may be adapted to other types of organoids.By leveraging the power of 3D organoid culture in a high-density plate format,we provide a physiologically relevant screening platform to model tumors to accelerate organoid-based research and drug discovery.

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

The transforming growth factor-beta(TGFβ)signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment,making anti-TGFβagents a significant area of interest in cancer immunotherapy.However,the clinical translation of current anti-TGFβagents that target upstream cytokines and receptors remains challenging.Therefore,the development of small-molecule inhibitors specifically targeting SMAD4,the downstream master regulator of the TGFβpathway,would offer an alternative approach with significant therapeutic potential for anti-TGFβsignaling.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 antiTGFβsignaling agents.