Compartmentalization of enhanced biomolecular interactions for high-throughput drug screening in test tubes

Abstract

Modification-dependent and -independent biomolecular interactions (BIs), including protein-protein, protein-DNA/RNA and protein-lipid, play crucial roles in all cellular processes. Dysregulation of BIs or malfunction of the associated enzymes results in various diseases, thus they are attractive targets for therapies. High-throughput screening (HTS) can greatly facilitate the discovery of drugs for these targets. Here we describe a HTS drug discovery method, called compartmentalization of enhanced biomolecular interactions in test tubes (CEBIT). CEBIT uses selective recruitment of biomolecules into phase separated compartments harboring their cognate binding partners as readouts.CEBIT were tailored to detect various BIs and associated modifying enzymes. Using CEBIT-based HTS assays, we successfully identified known inhibitors of the p53/MDM2 interaction and of SUV39H1 from a compound library. CEBIT is simple and versatile, and is likely to become a powerful tool for drug discovery and basic biomedical research.

Transcriptional condensates formed by phase-separated ALOG family proteins control shoot meristem maturation for flowering

“…Image analysis of heterogeneity of the fluorescence intensity and quantification of the cells with condensates indicated that almost all the combinations of interacting pairs induce formation of biological condensates, except the combination of TFAM1 and TFAM2, whose interaction displays homogenous fluorescence signals ( Figure 6A, 6B and Supplemental Figure 3). To validate the interactions in vitro and monitor the phase separation behavior during interactions without disturbance from other potentially interacting partners, we recombinantly expressed and purified mCherry-TMF (red fluorescence) and GFP-TFAM (green fluorescence) proteins to perform crossmixing phase separation reactions (Zhou et al, 2020). Apparently, TMF can coexist with itself to form the perfectly merged droplets ( Figure 6C).…”