Phase-separated condensate-aided enrichment of biomolecular interactions for high-throughput drug screening in test tubes

Min Zhou,Weiping Li,Jian Li,Leiming Xie,Rongbo Wu,Liang Wang,Shuai Fu,Wei Su,Jianyang Hu,Jing Wang,Pilong Li

doi:10.1074/jbc.ra120.012981

Abstract

Modification-dependent and -independent biomolecular interactions, including protein-protein, protein-DNA/RNA, protein-sugar, and protein-lipid interactions, play crucial roles in all cellular processes. Dysregulation of these biomolecular interactions or malfunction of the associated enzymes results in various diseases; therefore, these interactions and enzymes are attractive targets for therapies. High-throughput screening can greatly facilitate the discovery of drugs for these targets. Here, we describe a biomolecular interaction detection method, called phase-separated condensate-aided enrichment of biomolecular interactions in test tubes (CEBIT). The readout of CEBIT is the selective recruitment of biomolecules into phase-separated condensates harboring their cognate binding partners. We tailored CEBIT to detect various biomolecular interactions and activities of biomolecule-modifying enzymes. Using CEBIT-based high-throughput screening assays, we identified known inhibitors of the p53/MDM2 (MDM2) interaction and of the histone methyltransferase, suppressor of variegation 3-9 homolog 1 (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.

Highlights

Modification-dependent and -independent biomolecular interactions, including protein–protein, protein–DNA/RNA, protein–sugar, and protein–lipid interactions, play crucial roles in all cellular processes
We created two fusion proteins, one with GFP fused to the C terminus of SmF (SmF-GFP) and the other with the second Src homology 3 (SH3) domain of human NCK1 fused to the C terminus of SmF-GFP (SmF-GFP-SH3)
Size-exclusion chromatography coupled with multiangle light scattering (SEC-MALS) analysis indicated that SmF-GFP formed a 14-meric complex in solution, and further fusion of a SH3 domain to SmF-GFP did not alter the 14-meric status (Fig. 1a)

Summary

Introduction

Modification-dependent and -independent biomolecular interactions, including protein–protein, protein–DNA/RNA, protein–sugar, and protein–lipid interactions, play crucial roles in all cellular processes Dysregulation of these biomolecular interactions or malfunction of the associated enzymes results in various diseases; these interactions and enzymes are attractive targets for therapies. Cells use numerous membrane-enclosed and membraneless organelles to compartmentalize biochemical reactions Membraneless organelles, such as P granules [13], nucleoli [14], stress granules [15], and post-synaptic densities [16], are collectively referred to as biomolecular condensates. Inspired by the scaffold-client model, we explored the possibility of using a similar architecture to implement a system for assaying modification-dependent and -independent biomolecular interactions, which can be used for efficient discovery of drugs targeting these biomolecular interactions and the associated enzymes

Methods

Results

Conclusion