Ligand and Target Discovery by Fragment-Based Screening in Human Cells

Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands, and the full complement of proteins with potential to be targeted by small molecules remains unknown. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We advance this knowledge to create ligands that affect the activity of proteins heretofore lacking chemical probes and to identify by phenotypic screening small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets. PolyA+ Rnaseq profilling of 3T3-L1 stably overexpressing Gfp or PGRMC2 treated with dmso and induced to differentiate into adipocytes. RNA samples were collected 24 hours after induction of differentiation.

小分子文库（small-molecule libraries）的合成与筛选技术进展，加速了用于研究生物学过程的化学探针（chemical probes）的发现进程。但目前仅有一小部分人类蛋白质组（human proteome）拥有对应的化学配体（chemical ligands），且可被小分子靶向的蛋白质完整集合仍未明确。本研究报道了一种整合基于片段的配体发现（fragment-based ligand discovery）与定量化学蛋白质组学（quantitative chemical proteomics）的技术平台，可直接在人类细胞中绘制数千个可逆小分子-蛋白质相互作用图谱，其中多数相互作用可实现位点特异性鉴定。基于该研究成果，我们开发出可调控此前尚无化学探针的蛋白质活性的配体，并通过表型筛选（phenotypic screening）鉴定出通过结合功能尚未明确的膜蛋白（membrane protein）PGRMC2来促进脂肪细胞分化（adipocyte differentiation）的小分子。因此，在人类细胞中开展的基于片段的筛选，可获得覆盖全蛋白质组的蛋白质配体结合潜力（protein ligandability）图谱，并助力具有生物活性的小分子（bioactive small molecules）及其分子靶点（molecular targets）的协同发现。本数据集包含稳定过表达绿色荧光蛋白（GFP）或PGRMC2的3T3-L1细胞，经二甲基亚砜（DMSO）处理并诱导分化为脂肪细胞后的PolyA+ RNA测序（PolyA+ RNA-seq）分析结果。RNA样本于分化诱导24小时后收集。