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

Abstract

SUMMARY 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. 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 show that fragment hits can be advanced to furnish selective ligands that affect the activity of proteins heretofore lacking chemical probes. We further combine fragment-based chemical proteomics with phenotypic screening to identify 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.

Mapping Protein Targets of Bioactive Small Molecules Using Lipid-Based Chemical Proteomics

“…S2). Using this same assay, we also confirmed that the recently described PTGR2 inhibitor KNJ057, 24 but not the structurally related inactive analog KNJ051, also blocks PTGR2 interactions with the A-DA probe (Fig. S2).…”

Nitric oxide inhibits the mitochondrial carnitine/acylcarnitine carrier through reversible S -nitrosylation of cysteine 136

“…Such perspectives, among other reasons, support the great interest recently emerged, in searching for inhibitors of the CACT[51]. In conclusion, NO represents the physiological inhibitor acting via nitrosylation of C136, which was previously identified as a crucial residue of CACT byThis work was supported by funds from: Programma Operativo Nazionale [01_00937] -"Modelli sperimentali biotecnologici integrati per lo sviluppo e la selezione di molecole di interesse per la salute dell'uomo", MIUR (Italian Ministery of Instruction, University and Research).…”

Small Molecules and Their Role in Effective Preclinical Target Validation

“…Shown to indeed be possible, a clickable derivative was accessed which was used to understand the direct biological targets of the probe and assess BRD4 target engagement by (+)-JQ-1 [15,16]. Clickable photoaffinity labeling probes have also been used to map thousands of site-specific small-molecule protein interactions within human cells, from which more potent and selective small molecules can be optimized, presenting this technique as a versatile approach to small-molecule discovery as we advance into the future [17].…”