Chemical modulation of proteins enables a mechanistic understanding of biology and represents the foundation of most therapeutics. However, despite decades of research, 80% of the human proteome lacks functional ligands. Chemical proteomics has advanced fragment-based ligand discovery toward cellular systems, but throughput limitations have stymied the scalable identification of fragment-protein interactions. We report proteome-wide maps of protein-binding propensity for 407 structurally diverse small-molecule fragments. We verified that identified interactions can be advanced to active chemical probes of E3 ubiquitin ligases, transporters, and kinases. Integrating machine learning binary classifiers further enabled interpretable predictions of fragment behavior in cells. The resulting resource of fragment-protein interactions and predictive models will help to elucidate principles of molecular recognition and expedite ligand discovery efforts for hitherto undrugged proteins.

Folate metabolism is intricately linked to purine de novo synthesis through the incorporation of folate-derived one-carbon units into the purine scaffold. By investigating chemical and genetic dependencies caused by mutations in methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1 (MTHFD1), we discovered a key role for Nudix hydrolase 5 (NUDT5) in regulating purine de novo synthesis. Genetic depletion and selective chemical degradation showed that a scaffolding role, rather than NUDT5 enzymatic activity, was causing this phenotype. NUDT5 interacted with phosphoribosyl pyrophosphate amidotransferase (PPAT), the rate-limiting enzyme of purine de novo synthesis, to repress the pathway in response to increased purine abundance. Through this mechanism, loss of NUDT5 mediates resistance to purine analogs in cancer treatment and prevents adenosine toxicity in MTHFD1 deficiency.

Despite being considered druggable and attractive therapeutic targets, most of the solute carrier (SLC) membrane transporters remain pharmacologically underexploited. One of the reasons for this is a lack of reliable chemical screening assays, made difficult by functional redundancies among SLCs. In this study we leveraged synthetic lethality between the lactate transporters SLC16A1 and SLC16A3 in a screening strategy that we call paralog-dependent isogenic cell assay (PARADISO). The system involves five isogenic cell lines, each dependent on various paralog genes for survival/fitness, arranged in a screening cascade tuned for the identification of SLC16A3 inhibitors. We screened a diversity-oriented library of ∼90,000 compounds and further developed our hits into slCeMM1, a paralog-selective and potent SLC16A3 inhibitor. By implementing chemoproteomics, we showed that slCeMM1 is selective also at the proteome-wide level, thus fulfilling an important criterion for chemical probes. This study represents a framework for the development of specific cell-based drug discovery assays.

Regulated exocytosis controls key cellular functions ranging from neurotransmitter release to the secretion of immune mediators, and its disruption is associated with numerous pathologies. The cytotoxic activity of lymphocytes is particularly dependent on regulated and polarized lytic granule delivery toward infected or malignant cells. Although genetic and mechanistic studies have identified factors regulating exocytosis in cytotoxic lymphocytes, a systematic mapping of the relevant factors and their relationships is lacking. Through a genome-scale CRISPR knockout screen in a human natural killer cell line, we characterized a complex genetic network regulating cytotoxic granule exocytosis, with lipid metabolism and protein lipidation among the most prominent pathways. By combining global protein palmitoylation and lipidomic studies, we found that ZDHHC17 drives palmitoylation of the core SNARE complex protein SNAP23 to target cytotoxic granules to GM1-rich lipid rafts whose assembly is controlled by serine palmitoyltransferase. In summary, our study identifies previously unrecognized factors essential for cytotoxic function in human lymphocytes and uncovers how lipid metabolism and protein palmitoylation are involved in the process of regulated exocytosis.

Endothelial cells (ECs) are critical regulators of vascular function and exhibit tissue-specialized roles. During aging, these cells become dysfunctional, resulting in increased susceptibility to cardiovascular disease. Although single-cell transcriptomics has revealed extensive endothelial heterogeneity, a comprehensive atlas of human EC transcriptomes over the course of the adult human lifespan has been lacking. Here, we present the Human Aging Endothelial Cell Atlas (HAECA), a harmonized single-cell transcriptomic compendium of over 375,000 ECs from 12 human tissues throughout adulthood. We identified age-associated transcriptional shifts, including a decline in angiogenic gene expression in venous ECs and widespread alterations in extracellular matrix and mechanotransduction-associated signatures. Furthermore, we uncovered a p21-linked transcriptional program in ECs, which we linked to cellular senescence in both in vitro and in vivo models. Together, our study provides a high-resolution transcriptomic reference of the human endothelium across tissues and age.