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Assigning functionality to cysteines by base editing of cancer dependency genes

ABSTRACT: Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We finally demonstrate how measurements of reactivity in native versus denatured proteomes can further discriminate essential cysteines amendable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell In Vitro, Cell Culture

DISEASE(S): Cell Type Cancer

SUBMITTER: Haoxin Li

LAB HEAD: Benjamin Cravatt

PROVIDER: PXD038232 | Pride | 2023-09-06

REPOSITORIES: Pride

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Assigning functionality to cysteines by base editing of cancer dependency genes

Project description: Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in >110 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

2023-09-06 | PXD041314 | Pride

Assigning functionality to cysteines by base editing of cancer dependency genes

Project description:Chemical probes are lacking for most human proteins. Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse proteins. Determining which of these covalent binding events impact protein function, however, remains challenging. Here, we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cell proliferation. We show that the resulting atlas, which covers >13,800 cysteines on >1,750 cancer dependency proteins, correctly predicts the essentiality of cysteines targeted by cancer therapeutics and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines on >110 cancer dependency proteins. We finally demonstrate how measurements of reactivity in native versus denatured proteomes can further discriminate essential cysteines amendable to chemical modification from those buried in protein structures, providing a valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

2023-09-06 | PXD038239 | Pride

An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells

Project description:Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

2020-07-30 | GSE137756 | GEO

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Project description:Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFkB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting,and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity

2024-04-23 | PXD047840 | Pride

Direct mapping of ligandable tyrosines and lysines in cells with chiral sulfonyl fluoride probes

Project description:Advances in chemoproteomic technology have revealed covalent interactions between small molecules and protein nucleophiles, primarily cysteine, on a proteome-wide scale. Most chemoproteomic screening approaches are indirect, relying on competition between electrophilic fragments and a minimalist electrophilic probe with inherently limited proteome coverage. Here, we develop a chemoproteomic platform for direct electrophile-site identification based on enantiomeric pairs of clickable arylsulfonyl fluoride probes. Using stereoselective site modification as a proxy for ligandability in intact cells, we identified 634 tyrosines and lysines within functionally diverse protein sites, liganded by structurally diverse probes. Among multiple validated sites, we discovered a chiral probe that modifies Y228 in the MYC binding site of the epigenetic regulator WDR5, as revealed by a high-resolution crystal structure. A distinct chiral probe stimulates tumor cell phagocytosis by covalently modifying Y387 in the recently discovered immuno-oncology target, APMAP. Our work provides a deep resource of ligandable tyrosines and lysines for the development of covalent chemical probes.

2023-05-18 | PXD042307 | Pride

An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells

2024-02-12 | PXD049322 | Pride

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics

Project description:The nucleus controls cell growth and reproduction by well-orchestrated interactions between nuclear proteins and chromatin. Mutations that result in the dysregulation of these chromatin-associated proteins are known to lead to the onset of numerous diseases. Many of these nuclear proteins have remained recalcitrant to traditional non-covalent small-molecule ligand discovery. Covalent ligands can provide a promising approach for targeting proteins such as transcription factors that lack ordered small-molecule binding pockets. Here, we present a chemoproteomic platform that couples proximity labeling (PL) using a Histone-TurboID (His-TID) construct with competitive isoTOP-ABPP to identify ligandable nuclear cysteines. Application of covalent scout fragments, KB02 and KB05, revealed ligandable cysteine sites on diverse proteins involved in transcriptional regulation, spindle assembly, and DNA repair. To identify functional liganding events that alter target-protein association with chromatin, we monitor the effects of KB02 and KB05 on the chromatin-associated proteome. Importantly, we identify proteins that show both increased and decreased association with chromatin in the presence of the covalent fragments. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and association with chromatin upon covalent liganding at Cys106 by KB02. Together, our PL-assisted nuclear-focused chemoproteomic platform provides us insights into nuclear cysteine ligandability and the functional effects of ligand binding on chromatin association, thereby furthering our understanding of the potentially druggability of the nuclear proteome.

2024-01-24 | PXD043732 | Pride

A Proteome-Wide Atlas of Lysine-Reactive Chemistry

Project description:Recent advances in chemical proteomics have begun to characterize the reactivity and ligandability of lysines on a global scale. Yet, only a limited diversity of aminophilic electrophiles have been evaluated for interactions with the lysine proteome. Here, we report an in-depth profiling of >30 uncharted aminophilic chemotypes that greatly expands the content of ligandable lysines in human proteins. Aminophilic electrophiles showed disparate proteomic reactivities that range from selective interactions with a handful of lysines to, for a set of dicarboxaldehyde fragments, remarkably broad engagement of the covalent small molecule-lysine interactions captured by the entire library. We used these latter â€œscoutâ€� electrophiles to efficiently map ligandable lysines in primary human immune cells under stimulatory conditions. Finally, we show that aminophilic compounds perturb diverse biochemical functions through site-selective modification of lysines in proteins, including protein-RNA interactions implicated in innate immune responses. These findings support the broad potential of covalent chemistry for targeting functional lysines in the human proteome.

2021-09-20 | PXD025829 | Pride

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

Project description: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.

2017-01-24 | GSE90731 | GEO

NAIA for cysteine profiling in HepG2 cells, protein target identification of covalent ligand and studying cysteine oxidative modifications

Project description:New cysteine probes, N-acryloylindole-alkynes (NAIAs), have been developed. We have applied NAIA-5 for cysteine profiling in HepG2 cell lysates, and the results have been compared to the conventional cysteine probe iodoacetamide-alkyne (IAA). NAIA-5 allows identification of more ligandable Cys than IAA in our experiment, and more interesting, a unique pool of Cys which have not been profiled previously by any reported cysteine probe. We have further utilized NAIA-5 for covalent ligand screening experiment. This allows us to discover hit compounds targeting the newly identified cysteines and proteins. One of the hit compounds, CL1, demonstrated good bioactivity by arresting cell cycle at G1 phase through targeting proteins associated with cell cycle and regulations. In addition, NAIA-5 has been successfully coupled with NAI compound for studying cysteine oxidative modifications in HepG2 cells by MS experiments.

2023-06-14 | PXD041264 | Pride

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