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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):

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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			Action	DRS
	20190321_isoTMT_06_01.raw	Raw
	20190321_isoTMT_06_02.raw	Raw
	20190321_isoTMT_06_03.raw	Raw
	20190321_isoTMT_06_04.raw	Raw
	20190321_isoTMT_06_05.raw	Raw

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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

Redirecting the pioneering function of FOXA1 with covalent small molecules

Project description:Pioneer transcription factors (TFs) exhibit a special ability to bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report electrophilic small molecules that stereoselectively and site-specifically bind the pioneer TF, FOXA1, at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the covalent ligands relax the canonical DNA binding preference of FOXA1 by strengthening interactions with suboptimal ancillary sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

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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.

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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

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

2024-02-12 | PXD049322 | Pride

Project description:Assigning functionality to cysteines by base editing of cancer dependency genes

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A platform for mapping reactive cysteines within the immunopeptidome

Project description:The major histocompatibility complex class I (MHC-I) antigen presentation pathways play pivotal roles in orchestrating immune responses. Recent studies have begun to utilize cysteines within the immunopeptidome for therapeutic applications, such as using covalent ligands to create haptenated neoantigens for inducing an immune response. In this study, we report a platform for mapping reactive cysteines present on MHC-I-bound peptide antigens. We have developed cell-impermeable sulfonated maleimide probes capable of effectively capturing reactive cysteines on antigens. Utilizing these probes in chemoproteomic experiments, we discovered that cysteines on MHC-I-bound antigens exhibit various degrees of reactivity. Furthermore, interferon-gamma stimulation produces increased reactivity of cysteines at position 8 of 9-mer MHC-I-bound antigens. Our findings may open up new avenues for understanding the distinctive roles of cysteine within the MHC-I immunopeptidome and leveraging the differentially reactive cysteines for therapeutic intervention.

2024-10-17 | PXD054678 | Pride

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