Ligand and Target Discovery by Fragment-Based Screening in Human Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE90731 | GEO | 2017/01/24
SECONDARY ACCESSION(S): PRJNA355636
REPOSITORIES: GEO
Similar Datasets
Project description:A large swath of the human proteome is dedicated to RNA homeostasis, but most RNA-binding proteins lack chemical probes1,2. Here, phenotypic screening led to the discovery of electrophilic small molecules that swiftly (within 4 h) and stereospecifically decrease transcripts encoding the androgen receptor (AR) and its major V7 splice variant in human prostate cancer cells. We show by chemical proteomics that these compounds covalently engage cysteine-145 on the RNA-binding protein NONO. Transcriptomics and proteomics profiling revealed that covalent NONO ligands suppress a discrete set of transcripts and proteins, including multiple oncogenic transcription factors, and impair the proliferation of cancer cells. These effects were not observed following genetic disruption of NONO, which instead blocked ligand activity. The covalent ligands promote accumulation of NONO in nuclear foci and at the first 5 splice site of immature transcripts, pointing to a trapping mechanism that may prevent compensatory action by the related protein PSPC1, which was found to increase in cancer cells following genetic or chemical perturbation of NONO. These findings, taken together, designate NONO as a druggable RNA-binding protein that can be co-opted by covalent small molecules to suppress pro-tumorigenic transcriptional networks.
2023-03-03 | GSE198212 | GEO
Project description:A large swath of the human proteome is dedicated to mRNA homeostasis, but most RNA-binding proteins lack chemical probes. Here, we report the discovery through phenotypic screening of electrophilic small molecules that swiftly (within 4 h) and stereospecifically decrease transcripts encoding the androgen receptor (AR) and its major V7 splice variant in human prostate cancer cells. We show by chemical proteomics that these compounds covalently engage cysteine-145 of the RNA-binding protein NONO. Broader profiling revealed that covalent NONO ligands suppress a discrete set of transcripts and proteins, including multiple oncogenic transcription factors, and impair the proliferation of cancer cells. These effects were not observed following genetic disruption of NONO, which instead blocked ligand activity. The covalent ligands promote accumulation of NONO in nuclear foci and at the first 5’ splice site of immature transcripts, pointing to a trapping mechanism that may prevent the compensatory action of the paralogous proteins PSPC1 and SFPQ, which were found to increase in cancer cells following genetic or chemical perturbation of NONO. These findings, taken together, designate NONO as a druggable RNA-binding protein that can be co-opted by covalent small molecules to suppress pro-tumorigenic transcriptional networks.
2024-01-26 | PXD039283 | Pride
Project description:Efforts to expand the ligandable proteome have explored stereochemically defined fragments equipped with photoreactive and clickable handles (photo-stereoprobes) for mapping reversible small molecule-protein interactions in living systems. Here, we investigate the impact of structural elaboration of fragment photo-stereoprobes on interactions with the proteome of human cancer cells. We find that even conservative increases in size and complexity have profound effects on protein binding for fragment photo-stereoprobes, leading to enhanced stereoselective protein interactions, while abrogating others. We use stereochemically matched competitor ligands to assess the extent of protein binding in human cells, leading to the discovery of high-stoichiometry ligands for proteins from different functional classes, including a structurally novel set of epoxide hydrolase inhibitors. Our findings support the broad ligandability potential of the human proteome while also highlighting challenges in converting reversible small molecule-protein interaction maps into more advanced chemical probes.
2024-05-31 | PXD052692 | Pride
Project description:Chemical modulation of protein function enables a mechanistic understanding of biological circuits and represents the foundation of most medicines. However, despite major efforts over decades of research, around 80% of the human proteome still lack functional ligands. Chemical proteomics allowed the advancement of fragment-based ligand discovery (FBLD) towards cellular systems and emerged as a promising strategy to detect reversible fragment-protein interactions that can be furnished into bioactive chemical probes. Limitations in scale and throughput have, however, stymied the interpretation of the resulting ligandability maps, thus complicating the identification of specific and actionable fragment-protein interactions. Here, we report reversible, global ligandability maps for 435 structurally different fragments, collectively representing 6739 discrete interactions. We benchmark the dataset by showing that identified fragments can be advanced to active chemical probes. Integrating multi-layered ligandability portraits with artificial intelligence (AI) and machine learning (ML) enabled quantitative and qualitative predictions of fragment interactomes via chemically interpretable models. The resulting, interactive catalogue of fragment-protein interactions and predictive models is expected to expedite ligand discovery efforts in a community-wide fashion and should facilitate the pursuit of hitherto undrugged target proteins.
2024-05-07 | PXD041587 | Pride
Project description:A large swath of the human proteome is dedicated to mRNA homeostasis, but most RNA-binding proteins lack chemical probes1,2. Here, we report the discovery through phenotypic screening of electrophilic small molecules that swiftly (within 4 h) and stereospecifically decrease transcripts encoding the androgen receptor (AR) and its major V7 splice variant in human prostate cancer cells. We show by chemical proteomics that these compounds covalently engage cysteine-145 on the RNA-binding protein NONO. Broader profiling revealed that covalent NONO ligands suppress a discrete set of transcripts and proteins, including multiple oncogenic transcription factors, and impair the proliferation of cancer cells. These effects were not observed following genetic disruption of NONO, which instead blocked ligand activity. The covalent ligands promote accumulation of NONO in nuclear foci and at the first 5’ splice site of immature transcripts, pointing to a trapping mechanism that may prevent compensatory action by the related protein PSPC1, which was found to increase in cancer cells following genetic or chemical perturbation of NONO. These findings, taken together, designate NONO as a druggable RNA-binding protein that can be co-opted by covalent small molecules to suppress pro-tumorigenic transcriptional networks.
2024-01-26 | PXD032087 | Pride
Project description:Most proteins in the human proteome lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such “binding-first” assays affect protein function, nonetheless, often remains unclear. Here, we describe a “function first” proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disassemble the PA28 proteasome regulatory complex and remodel the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells. In this experiment, we probed the RNA-binding profile of DDX42 by eCLIP via an endogenously introduced HA-tag in the context of various SF3B1 ligands.
2023-04-13 | GSE220845 | GEO
Project description:Most proteins in the human proteome lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such “binding-first” assays affect protein function, nonetheless, often remains unclear. Here, we describe a “function first” proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disassemble the PA28 proteasome regulatory complex and remodel the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells. In this experiment, we probed the RNA-binding profile of DDX42 by eCLIP via an endogenously introduced HA-tag in the context of various SF3B1 ligands.
2023-04-13 | GSE220185 | GEO
Project description:Activity-based protein profiling (ABPP) has emerged as a powerful and versatile tool to enable annotation of protein functions and discovery of targets of bioactive ligands in complex biological systems. It utilizes chemical probes to covalently label functional sites in proteins so that they can be enriched for mass spectrometry (MS)-based quantitative proteomics analysis. However, the semi-stochastic nature of data-dependent acquisition (DDA) and high cost associated with isotopically encoded quantification reagents compromise the power of ABPP in multidimensional analysis and high-throughput screening, when a large number of samples need to be quantified in parallel. Here, we combine the data-independent acquisition (DIA) mass spectrometry with ABPP to develop an efficient label-free quantitative chemical proteomic method, DIA-ABPP, with good reproducibility and high accuracy for multiplex quantification. We demonstrated the power of DIA-ABPP for comprehensive profiling of functional cysteineome in three distinct applications, including dose-dependent quantification of cysteines’ sensitivity toward a reactive metabolite, screening of ligandable cysteines with a covalent fragment library, and profiling of cysteinome fluctuation in circadian clock cycles. DIA-ABPP will open new opportunities for in-depth and multidimensional profiling of functional proteomes and interactions with bioactive small molecules in complex biological systems.
2021-07-27 | PXD027578 |
Project description:Photoreactive fragment-like probes have been applied to discover target proteins that constitute novel cellular vulnerabilities and to identify viable chemical hits for drug discovery. Through forming covalent bonds, functionalized probes can achieve stronger target engagement and require less effort for on-target mechanism validation. However, the design of probe libraries, which directly affects the biological target space that is interrogated, and effective target prioritization remain critical challenges of such a chemical proteomic platform. In this study, we designed and synthesized a diverse panel of twenty fragment-based probes containing natural product-based privileged structural motifs for small-molecule lead discovery. These probes were fully functionalized with orthogonal diazirine and alkyne moieties and used for protein crosslinking in live lung cancer cells, target enrichment via “click chemistry,” and subsequent target identification through label-free quantitative LC-MS/MS analysis. Pair-wise comparison with a blunted negative control probe and stringent prioritization via individual cross-comparisons against the entire panel identified glutathione S-transferase zeta 1 (GSTZ1) as a specific and unique target candidate. DepMap database query, RNA interference-based gene silencing and proteome-wide tyrosine reactivity profiling suggested that GSTZ1 cooperated with different oncogenic alterations by supporting survival signaling in refractory NSCLC cells. This finding may form the basis for developing novel GSTZ1 inhibitors to improve the therapeutic efficacy of oncogene-directed targeted drugs. In summary, we designed a novel fragment-based probe panel and developed a target prioritization scheme with improved stringency, which allows for the identification of unique target candidates, such as GSTZ1 in refractory lung cancer.
2024-04-05 | PXD037894 | Pride
Project description:Chemical proteomics enables the global assessment of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, been limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically-defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these ‘photo-stereoprobes’ interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible nanoBRET assays. Integrated phenotypic analysis and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and discovering and characterizing bioactive small molecules by cell-based screening.
2024-05-31 | PXD050096 | Pride