Covalent Targeting of Splicing in T Cells
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ABSTRACT:
PROVIDER: PRJNA1171002 | ENA |
REPOSITORIES: ENA
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Project description:Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.
2024-10-09 | GSE279165 | GEO
Project description:Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and extensive ITK mRNA alternative splicing. We further introduce the most comprehensive list to date of proteins involved in splicing and leverage cysteine- and protein-directed activity-based protein profiling with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.
2024-10-21 | PXD056704 | Pride
Project description:Small cell lung cancer (SCLC) is an aggressive disease with high mortality. The identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library we observe that SCLC is sensitive to transcription-targeting drugs, and in particular to THZ1, a newly identified covalent inhibitor of cyclin-dependent kinase 7 (CDK7). We find that expression of super-enhancer associated transcription factor genes including MYC family proto-oncogenes and neuroendocrine lineage-specific factors are highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a novel treatment paradigm for targeted SCLC therapy. ChIP-Seq for H3K27ac in small cell lung cancer lines
2014-10-23 | E-GEOD-62412 | biostudies-arrayexpress
Project description:Targeting cellular RNA by small molecules has come to the forefront of biotechnology and holds great promise for therapeutic use. Strategies to identify, validate and optimize these molecules are essential, but are still lacking in some aspects. In particular, the site-specific covalent labeling and modification of RNA in living cells poses many challenges. Here, we describe a general structure-guided approach to engineer non-covalent RNA aptamer–ligand complexes into their covalent counterparts using a molecular tether. The key is to modify the native ligand with an electrophilic handle that allows it to react specifically with a guanine at the RNA ligand binding site. We show that site-specific cross-linking between ligand and RNA is achieved in mammalian cells upon transfection of a genetically encoded version of the preQ1-I riboswitch aptamer. Further, we showcase the versatility of the tether by engineering the first covalent fluorescent light-up aptamer (coFLAP) out of the non-covalent Pepper FLAP. The coPepper system maintains strong fluorescence in live-cell imaging even after repeated washing. Thus, any background signal arising from unspecific fluorophore accumulation in the cell can be eliminated. In addition, we generated a bifunctional Pepper ligand containing a second handle for bioorthogonal chemistry to allow for easily traceable and efficient pulldown of the covalently linked target RNA. Finally, we provide evidence for the suitability of this tethering strategy for specific drug targeting. Taken together, our results show that functionalized ligands generated by rational design can cross-link site-specifically with target RNAs in cells, and hence, open up a wide range of applications in RNA biology that require irreversible small molecule binding.
2024-09-30 | GSE271728 | GEO
Project description:Cyclin-dependent kinase 7 (CDK7) plays a critical role in the general regulation of RNA polymerase II-mediated transcription. However, the absence of selective CDK7 inhibitors has hindered the ability to investigate the consequences of acute and prolonged inhibition of CDK7 under normal and pathological conditions. Here we present the discovery and characterization of the first covalent CDK7 inhibitor, CDK7-IN-1, that has the unprecedented ability to target a unique cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7 amongst the 20 known CDKs. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-cell acute lymphoblastic leukemia (T-ALL), exhibit 100-fold greater sensitivity to CDK7-IN-1 over other tumor and normal cell lines. Genome-wide expression analysis in Jurkat T-ALL indicates that CDK7-IN-1 disproportionally affects RUNX1 as well as other components of the TAL1 transcriptional network and its targets, downregulating key regulators of transcription and apoptosis critical for the T-ALL state. These oncogenes are encoded by short-lived mRNA transcripts, are associated with super-enhancers, and exhibit a strong dependency on continuous transcription for sustained expression. Therefore, pharmacological modulation of CDK7 kinase activity may define a method for the identification and treatment of tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state. Jurkat cells were treated with various drugs including a covalent inhibitor of CDK7 (CDK7-IN-1), a reversible inhibitor of CDK7 (CDK7-IN-1), Flavopiridol, Actinomycin D, and DMSO controls. Replicates are annotated.
2013-12-23 | E-GEOD-50622 | biostudies-arrayexpress
Project description:Effective and convenient covalent protein conjugation could benefit diverse biological systems, stabilizing key protein protein interactions which are otherwise reversible. Neisseria meningitidis contains a protein which undergoes autoproteolysis via an anhydride. Here, we harness this spontaneously generated electrophile for covalent targeting of unmodified endogenous proteins. In ‘NeissLock’, a binding protein genetically fused to the self-processing module (SPM) docks to its target protein. Upon triggering with calcium, the aspartic anhydride generated at the C-terminus of the binding protein allows nucleophilic attack by nearby residues on the target protein, so ligating the proteins. We established a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We validated and optimized the NeissLock concept using the Ornithine Decarboxylase/Antizyme complex. A range of nucleophiles on the target (α-amine or ε-amines) could rapidly react with the anhydride, but reaction was blocked if the partner protein did not dock. Surprisingly, the optimal pH for covalent ligation was 7.0. We then armed Transforming Growth Factor-α with SPM and established covalent targeting to Epidermal Growth Factor Receptor at the surface of living cells. NeissLock harnesses exceptional protein chemistry to allow covalent targeting of endogenous proteins under mild conditions with up to 80% yield, allowing new possibilities for molecular engineering.
2021-02-01 | PXD023073 | Pride
Project description:Small cell lung cancer (SCLC) is an aggressive disease with high mortality. The identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library we observe that SCLC is sensitive to transcription-targeting drugs, and in particular to THZ1, a newly identified covalent inhibitor of cyclin-dependent kinase 7 (CDK7). We find that expression of super-enhancer associated transcription factor genes including MYC family proto-oncogenes and neuroendocrine lineage-specific factors are highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a novel treatment paradigm for targeted SCLC therapy. Microarray expression in small cell lung cancer lines treated with DMSO or THZ1
2014-10-23 | E-GEOD-62609 | biostudies-arrayexpress
Project description:Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast, however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing. Human U2OS cells were transfected with siRNAs to specifically knockdown the ubiquitn-like protein Hub1. Cells treated with non-targeting oligos (GL2) served as negative control. Total RNAs of three biological replicates of each knockdown experiment were isolated and changes of splicing patterns were subsequently analyzed by Affymetrix GeneChip Human Exon 1.0 ST arrays
2014-05-30 | E-GEOD-56878 | biostudies-arrayexpress
Project description:Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.
2016-05-19 | GSE72023 | GEO
Project description:Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors
| PRJNA292774 | ENA