Piperlongumine (PL) conjugates induce targeted protein degradation
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA851461 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19774160.fastq.gz | Fastqsanger.gz | |||
| SRR19774161.fastq.gz | Fastqsanger.gz | |||
| SRR19774162.fastq.gz | Fastqsanger.gz | |||
| SRR19774163.fastq.gz | Fastqsanger.gz | |||
| SRR19774164.fastq.gz | Fastqsanger.gz |
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Project description:PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-Ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.
2022-06-26 | GSE206612 | GEO
Project description:PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS-032, a CDK9 inhibitor. To evaluate the specificity of the PL-SNS-032 lead conjugate named 955, TMT-based proteomics were conducted to compare 955 with its warhead SNS-032 in MOLT4 cells. As expected, cells exhibited a significant reduction of CDK9 after treatment with 0.1 µM 955 for 1 h and 6 h while treatment with 1 µM SNS-032 for 6 h had no significant effect on CDK9. Interestingly, 955, but not SNS-032, can also potently degrade CDK10.
2024-05-24 | PXD039385 | Pride
Project description:Proteins can be targeted for degradation by using engineered molecular conjugates to enhance their interaction with eukaryotic ubiquitination machinery. For instance, the fusion of an E3 ubiquitin ligase to a suitable target binding domain creates a biological ‘Proteolysis-Targeting Chimera’ (bioPROTAC). Here we report an analogous approach where the target is recruited directly to an E2 ubiquitin-conjugating enzyme via an attached target binding domain. We demonstrate that E2 bioPROTACs can induce the degradation of the human proteins SHP2, KRAS and Human Antigen R (HuR). Taking advantage of both rational and unbiased combinatorial screening, we identified novel SHP2 degraders that possess unusually weak affinity. Finally, we explored the proteomic impact of E2 vs. E3 bioPROTACs to inform on their wider impact on the cellular proteome.
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Project description:Previous work has led us to examine the differences in the choroid plexus function in B10.pl WT mice versus B10.PL RAG-/- mice. We believe that there is a difference between those that are normal functioning and those that are lymphocyte deficient. To determine the gene expression profile of the choroid plexus in wild type mice as compared to those that are lymphocyte deficient. We hypothesize that there is altered expression in the genes that mediate cellular adhesion in choroid plexus from wild type mice as compared to those that are lymphocyte deficient. 8-10 week old animals (age and sex matched) were injected with Evan's blue post anesthetization. After waiting an hour the animals were euthanized and their brains were extracted and placed in RNALater for 24 hours. The brains were then sliced sagitally
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Project description:Targeting protein degradation with Proteolysis-Targeting Chimeras (PROTACs) is an area of great current interest in drug discovery. Nevertheless, although the high effectiveness of PROTACs against a wide variety of targets has been established, most degraders reported to date display limited intrinsic tissue selectivity and do not discriminate between cells of different types. Here, we describe a strategy for selective protein degradation in a specific cell type. We report the design and synthesis of a trastuzumab-PROTAC conjugate (Ab-PROTAC 3) in which E3 ligase-directed degrader activity is caged with an antibody linker which can be hydrolyzed following antibody-PROTAC internalization, releasing the active PROTAC and inducing catalytic protein degradation. We show that 3 selectively targets bromodomain-containing protein 4 (BRD4) for degradation only in HER2 positive breast cancer cell lines, while sparing HER2 negative cells. Using live cell confocal microscopy, we show internalization and lysosomal trafficking of the conjugate specifically in HER2 positive cells, leading to the release of active PROTAC in quantities sufficient to induce potent BRD4 degradation. These studies demonstrate proof-of-concept for tissue-specific BRD4 degradation, overcoming limitations of PROTAC selectivity, with significant potential for application to novel targets.
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Project description:To investigate the effect of PL on gene expression, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to suppress the cell proliferation of OSCC cell lines. SAS and HSC-3 cells were treated with PL (5 μmol/L) for 4 h in vitro.
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Project description:Using this approach the dasatinib-oligonucleotide conjugates was applied to planar arrays of >9,000 human proteins spotted in two technical replicates. All proteins in the commercially available ProtoArray® Human Protein MicroArray (Thermo Fisher Scientific) have been purified and arrayed under native conditions to allow such studies. We adopted this format for investigation of the binding profiles of the drug-oligonucleotide conjugates. Fluorophore-labeled drugs have previously been used to measure binding in protein arrays. The oligonucleotide-conjugated constructed allowed for locally amplified detection via RCA. Circularizing oligonucleotides (padlock probes) were designed with 5’ and 3’ ends complementary to adjacent segments of the oligonucleotides conjugated to the drug molecules. Once converted to oligonucleotide circles by ligation, the probes were replicated through localized RCA, primed by the drug-conjugated oligonucleotides, and visualized using fluorescence-labeled hybridization probes to the repeated sequence of the RCA products. RCA offers a signal enhancement of several hundredfold over singly fluorophore labeled compounds, permitting visualization of even single bound drug probes
2022-09-12 | E-MTAB-12192 | biostudies-arrayexpress
Project description:SPT6 is both, an important histone chaperone and POL2 elongation factor but its primary role on transcription in mammalian cells remains open, as no acute depletion system is available. We used targeted protein degradation to rapidly deplete SPT6 and analyzed defects in POL2 behavior by a multi-omics approach and estimated POL2 processivity, elongation rates and termination and compared it to gene transcription. Our data indicate that SPT6 is a crucial factor for POL2 elongation. Unexpectedly, SPT6 has also a vital role in POL2 termination, as acute depletion induces POL2 read through at most protein coding genes. In contrast, acute depletion did not induce spurious intragenic initiation, while this behavior can be induced by long-term depletion of SPT6 and can therefore be attributed to its function as a histone chaperone. In conclusion, targeted protein degradation of SPT6 allows the kinetic discrimination of its function as a histone chaperone and POL2 elongation factor.
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Project description:Previous work has led us to examine the differences in the choroid plexus function in B10.pl WT mice versus B10.PL RAG-/- mice. We believe that there is a difference between those that are normal functioning and those that are lymphocyte deficient. To determine the gene expression profile of the choroid plexus in wild type mice as compared to those that are lymphocyte deficient. We hypothesize that there is altered expression in the genes that mediate cellular adhesion in choroid plexus from wild type mice as compared to those that are lymphocyte deficient. 8-10 week old animals (age and sex matched) were injected with Evan's blue post anesthetization. After waiting an hour the animals were euthanized and their brains were extracted and placed in RNALater for 24 hours. The brains were then sliced sagitally Keywords: RAG mutant, choroid plexus
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