Yu Rao 
Publications
Merging PROTAC and molecular glue for degrading BTK and GSPT1 proteins concurrently.
Yang Zimo Z Sun Yonghui Y Ni Zhihao Z Yang Celi C Tong Yan Y Liu Yujie Y Li Haitao H Rao Yu Y
Cell research 20210820 12
Similar Datasets
Project description:Targeted protein degradation is a groundbreaking modality in drug discovery ; however, further improvement of the degradation efficacy is demanded. Here, we identify small molecules that enhance the targeted degradation of the anticancer target BRD4 and related hard-to-degrade targets BRD2/3 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4–PROTAC– CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. Combining the treatment with these chemicals further increases the efficacy of a highly potent PROTAC, SIM1. We propose that various cell intrinsic signaling pathways spontaneously counteract chemically induced target degradation, which can be liberated by specific inhibitors.
2024-05-17 | GSE243615 | GEO
Project description:Temporal control of proteins in cells and living animals is crucial to improving the understanding of protein function in the post-genomic era. In addition, technologies that offer such control for engineered proteins could be used in therapeutic applications. Since regulation of proteins at a genomic or transcriptional level can be irreversible or a slow process, these tools may not be useful in settings where rapid temporal control is required to achieve immediate knockdown effects. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a strategy to achieve rapid, post-translational control of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we developed several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a small molecule ligand of the E3 ligase Cereblon. These molecules induce degradation of various proteins of interest (POIs) genetically fused to E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We demonstrate that various eDHFR-tagged proteins, from fluorescent proteins to transcription factors and membrane-associated proteins, can be downregulated to 95% of maximum expression with our lead PROTAC molecule 7c. The data suggest that TMP-based PROTACs induce maximal degradation of POIs at drug concentrations that minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates. Finally, we show the ability to achieve multiplexed regulation with another known degron-PROTAC pair, and the formidable strength of our system for reversible protein regulation in a rodent model of metastatic cancer. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged protein and have a strong potential for translation to in vivo models as well as dual degradation strategies with existing technologies.
2023-12-06 | PXD045052 | Pride
Project description:Targeted protein degradation is a groundbreaking modality in drug discovery ; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related hard-to-degrade targets BRD2/3 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4–PROTAC–CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.
2024-05-17 | GSE262938 | GEO
Project description:MDM2 inhibitor remarkably induced biomineralization in hDPSCs but it remained the problem that p53 activation is insufficient due to MDM2-p53 autoregulatory feedback loop. To overcome the limitation of the MDM2 inhibitors, we applied proteolysis targeting chimera (PROTAC), a technology that degrades a protein of interest (POI) by intracellular ubiquitin-proteasome system. Hence, we propose a strategy to induce hard tissue regeneration by MDM2-targeting PROTAC technology. We selected Nutlin-3 of POI ligand among various MDM2 inhibitors based on the screening process, and the selected CRBN of E3 ligase ligand. The MDM2-PROTAC synthesis platform was designed by each ligand combination. By performing the degradation test for selected compounds, we evaluated the characteristics of the developed MDM2 PROTAC such as the maximal degradation concentration (DCmax), the half of maximal degradation concentration (DC50), and half-lifetime. To investigate gene expression profiling of MDM2-targeting small molecules, we conducted RNA-sequencing under MDM2 PROTAC and Nutlin-3 (POI ligand) treated conditions. We not only confirmed a robust effect on biomineralization by MDM2-targeting small molecules but also demonstrated the potent osteogenic differentiation ability of MDM2 PROTAC when compared to an MDM2 inhibitor. MDM2-PROTAC significantly increased mRNA levels of osteogenic differentiation marker genes. Also, the significant bone generation effect of MDM2 PROTAC was validated in an ovariectomy (OVX)-induced osteoporosis animal model. Through these results, it is expected that a new therapeutic modality for hard tissue regeneration will be possible, and the application range of the PROTAC system can be expanded.
2024-06-11 | GSE255297 | GEO
Project description:Scientists have developed several methods to control the activity of Cas protein in a timely way and hence reduce off-target effects, including anti-CRISPR proteins and small molecule inhibitors. The PROTAC is a new conception of using the natural ubiquitin-proteasome system to degrade the protein of interest in drug design and development. Here, we engineered Cas proteins (Cas9, Cas12, and Cas13) by inserting a Phe-Cys-Pro-Phe (FCPF) amino acid sequence (also known as the π-clamp system) and demonstrate that the modified CasFCPF proteins could be labeled in live cells by perfluoroaromatics carrying FITC fluorophore or were degraded by perfluoroaromatics-functionalized PROTAC (PROTAC-FCPF). Proteome-wide analysis of PROTAC-FCPF-mediated Cas9FCPF protein degradation revealed high target specificity, suggesting a wide application of chemically induced proximity in combination with site-specific protein modification in the regulation of protein stability, activity and functionality.
2022-10-18 | PXD022199 | Pride
Project description:A total of 12 WW domains from Saccharomyces cerevisiae were expressed and purified as fusion proteins to either GST or MBP. The fusion proteins were chemically biotinylated and applied to duplicate protein microarrays. Data processing revealed a total of 587 interactions between the domains and 207 proteins. Most of these interactions have not been previously observed. Keywords: Protein microarray analysis of yeast WW domains
2006-02-08 | GSE3758 | GEO
Project description:Müller glia (MG) stem cells are dormant retinal stem cells. While teleost fish MG re-enter the cell cycle after retinal damage and regenerate lost retinal neurons through asymmetric cell division of MG-derived progenitors, mammalian MG lack such ability. Here, we show that forced p27 downregulation and Cyclin D1 overexpression in MG using a single adeno-associated virus cell cycle activator (CCA) vector had a strong synergistic effect to induce MG proliferation. We used transcriptome profiling at single-cell level to demonstrate that CCA could induce MG proliferation and reprogram MG into neurogenic and rod-like cells. In addition, retinal injury combined with histone deacetylase inhibitor facilitates CCA to induce more rod-like cells, but not new cell types. Our study suggests new and powerful strategies for awakening the proliferative and neurogenic potential of MG in adult mammalian retinas.
2024-08-01 | GSE225142 | GEO
Project description:To address the limitation associated with degron based systems, we have developed iTAG, a novel synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localisations without exhibiting the well documented “hook effect” of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.
2023-06-27 | PXD038299 | Pride
Project description:PROTACs induce the degradation of target proteins by hijacking an E3 ligase. However, it is usually difficult to directly identify the E3 ligase recruited by a new E3 ligase ligand in a PROTAC using the co-immunoprecipitation method because the formation of the complex is transient and very dynamic. TurboID assay is a powerful proximity labeling method that can sensitively detect weak and transient protein-protein interactions by biotinylating proteins that interact with a bait protein fused with an engineered biotin ligase. Here, we adapted TurboID technology to identify and characterize the specific E3 ligase(s) recruited by 955, a potent piperlongumine (PL)-SNS-032 conjugated CDK9 PROTAC, to mediate CDK9 degradation.
2024-05-24 | PXD039401 | Pride
Project description:The different layers of complexity in PROTAC design have slowed the broad implementation of this novel pharmacological approach. Given the challenges and opportunities of CRL4CRBN hijacking PROTACs we set out to identify and develop broadly implementable strategies to generate PROTACs with improved specificity for their targets. We used structural analysis of known neo-substrate degrons to generate “degron blocked IMiD” analogues and validated that they did not degrade any known neo-substrates using quantitative proteomics. We then applied this strategy to a known BRD9 PROTAC (dBRD9-A) to generate a degron-blocking analogue with a specific degradation profile.
2023-07-10 | PXD038298 | Pride