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.
Project description:PTK6 PROTAC treatment of T47D breast cancer cells were analyzed with mass spectrometry for determining PTK6 PROTAC specificity and global proteomic changes.
Project description:The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation, and survival enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAFV600E have shown great efficacy in the clinic but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC termed P4B displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAFV600E cell lines. In addition, P4B displayed utility in two cell lines harboring alternate BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a rationale for optimizing the drug-like properties of P4B to enable proof of concept studies in vivo.
Project description:Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins which undermines the growth and survival of AML cells. However, BETi treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4, which potentially compromises the activity of BETi in AML cells. Unlike BETi, BET-PROTAC (proteolysis-targeting chimera) ARV-825 recruits and utilize an E3-ubiquitin ligase to effectively degrade BETPs in AML cells. BET-PROTACs induce more apoptosis than BETi of mtRUNX1 AML cells. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi. It was noted that treatment with BETi or BET-PROTAC caused significant and sustained depletion of RUNX1 in AML cells. We also determined the effects of global depletion of RUNX1 in mtRUNX1 expressing AML OCI-AML5 cells. We observed an overlap in the signature of RUNX1 knockdown by shRNA with that of OTX015 and ARV-825 in OCI-AML5 cells.
Project description:Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that promote cellular degradation of a protein of interest (POI). PROTACs act as molecular mediators, bringing an E3 ligase and a POI into proximity, thus promoting ubiquitination and degradation of the targeted POI. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs.
Project description:The role of signal transducer and activator of transcription protein 3 (STAT3) in AKI remains controversial. Our study demonstrated an upregulation of total STAT3 protein in AKI mouse models induced by cecal ligation and puncture (CLP) or ischemia-reperfusion (I/R), correlating with patient biopsy results. This increase may be attributed to histone H3K27 acetylation. STAT3 knockout in renal tubular epithelial cells significantly reduced AKI injury and inflammation in mice. Mechanistically, STAT3 induces the transcription of tripartite motif-containing protein 21 (TRIM21), triggering a cascade that activates gasdermin D (GSDMD), resulting in pyroptosis. Administration of the novel proteolysis-targeting chimera (PROTAC) compound E034, which selectively targets STAT3 for ubiquitination and degradation, significantly alleviated renal injury in a low-dose, single-dose regimen, underscoring its substantial therapeutic potential with infrequent dosing requirements. In the context of renal injury, PROTAC emerges as a promising modality by specifically targeting the STAT3/TRIM21/GSDMD axis, which our study has identified as a potential therapeutic target, thereby potentially endowing novel and clinically significant therapeutic strategies.
Project description:The role of signal transducer and activator of transcription protein 3 (STAT3) in AKI remains controversial. Our study demonstrated an upregulation of total STAT3 protein in AKI mouse models induced by cecal ligation and puncture (CLP) or ischemia-reperfusion (I/R), correlating with patient biopsy results. This increase may be attributed to histone H3K27 acetylation. STAT3 knockout in renal tubular epithelial cells significantly reduced AKI injury and inflammation in mice. Mechanistically, STAT3 induces the transcription of tripartite motif-containing protein 21 (TRIM21), triggering a cascade that activates gasdermin D (GSDMD), resulting in pyroptosis. Administration of the novel proteolysis-targeting chimera (PROTAC) compound E034, which selectively targets STAT3 for ubiquitination and degradation, significantly alleviated renal injury in a low-dose, single-dose regimen, underscoring its substantial therapeutic potential with infrequent dosing requirements. In the context of renal injury, PROTAC emerges as a promising modality by specifically targeting the STAT3/TRIM21/GSDMD axis, which our study has identified as a potential therapeutic target, thereby potentially endowing novel and clinically significant therapeutic strategies.
Project description:Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and NFkB target genes, which undermines the growth and survival of MCL cells. However, BETi treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4, which potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1, and the NFkB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the level of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared to ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Co-treatment of ARV-771 with ibrutinib or the BCL2-antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior pre-clinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or resistant MCL.