Cell Chemical Biology最新文献

Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand binding liberates a conserved sequence that acts as an intramolecular, tethered agonist (TA), yet this model has not been evaluated systematically for all aGPCRs. Here, we assessed the TA-dependent activities of all 33 aGPCRs in a suite of transcriptional reporter, G protein activation, and β-arrestin recruitment assays using a new fusion protein platform. Strikingly, only ∼50% of aGPCRs exhibited robust TA-dependent activation, and unlike other GPCR families, aGPCRs showed a notable preference for G_12/13 signaling. AlphaFold2 predictions assessing TA engagement in the predicted intramolecular binding pocket aligned with the TA dependence of the cellular responses. This dataset provides a comprehensive resource to inform the investigation of all human aGPCRs and for targeting aGPCRs therapeutically.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands joined by a linker, enabling them to simultaneously bind with an E3 ligase and a protein of interest (POI) and trigger proteasomal degradation of the POI. Limitations of PROTAC include lack of potent E3 ligands, poor cell selectivity, and low permeability. AS1411 is an antitumor aptamer specifically recognizing a membrane-nucleus shuttling nucleolin (NCL). Here, we repurpose AS1411 as a ligand for an E3 ligase mouse double minute 2 homolog (MDM2) via anchoring the NCL-MDM2 complex. Then, we construct an AS1411-NCL-MDM2-based PROTAC (ANM-PROTAC) by conjugating AS1411 with large-molecular-weight ligands for “undruggable” oncogenic STAT3, c-Myc, p53-R175H, and AR-V7. We show that the ANM-PROTAC efficiently penetrates tumor cells, recruits MDM2 and degrades the POIs. The ANM-PROTAC achieves tumor-selective distribution and exhibits excellent antitumor activity with no systemic toxicity. This is a PROTAC with built-in tumor-targeting and cell-penetrating capacities.

The hedgehog (Hh) signaling pathway has long been a hotspot for anti-cancer drug development due to its important role in cell proliferation and tumorigenesis. However, most clinically available Hh pathway inhibitors target the seven-transmembrane region (7TM) of smoothened (SMO), and the acquired drug resistance is an urgent problem in SMO inhibitory therapy. Here, we identify a sterol analog Q29 and show that it can inhibit the Hh pathway through binding to the cysteine-rich domain (CRD) of SMO and blocking its cholesterylation. Q29 suppresses Hh signaling-dependent cell proliferation and arrests Hh-dependent medulloblastoma growth. Q29 exhibits an additive inhibitory effect on medulloblastoma with vismodegib, a clinically used SMO-7TM inhibitor for treating basal cell carcinoma (BCC). Importantly, Q29 overcomes resistance caused by SMO mutants against SMO-7TM inhibitors and inhibits the activity of SMO oncogenic variants. Our work demonstrates that the SMO-CRD inhibitor can be a new way to treat Hh pathway-driven cancers.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are a class of cancer drugs that enzymatically inhibit PARP activity at sites of DNA damage. Yet, PARPi function mainly by trapping PARP1 onto DNA with a wide range of potency among the clinically relevant inhibitors. How PARPi trap and why some are better trappers remain unknown. Here, we show trapping occurs primarily through a kinetic phenomenon at sites of DNA damage that correlates with PARPi k_off. Our results suggest PARP trapping is not the physical stalling of PARP1 on DNA, rather the high probability of PARP re-binding damaged DNA in the absence of other DNA-binding protein recruitment. These results clarify how PARPi trap, shed new light on how PARPi function, and describe how PARPi properties correlate to trapping potency.

Stem cells remain quiescent in vivo and become activated in response to external stimuli. However, the mechanism regulating the quiescence-activation balance of bone-marrow-derived mesenchymal stem cells (BM-MSCs) is still unclear. Herein, we demonstrated that CYP7B1 was the common critical molecule that promoted activation and impeded quiescence of BM-MSCs under inflammatory stimulation. Mechanistically, CYP7B1 degrades 25-hydroxycholesterol (25-HC) into 7α,25-dihydroxycholesterol (7α,25-OHC), which alleviates the quiescence maintenance effect of 25-HC through Notch3 signaling pathway activation. CYP7B1 expression in BM-MSCs was regulated by NF-κB p65 under inflammatory conditions. BM-MSCs from CYP7B1 conditional knockout (CKO) mice had impaired activation abilities, relating to the delayed healing of bone defects. Intravenous infusion of BM-MSCs overexpressing CYP7B1 could improve the pathological scores of mice with collagen-induced arthritis. These results clarified the quiescence-activation regulatory mechanism of BM-MSCs through the NF-κB p65-CYP7B1-Notch3 axis and provided insight into enhancing BM-MSCs biological function as well as the subsequent therapeutic effect.

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small- or large-molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3.

Most BTB-containing E3 ligases homodimerize to recognize a single substrate by engaging multiple degrons, represented by E3 ligase KEAP1 dimer and its substrate NRF2. Inactivating KEAP1 to hinder ubiquitination-dependent NRF2 degradation activates NRF2. While various KEAP1 inhibitors have been reported, all reported inhibitors bind to KEAP1 in a monovalent fashion and activate NRF2 in a lagging manner. Herein, we report a unique bivalent KEAP1 inhibitor, biKEAP1 (3), that engages cellular KEAP1 dimer to directly release sequestered NRF2 protein, leading to an instant NRF2 activation. 3 promotes the nuclear translocation of NRF2, directly suppressing proinflammatory cytokine transcription. Data from in vivo experiments showed that 3, with unprecedented potency, reduced acute inflammatory burden in several acute inflammation models in a timely manner. Our findings demonstrate that the bivalent KEAP1 inhibitor can directly enable sequestered substrate NRF2 to suppress inflammatory transcription response and dampen various acute inflammation injuries.

The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not in vivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth in vivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for in vivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.

Cereblon (CRBN) is an E3 ligase substrate adapter widely exploited for targeted protein degradation (TPD) strategies. However, achieving efficient and selective target degradation is a preeminent challenge with ligands that engage CRBN. Here, we report that the cyclimids, ligands derived from the C-terminal cyclic imide degrons of CRBN, exhibit distinct modes of interaction with CRBN and offer a facile approach for developing potent and selective bifunctional degraders. Quantitative TR-FRET-based characterization of 60 cyclimid degraders in binary and ternary complexes across different substrates revealed that ternary complex binding affinities correlated strongly with cellular degradation efficiency. Our studies establish the unique properties of the cyclimids as versatile warheads in TPD and a systematic biochemical approach for quantifying ternary complex formation to predict their cellular degradation activity, which together will accelerate the development of ligands that engage CRBN.

Understanding the intricate network of biomolecular interactions that govern cellular processes is a fundamental pursuit in biology. Over the past decade, photocatalytic proximity labeling has emerged as one of the most powerful and versatile techniques for studying these interactions as well as uncovering subcellular trafficking patterns, drug mechanisms of action, and basic cellular physiology. In this article, we review the basic principles, methodologies, and applications of photocatalytic proximity labeling as well as examine its modern development into currently available platforms. We also discuss recent key studies that have successfully leveraged these technologies and importantly highlight current challenges faced by the field. Together, this review seeks to underscore the potential of photocatalysis in proximity labeling for enhancing our understanding of cell biology while also providing perspective on technological advances needed for future discovery.