RESUMEN
Ligand-Induced duplex-quadruplex transition within the c-MYC promoter region is one of the most studied and advanced ideas for c-MYC regulation. Despite its importance, there is a lack of methods for monitoring such process in cells, hindering a better understanding of the essence of c-MYC G-quadruplex as a drug target. Here we developed a new fluorescent probe ISCH-MYC for specific c-MYC G-quadruplex recognition based on GTFH (G-quadruplex-Triggered Fluorogenic Hybridization) strategy. We validated that ISCH-MYC displayed distinct fluorescence enhancement upon binding to c-MYC G-quadruplex, which allowed the duplex-quadruplex transition detection of c-MYC G-rich DNA in cells. Using ISCH-MYC, we successfully characterized the induction of duplex to G-quadruplex transition in the presence of G-quadruplex stabilizing ligand PDS and further monitored and evaluated the altered interactions of relevant transcription factors Sp1 and CNBP with c-MYC G-rich DNA. Thus, our study provides a visualization strategy to explore the mechanism of G-quadruplex stabilizing ligand action on c-MYC G-rich DNA and relevant proteins, thereby empowering future drug discovery efforts targeting G-quadruplexes.
Asunto(s)
G-Cuádruplex , Proteínas Proto-Oncogénicas c-myc , ADN/química , ADN/genética , Ligandos , Hibridación de Ácido Nucleico , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Mitochondrion-lysosome interactions have garnered significant attention in recent research. Numerous studies have shown that mitochondrion-lysosome interactions, including mitochondrion-lysosome contact (MLC) and mitophagy, are involved in various biological processes and pathological conditions. Single fluorescent probes are termed a pivotal chemical tool in unraveling the intricate spatiotemporal interorganelle interplay in live cells. However, current chemical tools are insufficient to deeply understand mitochondrion-lysosome dynamic interactions and related diseases, Moreover, the rational design of mitochondrion-lysosome dual-targeting fluorescent probes is intractable. Herein, we designed and synthesized a pH-sensitive fluorescent probe called INSA, which could simultaneously light up mitochondria (red emission) and lysosomes (green emission) for their internal pH differences. Employing INSA, we successfully recorded long-term dynamic interactions between lysosomes and mitochondria. More importantly, the increasing mitochondrion-lysosome interactions in ferroptotic cells were also revealed by INSA. Further, we observed pH variations in mitochondria and lysosomes during ferroptosis for the first time. In brief, this work not only introduced a pH-sensitive fluorescent probe INSA for the disclosure of the mitochondrion-lysosome dynamic interplays but also pioneered the visualization of the organellar pH alternation in a specific disease model.
Asunto(s)
Colorantes Fluorescentes , Lisosomas , Humanos , Colorantes Fluorescentes/metabolismo , Lisosomas/metabolismo , Mitocondrias , Células HeLa , Concentración de Iones de HidrógenoRESUMEN
Helicases are crucial enzymes in DNA and RNA metabolism and function by unwinding particular nucleic acid structures. However, most convenient and high-throughput helicase assays are limited to the typical duplex DNA. Herein, we developed an immunosorbent assay to monitor the Werner syndrome (WRN) helicase unwinding a wide range of DNA structures, such as a replication fork, a bubble, Holliday junction, G-quadruplex and hairpin. This assay could sensitively detect the unwinding of DNA structures with detection limits around 0.1 nM, and accurately monitor the substrate-specificity of WRN with a comparatively less time-consuming and high throughput process. Remarkably, we have established that this new assay was compatible in evaluating helicase inhibitors and revealed that the inhibitory effect was substrate-dependent, suggesting that diverse substrate structures other than duplex structures should be considered in discovering new inhibitors. Our study provided a foundational example for using this new assay as a powerful tool to study helicase functions and discover potent inhibitors.
Asunto(s)
RecQ Helicasas , Síndrome de Werner , Humanos , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Inmunoadsorbentes , Replicación del ADN , Helicasa del Síndrome de Werner/genética , Helicasa del Síndrome de Werner/metabolismo , Exodesoxirribonucleasas/metabolismo , ADN/química , Síndrome de Werner/genéticaRESUMEN
Mitochondria have a crucial role in regulating energy metabolism and their dysfunction has been linked to tumorigenesis. Cancer diagnosis and intervention have a great interest in the development of new agents that target biomolecules within mitochondria. However, monitoring and modulating mitochondria RNA (mtRNA), an essential component in mitochondria, in cells is challenging due to limited functional research and the absence of targeting agents. In this study, we designed and synthesized a fluorescent quinolinium derivative, QUCO-1, which actively lit up with mtRNA in both normal and cancer cells in vitro. Additionally, we evaluated the function of QUCO-1 as an mtRNA ligand and found that it effectively induced severe mitochondrial dysfunction and OXPHOS inhibition in RKO colorectal cancer cells. Treatment with QUCO-1 resulted in apoptosis, cell cycle blockage at the G2/M phase, and the effective inhibition of cell proliferation. Our findings suggest that QUCO-1 has great potential as a promising probe and therapeutic agent for mtRNA, with the potential for treating colorectal cancer.
Asunto(s)
Neoplasias Colorrectales , Mitocondrias , Humanos , ARN Mitocondrial/metabolismo , Mitocondrias/metabolismo , Proliferación Celular , Apoptosis , Colorantes Fluorescentes/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Línea Celular TumoralRESUMEN
Benzothiazolium and benzoxazolium are common groups for the construction of hemicyanine dyes; however, their isosteric analogue benzoselenazolium have rarely been studied. Here, we report the development of the first benzoselenazolium-based hemicyanine dye for the selective detection of G-quadruplexes. This molecule, SEMA-1, was validated as a red-emitting and activatable fluorescent probe whose fluorescence would only be activated in the presence of G-quadruplexes in buffer solution. Consistent with this, SEMA-1 was found to accumulate in nucleoli and could be used to detect the high abundance of nucleolar rDNA and rRNA G-quadruplexes in fixed HeLa cells. On the other hand, due to the retained mitochondrial membrane potential in live HeLa cells, SEMA-1 was captured by mitochondria and had the potential to detect the mitochondrial G-quadruplexes. Collectively, this work demonstrates the value of developing G-quadruplex-specific fluorescent probes from novel benzoselenazolium-based hemicyanine scaffold.
Asunto(s)
G-Cuádruplex , Carbocianinas , Colorantes Fluorescentes , Células HeLa , HumanosRESUMEN
RNA imaging is of great importance for understanding its complex spatiotemporal dynamics and cellular functions. Considerable effort has been devoted to the development of small-molecule fluorescent probes for RNA imaging. However, most of the reported studies have mainly focused on improving the photostability, permeability, long emission wavelength, and compatibility with live-cell imaging of RNA probes. Less attention has been paid to the selectivity and detection limit of this class of probes. Highly selective and sensitive RNA probes are still rarely available. In this study, a new set of styryl probes were designed and synthesized, with the aim of upgrading the detection limit and maintaining the selectivity of a lead probe QUID-1 for RNA. Among these newly synthesized compounds, QUID-2 was the most promising candidate. The limit of detection (LOD) value of QUID-2 for the RNA was up to 1.8 ng/mL in solution. This property was significantly improved in comparison with that of QUID-1. Further spectroscopy and cell imaging studies demonstrated the advantages of QUID-2 over a commercially available RNA staining probe, SYTO RNASelect, for highly selective and sensitive RNA imaging. In addition, QUID-2 exhibited excellent photostability and low cytotoxicity. Using QUID-2, the global dynamics of RNA were revealed in live cells. More importantly, QUID-2 was found to be potentially applicable for detecting RNA granules in live cells. Collectively, our work provides an ideal probe for RNA imaging. We anticipate that this powerful tool may create new opportunities to investigate the underlying roles of RNA and RNA granules in live cells.
Asunto(s)
Colorantes Fluorescentes , ARN , Colorantes Fluorescentes/química , Sondas ARN , Imagen MolecularRESUMEN
The mitochondrial DNA G-quadruplex (mtDNA G4) is a potential regulatory element for the regulation of mitochondrial functions; however, its relevance and specific roles in diseases remain largely unknown. Here, we engineered a set of chemical probes, including MitoISCH, an mtDNA G4-specific fluorescent probe, together with MitoPDS, a mitochondria-targeted G4-stabilizing agent, to thoroughly investigate mtDNA G4s. Using MitoISCH to monitor previously intractable dynamics of mtDNA G4s, we surprisingly found that their formation was prevalent only in endothelial and cancer cells that rely on glycolysis for energy production. Consistent with this, promotion of mtDNA G4 folding by MitoPDS in turn caused glycolysis-related gene activation and glycolysis enhancement. Remarkably, this close relationship among mtDNA G4s, glycolysis, and cancer cells further allowed MitoISCH to accumulate in tumors and label them in vivo. Our work reveals an unprecedented link between mtDNA G4s and cell glycolysis, suggesting that mtDNA G4s may be a novel cancer biomarker and therapeutic target deserving further exploration.
Asunto(s)
ADN Mitocondrial/metabolismo , Colorantes Fluorescentes/química , G-Cuádruplex , Glucólisis/fisiología , Células 3T3 , Animales , Línea Celular Tumoral , ADN Mitocondrial/genética , Células Endoteliales/metabolismo , Humanos , Ratones , Ratones Desnudos , Mitocondrias/metabolismoRESUMEN
Because of the lack of facile and accurate methods to track stress granule (SG) dynamics in live cells and in vivo, in-depth studies of the biological roles of this attractive membraneless organelle have been limited. Herein, we report the first small-molecule probe, TASG, for the selective, convenient and real-time monitoring of SGs. This novel molecule can simultaneously bind to SG RNAs, the core SG protein G3BP1, and their complexes, triggering a significant enhancement in fluorescence intensity, making TASG broadly applicable to SG imaging under various stress conditions in fixed and live cells, ex vivo and in vivo. Using TASG, the complicated endogenous SG dynamics were revealed in both live cells and C. elegans. Collectively, our work provides an ideal probe that has thus far been absent in the field of SG investigations. We anticipate that this powerful tool may create exciting opportunities to investigate the underlying roles of SGs in different organisms.
Asunto(s)
Condensados Biomoleculares , Caenorhabditis elegans , Animales , ADN Helicasas , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de EstrésRESUMEN
Because of the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Herein, we report a new red-emitting fluorescent probe, QUMA-1, for the selective, continuous, and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding, and movement of RNA G-quadruplexes and the visualization of the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes.
Asunto(s)
G-Cuádruplex , Pliegue del ARN , ARN/análisis , ARN/química , Supervivencia Celular , Colorantes Fluorescentes/química , Células HeLa , Humanos , Imagen ÓpticaRESUMEN
Mitochondrial DNA (mtDNA) G-quadruplexes (G4s) have important regulatory roles in energy metabolism, yet their specific functions and underlying regulatory mechanisms have not been delineated. Using a chemical-genetic screening strategy, we demonstrated that the JAK/STAT3 pathway is the primary regulatory mechanism governing mtDNA G4 dynamics in hypoxic cancer cells. Further proteomic analysis showed that activation of the JAK/STAT3 pathway facilitates the translocation of RelA, a member of the NF-κB family, to the mitochondria, where RelA binds to mtDNA G4s and promotes their folding, resulting in increased mtDNA instability, inhibited mtDNA transcription, and subsequent mitochondrial dysfunction. This binding event disrupts the equilibrium of energy metabolism, catalyzing a metabolic shift favoring glycolysis. Collectively, the results provide insights into a strategy employed by cancer cells to adapt to hypoxia through metabolic reprogramming.
RESUMEN
Hyperactivated KRAS mutations fuel tumorigenesis and represent attractive targets for cancer treatment. While covalent inhibitors have shown clinical benefits against the KRASG12C mutant, advancements for non-G12C mutants remain limited, highlighting the urgent demand for pan-KRAS inhibitors. RNA G-quadruplexes (rG4s) in the 5'-untranslated region of KRAS mRNA can regulate KRAS translation, making them promising targets for pan-KRAS inhibitor development. Herein, we designed and synthesized 50 novel coumarin-quinolinium derivatives, leveraging our previously developed rG4-specific ligand, QUMA-1. Notably, several compounds exhibited potent antiproliferative activity against cancer cells as pan-KRAS translation inhibitors. Among them, 15a displayed exceptional capability in stabilizing KRAS rG4s, suppressing KRAS translation, and consequently modulating MAPK and PI3K-AKT pathways. 15a induced cell cycle arrest, prompted apoptosis in KRAS-driven cancer cells, and effectively inhibited tumor growth in a KRAS mutant xenograft model. These findings underscore the potential of 15a as a pan-KRAS translation inhibitor, offering a novel and promising approach to target various KRAS-driven cancers.
Asunto(s)
G-Cuádruplex , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Línea Celular Tumoral , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de la Síntesis de la Proteína , MutaciónRESUMEN
Developing c-MYC transcription inhibitors that target the G-quadruplex has generated significant interest; however, few compounds have demonstrated specificity for c-MYC G-quadruplex and cancer cells. In this study, we designed and synthesized a series of benzoazole derivatives as potential G-quadruplex ligand-based c-MYC transcription inhibitors. Surprisingly, benzoselenazole derivatives, which are rarely reported as G-quadruplex ligands, demonstrated greater c-MYC G-quadruplex selectivity and cancer cell specificity compared to their benzothiazole and benzoxazole analogues. The most promising compound, benzoselenazole m-Se3, selectively inhibited c-MYC transcription by specifically stabilizing the c-MYC G-quadruplex. This led to selective inhibition of hepatoma cell growth and proliferation by affecting the MYC target gene network, as well as effective tumor growth inhibition in hepatoma xenografts. Collectively, our study demonstrates that m-Se3 holds significant promise as a potent and selective inhibitor of c-MYC transcription for cancer treatment. Furthermore, our findings inspire the development of novel selenium-containing heterocyclic compounds as c-MYC G-quadruplex-specific ligands and transcription inhibitors.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Ligandos , Genes myc , Proliferación CelularRESUMEN
Inter-organelle interactions play a vital role in diverse biological processes. Thus, chemical tools are highly desirable for understanding the spatiotemporal dynamic interplay among organelles in live cells and in vivo. However, designing such tools is still a great challenge due to the lack of universal design strategies. To break this bottleneck, herein, a novel unimolecular platform integrating the twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE) dual mechanisms was proposed. As a proof of concept, two organelles, lipid droplets (LDs) and mitochondria, were selected as models. Also, the first TICT-AIE integration molecule, BETA-1, was designed for simultaneous and dual-color imaging of LDs and mitochondria. BETA-1 can simultaneously target LDs and mitochondria due to its lipophilicity and cationic structure and emit cyan fluorescence in LDs and red fluorescence in mitochondria. Using BETA-1, for the first time, we obtained long-term tracking of dynamic LD-mitochondrion interactions and identified several impressive types of dynamic interactions between these two organelles. More importantly, the increase in LD-mitochondrion interactions during ferroptosis was revealed with BETA-1, suggesting that intervening in the LD-mitochondrion interactions may modulate this cell death. BETA-1 was also successfully applied for in vivo imaging of LD-mitochondrion interactions in C. elegans. This study not only provides an effective tool for uncovering LD-mitochondrion interactions and deciphering related biological processes but also sheds light on the design of new probes with an integrated TICT-AIE mechanism for imaging of inter-organelle interactions.
Asunto(s)
Caenorhabditis elegans , Gotas Lipídicas , Animales , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Mitocondrias/metabolismo , Diagnóstico por ImagenRESUMEN
Mutations in NRAS promote tumorigenesis and drug resistance. As this protein is often considered an undruggable target, it is urgent to develop novel strategies to suppress NRAS for anticancer therapy. Recent reports indicated that a G-quadruplex (G4) structure formed in the untranslated region of NRAS mRNA can downregulate NRAS translation, suggesting a potential NRAS suppression strategy. Here, we developed a novel cell-based method for large-scale screening of NRAS G4 ligand using the G-quadruplex-triggered fluorogenic hybridization probe and successfully identified the clinically used agent Octenidine as a potent NRAS repressor. This compound suppressed NRAS translation, blocked the MAPK and PI3K-AKT signaling, and caused concomitant cell cycle arrest, apoptosis, and autophagy. It exhibited better antiproliferation effects over clinical antimelanoma agents and could inhibit the growth of NRAS-mutant melanoma in a xenograft mouse model. Our results suggest that Octenidine may be a prominent anti-NRAS-mutant melanoma agent and represent a new NRAS-mutant melanoma therapy option.