Mitochondrial RelA empowers mtDNA G-quadruplex formation for hypoxia adaptation in cancer cells.

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.

Revealing Mitochondrion-Lysosome Dynamic Interactions and pH Variations in Live Cells with a pH-Sensitive Fluorescent Probe.

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.

Development and Characterization of Benzoselenazole Derivatives as Potent and Selective c-MYC Transcription Inhibitors.

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.

Discovery of Clinically Used Octenidine as NRAS Repressor That Effectively Inhibits NRAS-Mutant Melanoma.

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.

Construction of a TICT-AIE-Integrated Unimolecular Platform for Imaging Lipid Droplet-Mitochondrion Interactions in Live Cells and In Vivo.

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.

Development of a Highly Selective and Sensitive Fluorescent Probe for Imaging RNA Dynamics in Live Cells.

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.

Monitoring and Modulating mtDNA G-Quadruplex Dynamics Reveal Its Close Relationship to Cell Glycolysis.

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.

Tracking Stress Granule Dynamics in Live Cells and In Vivo with a Small Molecule.

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.