Lysosomal Targeting Research Articles

Thio-ProTide strategy: a novel H2S donor–drug conjugate (DDC) alleviates hepatic injury via innate lysosomal targeting

Hydrogen sulfide (H2S) is a gas signaling molecule with versatile bioactivities; however, its exploitation for disease treatment appears challenging. This study describes the design and characterization of a novel type of H2S donor–drug conjugate (DDC) based on the thio-ProTide scaffold, an evolution of the ProTide strategy successfully used in drug discovery. The new H2S DDCs achieved hepatic co-delivery of H2S and an anti-fibrotic drug candidate named hydronidone, which synergistically attenuated liver injury and resulted in more sufficient intracellular drug exposure. The potent hepatoprotective effects were also attributed to the H2S-mediated multipronged intervention in lipid peroxidation both at the whole cellular and lysosomal levels. Lysosomal H2S accumulation and H2S DDC activation were facilitated by the hydrolysis through the specific lysosomal hydrolase, representing a distinct mechanism for lysosomal targeting independent of the classical basic moieties. These findings provided a novel pattern for the design of optimally therapeutic H2S DDC and organelle-targeting functional molecules.

Chitosan-naphthalimide probes for dual channel recognition of HClO and H2S in cells and their application in photodynamic therapy

The combination of bio-imaging with photodynamic therapy (PDT) to accomplish theranostics is promising in cancer treatment. Three chitosan-naphthalimide probes were studied in this work. 4-(5-Bromothiophen-2-yl)-1,8-naphthalic anhydride was first synthesized, and then reacted with chitosan to obtain the macromolecules (CS-N-Br). The recognition group thiomorpholine or its derivatives were introduced into CS-N-Br to obtain nano-probes (CS-N-ML, CS-N-BSZ, CS-N-FSQ) eventually. The studies revealed that CS-N-ML and CS-N-FSQ exhibit high selectivity and can specifically recognize HClO and H2S. CS-N-ML and CS-N-FSQ can perform exogenous and endogenous confocal imaging of HClO and H2S in cells also. CS-N-ML's ability to target lysosomes positions indicated it could act as a lysosome-specific probe. It was discovered that the probes generate superoxide anions (O2•−) via a Type I mechanism. This discovery endows the probes with high photosensitizing activity even under hypoxic conditions. There is a positive correlation between the extent of the conjugated system and the photosensitivity of the probes, indicating that an enhanced conjugation leads to increased photosensitivity. Upon light irradiation, the probes generate ROS within HeLa cells. These results suggested that these probes can achieve theranostics for diseases associated with abnormal levels of HClO and H2S.

Lanthanide conjugate Pr-MPO elicits anti-cancer activity by targeting lysosomal machinery and inducing zinc-dependent cataplerosis.

Acquired drug resistance is a major challenge in the management of cancer, which underscores the need for discovery and development of novel therapeutic strategies. We report here the mechanism of the anti-cancer activity of a small coordinate complex composed of the rare earth metal praseodymium (Pr) and mercaptopyridine oxide (MPO; pyrithione). Exposure of cancer cells to relatively low concentrations of the conjugate Pr-MPO (5 µM) significantly impairs cell survival in a p53-independent manner and irrespective of the drug resistant phenotype. Mechanistically, Pr-MPO-induced cell death is caspase-independent, not inhibitable by necrostatin, but associated with the appearance of autophagy markers. However, further analysis revealed incomplete autophagic flux, thus suggesting altered integrity of lysosomal machinery. Supporting the lysosomal targeting activity are data demonstrating increased lysosomal Ca2+ accumulation and alkalinization, which coincides with cytosolic acidification (drop in pHc from 7.75 to 7.00). In parallel, an increase in lysosomal activity of glycosidase alpha acid (GAA), involved in passive glycogen breakdown, correlates with rapid depletion of glucose stores upon Pr-MPO treatment. This is associated with swift cataplerosis of TCA cycle intermediates, loss of NAD+/NADH and increase in pyruvate dehydrogenase (PDH) activity to compensate for pyruvate loss. Addition of exogenous pyruvate rescued cell survival. Notably, lysosomal impairment and metabolic catastrophe triggered by Pr-MPO are suggestive of Zn2+-mediated cytotoxicity, which is confirmed by the ability of Zn2+ chelator TPEN to block Pr-MPO-mediated anti-tumor activity. Together, these results highlight the ability of the small molecule lanthanide conjugate to target the cells' waste clearing machinery as well as mitochondrial metabolism for Zn2+-mediated execution of cancer cells, which could have therapeutic potential against cancers with high metabolic activity.

Chaperone-mediated autophagy modulates Snail protein stability: implications for breast cancer metastasis

Breast cancer remains a significant health concern, with triple-negative breast cancer (TNBC) being an aggressive subtype with poor prognosis. Epithelial-mesenchymal transition (EMT) is important in early-stage tumor to invasive malignancy progression. Snail, a central EMT component, is tightly regulated and may be subjected to proteasomal degradation. We report a novel proteasomal independent pathway involving chaperone-mediated autophagy (CMA) in Snail degradation, mediated via its cytosolic interaction with HSC70 and lysosomal targeting, which prevented its accumulation in luminal-type breast cancer cells. Conversely, Snail predominantly localized to the nucleus, thus evading CMA-mediated degradation in TNBC cells. Starvation-induced CMA activation downregulated Snail in TNBC cells by promoting cytoplasmic translocation. Evasion of CMA-mediated Snail degradation induced EMT, and enhanced metastatic potential of luminal-type breast cancer cells. Our findings elucidate a previously unrecognized role of CMA in Snail regulation, highlight its significance in breast cancer, and provide a potential therapeutic target for clinical interventions.

The lysosomal lipid transporter LIMP-2/SCARB2 is part of lysosome-endoplasmic reticulum STARD3-VAPB-dependent contact sites.

SCARB2/LIMP-2 is an abundant lysosomal membrane protein. Previous studies have shown LIMP-2 functions as a virus receptor, a chaperone for lysosomal enzyme targeting, and a lipid transporter. The large luminal domain of LIMP-2 contains a hydrophobic tunnel that enables transport of phospholipids, sphingosine and cholesterol from the lysosomal lumen to the membrane. The question about the fate of the lipids after LIMP-2-mediated transport is largely unexplored. To elucidate whether LIMP-2 is part of contact sites between lysosomes and the endoplasmic reticulum (ER), we performed a proximity-based interaction screen. This revealed that LIMP-2 interacts with the endosomal protein STARD3 and the ER-resident protein VAPB. Using imaging and co-immunoprecipitation, we demonstrated colocalization and physical interaction between LIMP-2 and these proteins. Moreover, we found that interaction of LIMP-2 with VAPB required the presence of STARD3. Our findings suggest that LIMP-2 is part of ER-lysosome contact sites, possibly facilitating cholesterol transport from the lysosomal to the ER membrane. This suggests a novel mechanism for inter-organelle communication and lipid trafficking mediated by LIMP-2.

Two strategies of enhancing the therapeutic potential of a coumarin probe: Integration of a protonatable moiety and BSA-based nanoparticle formation

Photodynamic therapy (PDT) represents a promising non-invasive approach for cancer treatment, relying on photosensitizers (PSs) to generate reactive oxygen species (ROS) upon light activation. This study aims to enhance the therapeutic potential of a coumarin-based fluorescent probe by employing two strategic modifications: the integration of a protonatable lysosome-targeting moiety and the formation of bovine serum albumin (BSA)-based nanoparticles. The COM molecule was synthesized with a morpholine moiety to facilitate lysosome targeting and protonation in acidic environments, thereby enhancing ROS production and promoting lysosomal dysfunction for increased cancer cell apoptosis. Additionally, COM was encapsulated in BSA nanoparticles (COM-BSA NPs) to leverage hydrophobic interactions and hydrogen bonding, which enhance ROS generation by promoting the intersystem crossing (ISC) process. Comprehensive photophysical studies confirmed the enhanced ROS generation under acidic conditions and within BSA-based NPs. Cellular experiments demonstrated effective lysosome targeting and anticancer efficacy of both COM and COM-BSA NPs. These findings highlight the dual benefits of maintaining the bioimaging capabilities of coumarin while significantly improving its therapeutic efficacy in PDT applications.

3D DNAzyme Motor Nanodevice With Self-Powered FRET Amplifier and Self-Supplied H2O2 for Enhancing Human Neutrophil Elastase Profiling and Chemodynamic Therapy in Lung Tumor.

The development of theragnostic nanosystems integrating FRET (fluorescence resonance energy transfer) imaging and chemodynamic therapy (CDT) for accurate diagnosis and effective treatment of lung tumors is still a big challenge. Herein, a peptide-assembled 3D DNAzyme motor nanodevice is engineered for a self-powered FRET amplifier profiling human neutrophil elastase (HNE) and self-supplied H2O2 enhancing CDT. The nanodevice is prepared by depositing AuNPs on ZIF-8, in which ZIF-8 co-loaded the lysosomal targeting peptide-modified copper peroxides (PCPs) and hairpins (H1, H2, and H3), AuNPs are co-labeled by DNAzyme-peptide (DP) conjugate and H3. In the tumor micro-environment, HNE driven 3D DNAzyme walker followed by an exponential amplification constructed by a synergistic cross-activation between hybridization chain reaction and DNAzyme, generating a self-powered FRET amplifier. The FRET amplifier specifically measures HNE with a sensitivity of 0.026pM, and successfully images exogenous HNE in living cells and monitors HNE in mouse models. Moreover, the PCPs can target lysosomes, reducing lysosome escape. The self-supplying H2O2 undertaken by PCPs improves the Cu (II)-catalyzed Fenton-like reaction, effectively causing cell apoptosis to inhibit tumor growth. Significantly, the nanodevice successfully screens inhibitors and discriminates the HNE level in normal and lung cancer tissues, suggesting that the nanodevice provides an effective tool for the diagnosis and treatment of lung tumors.

Designed endocytosis-inducing proteins degrade targets and amplify signals.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificitytargeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates.

Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism.

The lysosome integrates anabolic signalling and nutrient-sensing to regulate intracellular growth pathways. The leucine-rich repeat containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signalling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show LRRC8A regulates leucine-stimulated mTOR, lysosome size, number, pH, and expression of lysosomal proteins LAMP2, P62, LC3B, suggesting impaired autophagic flux. Mutating a LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signalling and altered lysosomal morphology and pH observed in LRRC8A KO cells. In vivo , LRRC8A-L706A;L707A KI mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance characterized by reduced skeletal muscle glucose-uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8 mediated metabolic signalling function that regulates lysosomal activity, systemic glucose homeostasis and insulin-sensitivity.

Fluorinated triphenylamine phthalocyanine @ silica-coated gold nanorods: A photoactivated lysosome escape and targeting mitochondria two-photon probe for imaging-guided photothermal synergistic photodynamic therapy in cancer cells

The timely evasion of nanomedicines from lysosomes is essential to avert premature degradation under the acidic and hydrolytic conditions characteristic of these cellular compartments. However, the development of effective strategies has been hindered by the complexity of design material and the scarcity of practical methods. In this study, we have synthesized a novel nanoparticle, designated as TPA-BPAF-SiPc@AuNR@SiO2. This nanoparticle was prepared by encapsulating near-infrared fluorinated triphenylamine-substituted silicon phthalocyanines (TPA-BPAF-SiPc) within mesoporous silica-coated gold nanorods (AuNR@SiO2). TPA-BPAF-SiPc@AuNR@SiO2 functions as a dual-function two-photon probe, facilitating photoactivated lysosome escape and targeting mitochondria. The inherent aggregation-induced emission (AIE) two-photon fluorescence of TPA-BPAF-SiPc is notably bright when encapsulated in AuNR@SiO2 nanocarriers, a phenomenon not observed in polymer nanocarriers composed of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) or in THF/water mixtures. Upon irradiation, this nanoparticle autonomously escapes from lysosomes and selectively targets mitochondria, a process can be visually monitored in real-time through the two-photon AIE fluorescence of TPA-BPAF-SiPc. Moreover, upon activation, TPA-BPAF-SiPc@AuNR@SiO2 produces a substantial quantity of reactive oxygen species (ROS) and induces hyperthermia effects, showcasing its potential for effective photodynamic therapy (PDT) in conjunction with synergistic hyperthermia. Flow cytometry data corroborate the induction of tumor cell death through both necrosis and apoptosis pathways by TPA-BPAF-SiPc@AuNR@SiO2. This study underscores the potential of TPA-BPAF-SiPc@AuNR@SiO2 as a multifunctional probe capable of enabling lysosome escape, mitochondria targeting, and two-photon fluorescence imaging-guided photothermal synergistic photodynamic therapy, specifically tailored for the treatment of breast cancer.

Divergent effects of acute and chronic PPT1 inhibition in melanoma

ABSTRACT Macroautophagy/autophagy-lysosome function promotes growth and survival of cancer cells, making them attractive targets for cancer therapy. One intriguing lysosomal target is PPT1 (palmitoyl-protein thioesterase 1). PPT1 inhibitors derived from chloroquine block autophagy, have significant antitumor activity in preclinical models and are being developed for clinical trials. However, the role of PPT1 in tumorigenesis remains poorly understood. Here we report that in melanoma cells, acute siRNA or pharmacological PPT1 inhibition led to increased ferroptosis sensitivity and significant loss of viability, whereas chronic PPT1 knockout using CRISPR-Cas9 produced blunted ferroptosis that led to sustained viability and growth. Each mode of PPT1 inhibition produced lysosome-autophagy inhibition but distinct proteomic changes, demonstrating the complexity of cellular adaptation mechanisms. To determine whether total genetic loss of Ppt1 would affect tumorigenesis in vivo, we developed a Ppt1 conditional knockout mouse model. We then crossed it into the BrafCA, PtenloxP, Tyr:CreERT2 melanoma mouse model to investigate the impact of Ppt1 loss on tumorigenesis. Loss of Ppt1 had no impact on melanoma histology, time to tumor initiation, or survival of tumor-bearing mice. These results suggest that chemical PPT1 inhibitors produce different adaptations than genetic PPT1 inhibition, and additional studies are warranted to fully understand the mechanism of chloroquine derivatives that target PPT1 in cancer.Abbreviations: 4-HT: 4-hydroxytamoxifen; BRAF: B-Raf proto-oncogene, serine/threonine kinase; cKO: conditional knockout; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9; DC661: A specific PPT1 inhibitor; DMSO: dimethyl sulfoxide; Dox; doxycycline hyclate; Easi-CRISPR: efficient additions with ssDNA inserts-CRISPR; GNS561/ezurpimtrostat: A PPT1 inhibitor; Hug: human guide; iCas: inducible CRISPR-Cas9; KO: knockout; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LDLR: low density lipoprotein receptor; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NT: non-target; PTEN: phosphatase and tensin homolog; PPT1: palmitoyl-protein thioesterase 1; RSL3: RAS-selective lethal small molecule 3; SCRIB/SCRB1: scribble planar cell polarity protein; Tyr:CreERT2: tyrosinase-driven Cre recombinase fused with the tamoxifen-inducible mutant ligand binding domain of the human estrogen receptor; UGCG: UDP-glucose ceramide glucosyltransferase; WT: wild-type.

Development of Integrin Targeting Chimeras (ITACs) for the Lysosomal Degradation of Extracellular Proteins.

The emerging of lysosomal targeting chimera (LYTAC) expands the field of targeted protein degradation (TPD) to include the extracellular proteins for precise depletion. However, most of the reported LYTACs either induce ubiquitous degradation of the protein of interest (POI) in a broad range of tissues or specifically target liver cells. More tissue-selective degraders are highly desirable. Herein, we describe the development of cyclic RGD (cRGD) peptide-antibody conjugates as a novel class of integrin targeting chimeras (ITACs) with potential cancer selectivity. Our results indicate that the ITACs are able to recruit integrin to induce the degradation of both soluble and membrane targets in the lysosome. We observed higher efficiency of ITACs on degrading membrane protein in cancer cells, providing a promising platform for cancer-selective TPD strategy.

Observation of polarity changes in Sjogren's syndrome mice using a targeting lysosomes and ratiometric fluorescent probe

Utilizing non-invasive, real-time dynamic imaging and high-resolution detection tools to track polarity changes in Sjögren's syndrome (SS) contributes to a better understanding of the disease progression. Herein, a ratiometric polarity-sensitive fluorescent probe (DIM) was designed and synthesized, DIM consisted of dicyanoisophorone as the fluorophore and morpholine moiety as lysosome targeting. DIM showed a ratiometric response to polarity and high selectivity (unaffected by viscosity, pH, ROS, RNS, etc.), offering a more accurate analysis of intracellular polarity through a built-in internal reference calibration. The polarity abnormality of submandibular glands in non-obese diabetic (NOD) mice was revealed and verified by in vivo ratiometric fluorescence imaging of DIM, suggesting that fluorescent probe have great potential in the diagnosis of salivary gland abnormalities.

A lysosome-targeted fluorescent probe for thiol detection in drug analysis and multiple biological systems.

Biothiols, characterized by their unique sulfhydryl (-SH) groups, possess excellent antioxidant properties, effectively neutralizing the damage to cellular structures caused by reactive oxygen species (ROS) in living organisms. Additionally, lysosomes play a crucial role in decomposing damaged biomolecules through the action of their internal enzymes, regulating the cellular redox state, and mitigating oxidative stress. To facilitate rapid monitoring of intracellular biothiols, particularly within lysosomes, we constructed a lysosome-targeted biothiol fluorescent probe, PHL-DNP, in this study. PHL-DNP exhibited excellent photophysical properties in an aqueous test system, including strong fluorescence enhancement response, excellent selectivity, and low detection limits (Cys 16.5nM, Hcy 16.8nM, GSH 21.3nM, Cap 26.6nM). These attributes enabled easy and efficient qualification of Cys on test strips and accurate determination of the effective content of captopril tablets. Notably, PHL-DNP demonstrated low cytotoxicity and precise lysosomal targeting. Through bioimaging, PHL-DNP not only monitored changes in biothiol levels under oxidative stress but also assessed biothiols in complex biological systems such as live HeLa cells, zebrafish, tumor tissue sections, and radish roots. This provides a promising tool for quantitative analysis of biothiols, disease marker detection, and drug testing.

Tailoring the pKa of Fluorescence Lifetime Imaging Probes to Visualize Aggrephagy and Resolve Its Microenvironmental Viscosity.

Aggrephagy describes lysosomal transport and degradation of protein aggregates via cellular macroautophagy, a key mechanism to prevent neurodegenerative diseases. Here, we develop a dual-probe method to visualize the aggrephagy process and resolve its viscosity heterogeneity using fluorescence lifetime imaging (FLIM). The dual-probe system consists of (1) a near-infrared lysosomal targeting FLIM probe (Lyso-P1) that is derived from a rhodamine scaffold with a tailored pKa value to accommodate an acidic lysosomal environment and (2) a green BODIPY-based FLIM probe (Agg-P2) that reports on degradation of cellular aggregates via HaloTag. Both probes exhibit acid-resistant, viscosity-dependent fluorescence intensity and lifetime (τ) responses, which are ready for intensity- and FLIM-based imaging. Photochemical, theoretical, and biochemical characterizations reveal the probes' mechanism-of-actions. In cells, we exploit Lyso-P1 and Agg-P2 to simultaneously quantify both lysosomal and protein aggegates' viscosity changes upon the aggrephagy process via FLIM. We reveal orthogonal changes in microenvironmental viscosities and morphological heterogeneity upon various cellular stresses. Overall, we provide an imaging toolset to quantitatively study aggrephay, which may benefit screening of aggrephay modulators for disease intervention.

Design of an Acidic pH-Activated NIR Fluorescent Convertible Rhodamine-Hemicyanine Probe-Peptide Conjugate for Living Cancer Cell Active Targeted Selective Tracking of Lysosomes.

We have synthesized an acidic pH-activatable dual targeting ratiometric fluorescent probe-peptide conjugate using the SPPS protocol on Rink amide AM resin. Living carcinoma cell specific active targeting, successive cell penetration, and selective staining of lysosomes are accomplished. Real-time monitoring of lysosomes, 3D, and multicolor cancer cell imaging are also attained. The de novo design consists of the integration of multifunctionality into a single molecular scaffold, e. g., RGDS peptide residue to target cancer cell surface overexpressed receptor αVβ3 integrin, live-cell penetrating organic unsymmetrical rhodamine-hemicyanine chromophore comprising a lysosome targeting morpholine group, and an acidic pH openable spiro-lactam ring for a visible-to-NIR switchable ratiometric response. Water-soluble fluorescent probe-peptide conjugate exhibits intramolecular spirolactamization at basic pH through Arg amide N. The visible spirolactam state predominantly exists at physiological and basic pH and can be switched to the highly conjugated NIR open amide state (λem=735 nm) through spiro-lactam ring opening triggered by acidic pH with a huge bathochromic shift (Δλabs=336 nm, ΔλFL=265 nm). Moreover, pH-sensitive ratiometric optical switching is achieved. This in situ acidic cancer cell lysosome activatable multifunctional fluorophore-peptide conjugate shows augmented molar absorptivity, enhanced quantum yield, and improved fluorescence lifetime at acidic lysosomal pH; negligible cytotoxicity; and dual targeted ratiometric imaging capability of living cancer cell selective lysosomes with a pKa value of 5.1.

Two Birds with One Stone: Guanidyl Carbon Dots with Enhanced Antioxidative and Lipolytic Functions in Metabolic Associated Fatty Liver

AbstractMetabolic‐associated fatty liver disease (MAFLD) has become one of the most widespread metabolic diseases nowadays, while no definitive agent is approved for practical treatment. Given excessive lipid deposition and cellular oxidative stress are two main inducers of MAFLD, developing synergistic therapeutic agents that enhance lipid metabolism and antioxidative ability are effective routes for MAFLD therapy. Herein, a novel kind of bio‐functional carbon dots (MTCDs) derived from metformin and tea polyphenols based on a fused pharmacophore strategy is developed. The surface of MTCDs retains abundant phenolic hydroxyl and guanidine groups, which are designed to reduce both lipid deposition and oxidative stress in the liver. Moreover, by finely controlling the surface charge, MTCDs exhibit excellent lysosome targeting functionality with favorable biocompatibility and low biotoxicity. Mechanistical studies further reveal that MTCDs treatment protects lysosomes from oxidative stress damage and activates hepatocyte AMP‐activated protein kinase signaling, which initiates lipophagy progress to accelerate lipolysis and lipid consumption. Collectively, these studies propose an effective “two birds with one stone” tool for MAFLD synergistic therapy and suggest a viable strategy for multi‐pharmacophore agent development.

Anticancer Activity of Benzo[a]phenoxazine Compounds Promoting Lysosomal Dysfunction.

Specific cancer therapy remains a problem to be solved. Breast and colorectal cancer are among the cancers with the highest prevalence and mortality rates. Although there are some therapeutic options, there are still few effective agents for those cancers, which constitutes a clinical problem that requires further research efforts. Lysosomes play an important role in cancer cells' survival, and targeting lysosomes has gained increased interest. In recent years, our team has been synthetizing and testing novel benzo[a]phenoxazine derivatives, as they have been shown to possess potent pharmacological activities. Here, we investigated the anticancer activity of three of the most potent derivatives from our library, C9, A36, and A42, on colorectal- and breast-cancer-derived cell lines, and compared this with the effect on non-neoplastic cell lines. We observed that the three compounds were selective for the cancer cells, namely the RKO colorectal cancer cell line and the MCF7 breast cancer cell line. In both models, the compounds reduced cell proliferation, cell survival, and cell migration, accumulated on the lysosome, and induced cell death accompanied by lysosomal membrane permeabilization (LMP), increasing the intracellular pH and ROS accumulation. Our results demonstrated that these compounds specifically target lysosomes from cancer cells, making them promising candidates as LMP inducers for cancer therapy.

Visual Tracking of Hydrogen Sulfide: Application of a Novel Lysosome-Targeted Fluorescent Probe for Bioimaging and Food Safety Assessment.

The equilibrium state of hydrogen sulfide (H2S), a gaseous signaling molecule produced by lysosomal metabolites, in vivo is crucial for cellular function. Abnormal fluctuations in H2S concentration can interfere with the normal function of lysosomes, which has been closely linked to the pathogenesis of a variety of diseases. In view of this, a novel fluorescent probe Lyso-DPP based on 1,3,5-triarylpyrazolines was developed for the precise detection of H2S in lysosomes by using the hydrophilic morpholine moiety as a lysosomal targeting unit, and 2,4-dinitroanisole as a fluorescence-quenching and H2S-responsive unit. The probe cleverly combines the advantages of simple synthesis, sensitive blue fluorescence turn-on with a limit of detection, LOD, of 97.3 nM, good stability, and fast response time (10 min), which makes Lyso-DPP successful in portable monitoring of meat freshness in the form of test strips. Moreover, the excellent biocompatibility and precise targeting capability of the probe Lyso-DPP make it perform well in the monitoring of H2S in lysosomes, living cells, and zebrafish. This work not only provides new technical tools for food quality control but also paves up new ideas for early diagnosis and treatment of H2S-related diseases.

Metformin Lysosomal Targeting: A Novel Aspect to Be Investigated for Metformin Repurposing in Neurodegenerative Diseases?

Metformin is a widely employed drug in type 2 diabetes. In addition to warranting good short- and long-term glycemic control, metformin displays many intriguing properties as protection against cardiovascular and neurodegenerative diseases, anti-tumorigenic and longevity promotion. In addition to being a low-cost drug, metformin is generally well tolerated. However, despite the enthusiastic drive to aliment these novel studies, many contradictory results suggest the importance of better elucidating the complexity of metformin action in different tissues/cells to establish its possible employment in neurodegenerative diseases. This review summarises recent data identifying lysosomal-dependent processes and lysosomal targets, such as endosomal Na+/H+ exchangers, presenilin enhancer 2 (PEN2), the lysosomal pathway leading to AMP-activated protein kinase (AMPK) activation, and the transcription factor EB (TFEB), modulated by metformin. Lysosomal dysfunctions resulting in autophagic and lysosomal acidification and biogenesis impairment appear to be hallmarks of many inherited and acquired neurodegenerative diseases. Lysosomes are not yet seen as a sort of cellular dump but are crucial in determining key signalling paths and processes involved in the clearance of aggregated proteins. Thus, the possibility of pharmacologically modulating them deserves great interest. Despite the potentiality of metformin in this context, many additional important issues, such as dosing, should be addressed in the future.