Programming DNA Nanoassemblies into Polyvalent Lysosomal Degraders for Potent Degradation of Pathogenic Membrane Proteins.

Lysosome-targeting chimera (LYTAC) shows great promise for protein-based therapeutics by targeted degradation of disease-associated membrane or extracellular proteins, yet its efficiency is constrained by the limited binding affinity between LYTAC reagents and designated proteins. Here, we established a programmable and multivalent LYTAC system by tandem assembly of DNA into a high-affinity protein degrader, a heterodimer aptamer nanostructure targeting both pathogenic membrane protein and lysosome-targeting receptor (insulin-like growth factor 2 receptor, IGF2R) with adjustable spatial distribution or organization pattern. The DNA-based multivalent LYTACs showed enhanced efficacy in removing immune-checkpoint protein programmable death-ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) in tumor cell membrane that respectively motivated a significant increase in T cell activity and a potent effect on cancer cell growth inhibition. With high programmability and versatility, this multivalent LYTAC system holds considerable promise for realizing protein therapeutics with enhanced activity.

Ratiometric lysosome-targeting luminescent probe based on a coumarin-ruthenium(II) complex for formaldehyde detection and imaging in living cells and mouse brain tissues.

Ratiometric luminescence probes have attracted widespread attention because of their self-calibration capability. However, some defects, such as small emission shift, severe spectral overlap and poor water solubility, limit their application in the field of biological imaging. In this study, a unique luminescence probe, Ru-COU, has been developed by combining tris(bipyridine)ruthenium(II) complex with coumarin derivative through a formaldehyde-responsive linker. The probe exhibited a large emission shift (Δλ > 100 nm) and good water solubility, achieving ratiometric emission responses at 505 nm and 610 nm toward formaldehyde under acidic conditions. Besides, ratiometric luminescence imaging of formaldehyde in living cells and Alzheimer disease mouse's brain slices demonstrates the potential value of Ru-COU for the diagnosis and treatment of formaldehyde related diseases.

A near-infrared fluorescent probe with viscosity sensitivity in lysosome for cancer visualization.

A viscosity-sensitive, lysosome-targeted near-infrared fluorescent probe (PYATT) was reported in this paper. The fluorescent spectra of PYATT are strongly dependent on viscosity, resulting in a Stokes shift of about 190 nm. Given its photostability, low cytotoxicity, and high fluorescence quantum yield, PYATT is expected to be used in cell imaging. Due to the higher viscosity of tumor cells than normal cells, the fluorescence intensity of PYATT in tumor cells is higher than normal cells, which can realize the visualization of tumors. The near-infrared probe (PYATT) is viscosity-dependent in lysosomes, which is valuable in early diagnosis and treatment of tumor.

Targeted degradation of membrane and extracellular proteins with LYTACs.

Targeted protein degradation technology has gained substantial momentum over the past two decades as a revolutionary strategy for eliminating pathogenic proteins that are otherwise refractory to treatment. Among the various approaches developed to harness the body's innate protein homeostasis mechanisms for this purpose, lysosome targeting chimeras (LYTACs) that exploit the lysosomal degradation pathway by coupling the target proteins with lysosome-trafficking receptors represent the latest innovation. These chimeras are uniquely tailored to degrade proteins that are membrane-bound and extracellular, encompassing approximately 40% of all proteome. Several novel LYTAC formulas have been developed recently, providing valuable insights for the design and development of therapeutic degraders. This review delineates the recent progresses of LYTAC technology, its practical applications, and the factors that dictate target degradation efficiency. The potential and emerging trends of this technology are discussed as well. LYTAC technology offers a promising avenue for targeted protein degradation, potentially revolutionizing the therapeutic landscape for numerous diseases.

Platinum-Metformin Conjugates Acting as Promising PD-L1 Inhibitors through the AMP-Activated Protein Kinase Mediated Lysosomal Degradation Pathway.

With the development of metalloimmunology, the potential of platinum drugs in cancer immunotherapy has attracted extensive attention. Although immunochemotherapy combining PD-1/PD-L1 antibodies with platinum drugs has achieved great success in the clinic, combination therapy commonly brings new problems. Herein, we have developed a platinum-metformin conjugate as a promising alternative to antibody-based PD-L1 inhibitors, not only disrupting PD-1/PD-L1 axis on cell surface but also down-regulating the total PD-L1 levels in non-small cell lung cancer (NSCLC) cells comprehensively, thus achieving highly efficient immunochemotherapy by a single small molecule. Mechanism studies demonstrate that Pt-metformin conjugate can selectively accumulate in lysosomes, promote lysosomal-dependent PD-L1 degradation via the AMPK-TFEB pathway, and modulate the upstream regulatory proteins related to PD-L1 expression (e.g. HIF-1α and NF-κB), eventually decreasing the total abundance of PD-L1 in NSCLC, overcoming tumor hypoxia, and activating anti-tumor immunity in vivo. This work suggests an AMPK-mediated lysosomal degradation pathway of PD-L1 for the first time and provides a unique design perspective for the development of novel platinum drugs for immunochemotherapy.

Aggregation-Induced emission photosensitizer with lysosomal response for photodynamic therapy against cancer.

Photosensitizers play a key role in bioimaging and photodynamic therapy (PDT) of cancer. However, conventional photosensitizers usually do not achieve the desired efficacy in PDT due to their poor photostability, targeting ability, and responsiveness. Herein, we designed a series of photosensitizers with aggregation-induced emission (AIE) effect using benzothiazole- triphenylamine (BZT-triphenylamine) as the parent nucleus. The synthesized compound SIN ((E)-2-(4-(diphenylamino)styryl)-3-(4-iodobutyl)benzo[d]thiazol-3-ium) exhibits good biocompatibility, photostability, and bright emission in the near-infrared range (600-800 nm). The fluorescence emission intensity is responsive to viscosity, with significant fluorescence enhancement (48 times) and high fluorescence quantum yield (4.45 %) at high viscosity. Moreover, SIN has particular lysosome targeting properties with a Pearson correlation coefficient (PCC) of 0.97 and has good 1O2 generation ability under white light irradiation, especially in a weak acidic environment. Thus, SIN can realize good bioimaging ability and photodynamic therapeutic efficacy under the highly viscous and weakly acidic environment of lysosomes in the tumor cells. This study indicates that SIN has potential as a multifunctional organic photosensitizer for bioimaging and PDT of tumor.

Water-soluble dual lysosome/mitochondria-targeted fluorescent probe for detection of SO2 in water, food, herb, and live cells.

In this paper, we present a new donor-π bridge-acceptor type fluorescent probe, MIB, which bears two organelle-targeted groups, namely positively charged benzothiazole group for mitochondria and morpholine moiety for lysosomes. In aqueous solution, the nucleophilic addition of HSO3- (as SO2 donor) to MIB blocked its long-range π-conjugation and ICT process and resulted in significant optical signal changes (blue-shifted UV absorbance and fluorescence), which enabled colorimetric and ratiometric fluorescent detection of HSO3- with high selectivity and sensitivity (detection limit of 63.15 nM). MIB offers obvious advantages of good water-solubility, fast response time (within 1 min), unique dual lysosome/mitochondria targeting capability and has been applied to the sensing of endogenous and exogenous SO2 in live cells through fluorescent imaging. In addition, the proposed probe has been utilized for the determination of bisulfite in real water, food and herbal medicine samples, showing good recovery (91.45 % - 109.3 %) and precision.

Lysosome-targetable selenium-doped carbon nanodots for in situ scavenging free radicals in living cells and mice.

Lysosome-targetable selenium-doped carbon nanodots (Lyso-Se-CDs) that can efficiently scavenge lysosomal •OH in living cells and mice were designed in this research. Se-CDs with redox-responsive fluorescence (λex = 379 nm, λem = 471 nm, quantum yield = 7.1%) were initially synthesized from selenocystine by a facile hydrothermal method, followed by the surface modification with morpholine, a lysosome targeting moiety. The as-synthesized Lyso-Se-CDs exhibited excellent colloidal stability, efficient scavenging abilities towards •OH, low biotoxicity, as well as good biocompatibility and lysosome targetability. Due to these desirable properties, Lyso-Se-CDs had been successfully utilized for rescuing cells from elevated lysosomal •OH levels. More importantly, Lyso-Se-CDs efficiently relieved phorbol 12-myristate 13-acetate (PMA) triggered ear inflammation in live mice. These findings reveal that Lyso-Se-CDs are potent candidates for treating •OH-related inflammation.

A Bioresponsive Near-Infrared Fluorescent Probe for Facile and Persistent Live-Cell Tracking.

Molecular imaging significantly transforms the field of biomedical science and facilitates the visualization, characterization, and quantification of biologic processes. However, it is still challenging to monitor cell localization in vivo, which is essential to the study of tumor metastasis and in the development of cell-based therapies. While most conventional small-molecule fluorescent probes cannot afford durable cell labeling, transfection of cells with fluorescent proteins is limited by their fixed fluorescence, poor tissue penetration, and interference of autofluorescence background. Here, a bioresponsive near-infrared fluorescent probe is reported as facile and reliable tool for real-time cell tracking in vivo. The design of this probe relies on a new phenomenon observed upon fluorobenzene-conjugated fluorescent dyes, which can form complexes with cytosolic glutathione and actively translocates to lysosomes, exhibiting enhanced and stable cell labeling. Fluorobenzene-coupled hemicyanine, a near-infrared fluorophore manifests to efficiently staining tumor cells without affecting their invasive property and enables persistent monitoring of cell migration in metastatic tumor murine models at high resolution for one week. The method of fluorobenzene functionalization also provides a simple and universal "add-on" strategy to render ordinary fluorescent probes suitable for long-term live-cell tracking, for which currently there is a deficit of suitable molecular tools.

Processing and targeting of cathepsin L (TbCatL) to the lysosome in Trypanosoma brucei.

Cathepsin L (TbCatL) is an essential lysosomal thiol protease in African trypanosomes. TbCatL is synthesized as two precursor forms (P/X) that are activated to mature form (M) with the removal of the prodomain upon arrival in the lysosome. We examine TbCatL trafficking in a novel system: truncated TbCatL reporter without the C-terminal domain (CTD; TbCatL∆) ectopically expressed in an RNA interference (RNAi) cell line targeting the CTD/3' untranslated region (UTR) of endogenous mRNA. TbCatL∆ is synthesized as P'/X'/M' species, localizes to the lysosome, and rescues the lethal TbCatL RNAi phenotype. Inactive TbCatLΔ:C150A is only processed to M' in the presence of endogenous TbCatL indicating trans-auto-catalytic activation. X' is formed with active endoplasmic reticulum (ER)-retained TbCatLΔ:MDDL, but not with TbCatLΔ:C150A, indicating stochastic generation in the ER by cis-auto-cleavage within the prodomain of newly synthesized P'. Modelling the TbCatL prodomain on the human CatL structure suggests three solvent accessible features that could contain post-Golgi targeting signals: the N-terminus, the helix 1/turn 1 junction, and a separate turn (T3). We demonstrate that the critical motif for lysosomal targeting is an asparagine-proline dipeptide in T3 that is strictly conserved in all Kinetoplastida. These findings show novel insights on the maturation of TbCatL, which is a critical virulence factor in mammalian infection.

Synthesis and biological effect of lysosome-targeting fluorescent anion transporters with enhanced anionophoric activity.

Two lysosome-targeting fluorescent anion transporters derived from coumarins, trifluoromethylated arylsquaramides and morpholines were synthesized, and their specificity and efficiency to target and alkalize lysosomes were investigated. They are able to target lysosomes specifically. Compared with the previous analogue without trifluoromethyl substituents, these two conjugates, in particular the one having a 3,5-bis(trifluoromethyl) substituent, exhibit significantly higher ability to facilitate the transport of chloride anions, alkalize lysosomes and reduce the activity of lysosomal Cathepsin B enzyme. The present finding suggests that improving the anionophoric activity of lysosome-targeting fluorescent anion transporters is favorable to the efficiency to alkalize lysosomes and deactivate lysosomal Cathepsin B enzyme.

Intracellular Fate of Nanoparticles with Polydopamine Surface Engineering and a Novel Strategy for Exocytosis-Inhibiting, Lysosome Impairment-Based Cancer Therapy.

Polydopamine (PDA) coating as a bioinspired strategy for nanoparticles (NPs) has been extensively applied in cancer theranostics. However, a cellular-level understanding of nano-biointeraction of these PDA-coated NPs (PDNPs), which drives the fate of them and acts as a critical step to determine their efficacy, still remains unknown. Herein, we utilized the representative mesoporous silica NPs (MSNs) to be coated with PDA and study their nano-bioactivities in cancer cells. HeLa cell line was utilized as a model in this study. The PDNPs were discovered to be internalized through three specific pathways, that is, Caveolae-, Arf6-dependent endocytosis, and Rab34-mediated macropinocytosis (55%, 20% and 37% of uptake inhibition by nystatin, Arf6 knockdown, and rottlerin, respectively). Autophagy-mediated accumulation of PDNPs in lysosomes was observed and the formed PDA shells shedded in the lysosomes. Almost 40% of the NPs were transported out of cells via Rab8/10- and Rab3/26-mediated exocytosis pathways at our tested level. On the basis of these results, a novel combined cancer treatment strategy was further proposed using drug-loaded MSNs-PDA by (i) utilizing naturally intracellular mechanism-controlled PDA shedding for organelle-targeted release of drugs in lysosomes to generate lysosome impairment and (ii) blocking the demonstrated exocytosis pathways for enhanced therapeutic efficacy.

NIR-Absorbing Tetraphenylethene-Containing bisBODIPY Nanoplatforms Demonstrate Effective Lysosome-Targeting and Combinational Phototherapy.

Photosensitizer-based phototherapies, including photodynamic therapy (PDT) and photothermal therapy (PTT), offer safe treatment modalities for tumor ablation with spatiotemporal precision. After photons are absorbed, PDT creates localized chemical damage by generating reactive oxygen species (ROS), while PTT induces localized thermal damage. However, PDT still faces hypoxic tumor challenges, while PTT encounters issues related to heat resistance and potential overheating. The combination of PDT and PTT shows great potential as an effective anticancer strategy. By targeting lysosomes with carefully designed phototherapeutic reagents for combined phototherapy, rapid dysfunction and cell death in cancer cells can be induced, showing promise for cancer treatment. Herein, two α-α-linked bisBODIPYs with tetraphenylethene (TPE) moieties are designed and synthesized. These TPE-substituted bisBODIPYs expand the absorption into NIR range (λmaxabs/λmaxem ∼ 740/810 nm) and confer aggregation-induced emission (AIE) activity (λmaxem ∼ 912 nm). Moreover, these bisBODIPYs self-assemble with surfactant F-127 into nanoparticles (NPs), which efficiently generate ROS (1O2 and •OH) in both solution and cellular environments and demonstrate superior photothermal conversion efficiencies (η ∼ 68.3%) along with exceptional photothermal stability. More importantly, these NPs showed lysosomal targeting and remarkable tumor ablation in cellular and murine models, indicating their potential in precision tumor therapy.

Visualizing lysosomes hypochlorous acid in Parkinson's disease models by a novel fluorescent probe.

Heightened oxidative stress is the principal driver behind the altered metabolism of neurotransmitters within the brains of Parkinson's disease (PD). Hypochlorous acid (HClO), a variant of reactive oxygen species (ROS), plays a crucial role in several lysosomal activities. An irregular concentration of HClO may result in significant molecular damage and contribute to the onset of neurodegenerative disorders. Despite this, the precise role of lysosomal HClO in PD remains unclear, due to its fast reactivity and low levels. This is further complicated by the lack of effective in situ imaging techniques for accurately tracking its dynamics. Therefore, it is of great significance to use effective tools to map the lysosomal HClO during the pathological process of PD. In this study, we propose a fluorogenic probe named Lys-PTZ-HClO for the specific and sensitive detection of HClO. Lys-PTZ-HClO exhibits features like a fast response time (10 s) and a low detection limit (0.72 µM). Benefiting from its superior properties, the probe was used to visualize the basal HClO levels, and the variation of HClO levels in lysosomal of living cells. More importantly, this probe was successfully applied for the first time to reveal increased lysosomal HClO in a cellular model of PD.

A lysosomal-targeted and viscosity-ultrasensitive near-infrared fluorescent probe for sensing viscosity in cells and a diabetic mice model.

Diabetes, as a metabolic disorder, has been implicated in organ dysfunction, often correlated with aberrant changes in viscosity. Lysosomal viscosity serves as an indicator of the lysosome's condition and activity, as it always varies synchronously with the change of lysosome's positioning, structure, and internal constituents. Diabetes, a condition within the metabolic disease category, has the potential to disrupt organ function due to irregular changes in viscosity. Therefore, early and precise diagnosis of diabetes is crucial for the prevention and management of diabetic conditions. Understanding the correlation between viscosity variations and lysosomal changes in vivo is vitally important for researching associated diseases. In this study, we developed Lyso-V, a near-infrared (NIR) fluorescent probe targeting lysosomes, with ultrasensitivity to viscosity changes. This probe, designed with a donor-π-bridge-acceptor (D-π-A) structure, exhibits a significant increase in NIR fluorescence intensity (approximately 690 times) when responding to viscosity, due to a twisted intramolecular charge transfer (TICT) mechanism. Furthermore, the probe designed specifically for lysosomes, enables the detection of changes in lysosomal viscosity as well as autophagy processes. Notably, through the application of this probe, we have detected an increased viscosity within the pathological model of the diabetic mouse. Moreover, Lyso-V was employed to measure the viscosity in diabetic mice. Owing to the multifaceted nature of the Lyso-V probe, it is anticipated to act as a practical and potent resource for deepening our understanding of the pathophysiological aspects of diabetes and aiding in its early detection.

Comparative study of the steady-state subcellular distribution of lysosome-associated membrane glycoprotein-2 (LAMP-2) isoforms with GYXXΦ-type tyrosine-based motifs that interact differently with four adaptor protein (AP) complexes.

Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and µ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with µ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four µ-subunits (µ1, µ2, µ3A and µ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with µ3A was more robust than that with other µ-subunits. LAMP-2B-CT interacted exclusively and moderately with µ3A. LAMP-2C-CT did not detectably interact with any of the four µ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.

A novel lysosomal targeted near-infrared probe for ratio detection of carbon monoxide in cells and in vivo.

Carbon monoxide (CO) as an endogenous gas signaling molecule possesses important physiological functions and is of great significance in the treatment of various diseases. Real-time tracking of CO in living organisms has become a research hotspot in recent years. This article presents a lysosomal targeted near-infrared ratio fluorescence probe (TBM-CO) for selective detection of CO based on the dicyanoisophorone skeleton and morpholine fragment. The probe TBM-CO with weak ICT effect can be transformed to precursor TBM-NH2 with strong ICT effect by the traditional Tsuji-Trost reaction procession in the presence of Pd2+ ions. The mechanism was proved by DFT calculation or the MS and HPLC results respectively. In the near-infrared region an obvious ratio fluorescence intensity change (F686 / F616) is observed in vitro spectral experiments. The concentration titration experiments indicate that there is a good liner relationship between the ratio fluorescence intensity and the concentration in the range of 0 to 50 µM (R2 = 0.996) and the detection limit is calculated as 0.38 µM. The cell fluorescence imaging and co-localization experiments further demonstrate that TBM-CO is able to detect the exogenous and endogenous CO in lysosomal subcellular organelle. Finally, it was used to detect the changes of CO concentration in living mice successfully. In short, a probe with three advantages of near-infrared emission, ratiometric fluorescence and organelle targeting was reported and used to detect CO successfully in cells and in living mice.

Revolutionizing viral disease treatment: Phase separation and lysosome/exosome targeting as new areas and new paradigms for antiviral drug research.

With the advancement of globalization, our world is becoming increasingly interconnected. However, this interconnection means that once an infectious disease emerges, it can rapidly spread worldwide. Specifically, viral diseases pose a growing threat to human health. The COVID-19 pandemic has underscored the pressing need for expedited drug development to combat emerging viral diseases. Traditional drug discovery methods primarily rely on random screening and structure-based optimization, and new approaches are required to address more complex scenarios in drug discovery. Emerging antiviral strategies include phase separation and lysosome/exosome targeting. The widespread implementation of these innovative drug design strategies will contribute towards tackling existing viral infections and future outbreaks.

Modular synthesis of pentacyclic-fused pyranoquinoliziniums as organelle-selective fluorescent probes.

On basis of their unique chemical and photophysical properties, and excellent biological activities, quinoliziniums have been widely used in various research fields. Herein, modular synthetic strategies for efficient synthesis of novel fluorescent quinoliziniums by using one-pot and stepwise rhodium(III)-catalyzed C-H annulations were developed. In the one-pot synthesis, the reaction between 2-aryl-4-quinolones (1) and 1,2-diarylalkynes (2) proceeded in a chemo- and regioselective manner to give quinolinone-fused isoquinolines (3) and pentacyclic-fused pyranoquinoliziniums (4). The structural diversity of pentacyclic-fused pyranoquinoliziniums (4) was expanded by the stepwise synthesis from 3 and 2, allowing the strategic incorporation of electron-donating (OMe and OH) and electron-withdrawing (Cl) substituents on the top and bottom parts of the pyranoquinoliziniums (4). These newly synthesized pyranoquinoliziniums (4) exhibited tunable absorptions (455-532 nm), emissions (520-610 nm), fluorescence lifetime (0.3-5.6 ns), large Stokes shifts (up to 120 nm), and excellent fluorescence quantum yields (up to 0.73) upon adjusting the different substituents. The the unique arrangement of N and O atoms and extended π-conjugation of 4 could cause the relocation of HOMO comparing with our previous quinoliziniums. Importantly, pyranoquinoliziniums (4a-4g and 4i) targeted the mitochondria, while 4h was localized in lysosome. Due to the remarkable photophysical properties and the potential for organelle targeting of the novel class of quinoliziniums, they could be further applied for biological, chemical and material applications.

Lysosome targeting fluorescent probe for NAAA imaging and its applications in the drug development for anti-inflammatory.

N-acylethanolamine acid amidase (NAAA) is a nucleophilic lysosomal cysteine hydrolase, which primarily mediates the hydrolytic inactivation of endogenous palmitoylethanolamide (PEA), which further influences the inflammatory process by regulating peroxisome proliferator-activated receptor-α (PPAR-α). Herein, a novel lysosome (Lyso)-targeting fluorescent probe (i.e., PMBD) was designed and synthesized for detecting endogenous NAAA selectively and sensitively, allowing real-time visual monitoring of endogenous NAAA in living cells. Moreover, PMBD can target Lyso with a high colocalization in Lyso Tracker. Finally, a high-throughput assay method for NAAA inhibitor screening was established using PMBD, and the NAAA-inhibitory effects of 42 anti-inflammatory Traditional Chinese medicines were evaluated. A novel potent inhibitor of NAAA, ellagic acid, was isolated from Cornus officinalis, which can suppress LPS-induced iNOS upregulation and NO production in RAW264.7 cells that display anti-inflammatory activities. PMBD, a novel Lyso-targeting fluorescent probe for visually imaging NAAA, could serve as a useful molecular tool for exploring the physiological functions of NAAA and drug development based on NAAA-related diseases.