Wheat ESCRT-III protein TaSAL1 regulates male gametophyte transmission and controls tillering and heading date.

Charged multivesicular protein 1 (CHMP1) is a member of the endosomal sorting complex required for transport-III (ESCRT-III) complex that targets membrane localized signaling receptors to intralumenal vesicles in the multivesicular body of the endosome and eventually to the lysosome for degradation. Although CHMP1 plays roles in various plant growth and development processes, little is known about its function in wheat. In this study, we systematically analysed the members of the ESCRT-III complex in wheat (Triticum aestivum) and found that their orthologs were highly conserved in eukaryotic evolution. We identified CHMP1 homologous genes, TaSAL1s, and found that they were constitutively expressed in wheat tissues and essential for plant reproduction. Subcellular localization assays showed these proteins aggregated with and closely associated with the endoplasmic reticulum when ectopically expressed in tobacco leaves. We also found these proteins were toxic and caused leaf death. A genetic and reciprocal cross analysis revealed that TaSAL1 leads to defects in male gametophyte biogenesis. Moreover, phenotypic and metabolomic analysis showed that TaSAL1 may regulate tillering and heading date through phytohormone pathways. Overall, our results highlight the role of CHMP1 in wheat, particularly in male gametophyte biogenesis, with implications for improving plant growth and developing new strategies for plant breeding and genetic engineering.

Insights into the function of ESCRT complex and LBPA in ASFV infection.

The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by the endosomal sorting complexes required for transport (ESCRT). The ESCRT machinery is mainly involved in the coordination of membrane dynamics; hence, several viruses exploit this complex and its accessory proteins VPS4 and ALIX for their own benefit. In this work, we found that shRNA-mediated knockdown of VPS4A decreased ASFV replication and viral titers, and this silencing resulted in an enhanced expression of ESCRT-0 component HRS. ASFV infection slightly increased HRS expression but not under VPS4A depletion conditions. Interestingly, VPS4A silencing did not have an impact on ALIX expression, which was significantly overexpressed upon ASFV infection. Further analysis revealed that ALIX silencing impaired ASFV infection at late stages of the viral cycle, including replication and viral production. In addition to ESCRT, the accessory protein ALIX is involved in endosomal membrane dynamics in a lysobisphosphatydic acid (LBPA) and Ca2+-dependent manner, which is relevant for intraluminal vesicle (ILV) biogenesis and endosomal homeostasis. Moreover, LBPA interacts with NPC2 and/or ALIX to regulate cellular cholesterol traffic, and would affect ASFV infection. Thus, we show that LBPA blocking impacted ASFV infection at both early and late infection, suggesting a function for this unconventional phospholipid in the ASFV viral cycle. Here, we found for the first time that silencing of VPS4A and ALIX affects the infection later on, and blocking LBPA function reduces ASFV infectivity at early and later stages of the viral cycle, while ALIX was overexpressed upon infection. These data suggested the relevance of ESCRT-related proteins in ASFV infection.

[Electroacupuncture at "Baihui"（GV20） and "Yongquan"（KI1） improves learning-memory ability of APP/PS1 double transgenic mice by regulating endosomal-lysosomal system].

OBJECTIVE: To explore the mechanism of electroacupuncture（EA） in improving learning-memory ability in Alzheimer's disease （AD） mice from the perspective of endosomal-lysosomal system. METHODS: Male APP/PS1 transgenic mice were randomly divided into model group and EA group （n=10 in each group） and 10 male C57BL/6 wild mice were taken as the normal group. EA （1 Hz/50 Hz, 1 mA） was applied at bilateral "Yongquan"（KI1） and acupuncture was applied at "Baihui" （GV20） for 15 min. The mice of the model and normal groups were subjected to restriction with the same method as those of the EA group for 15 min. The treatment was conducted once every other day for 6 weeks. The spatial learning-memory ability （shown by escape latency of place navigation test and the time of crossing the target platform and total swimming distance in the target quadrant in 1 min of spatial probe test ） was detected by Morris water maze test. The immunoactivity of senile plaques （SP） in the hippocampus tissue was detected by immunohistochemistry. The ultrastructural characters of hippocampal neurons were observed by transmission electron microscope, and the expression levels of Ras-related protein 5 （Rab5）, Ras-related protein 7 （Rab7） and cathepsin D （CTSD） in the hippocampus were detected by Western blot, separately. RESULTS: Compared with the normal group, the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD were significantly increased （P<0.05, P<0.01）, while the number of crossing the original platform and the total swimming distance in the platform quadrant were considerably reduced （P<0.05） in the model group. In contrast to the model group, the EA group had a marked decrease in the escape latency, SP immunoactivity, and protein expression levels of Rab5, Rab7 and CTSD （P<0.05, P<0.01）, and a striking increase in the number of crossing the original platform and the swimming distance in the platform quadrant （P<0.05）. Results of transmission electron microscope showed an accumulation of endosome, lysosome, and endolysosomes in the hippocampal neurons in the model group, which was evidently milder in the EA group. CONCLUSION: EA of GV20 and KI1 can improve the learning-memory ability of AD mice, which may be related to its function in reducing hippocampal Aß deposition and down-regulating endosomal-lysosomal system activity.

Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.

Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.

Biodegradable hollow mesoporous organosilica nanotheranostics (HMONs) as a versatile platform for multimodal imaging and phototherapeutic-triggered endolysosomal disruption in ovarian cancer.

A major impediment in the development of nanoplatform-based ovarian cancer therapy is endo/lysosome entrapment. To solve this dilemma, a hollow mesoporous organosilica-based nanoplatform (HMON@CuS/Gd2O3) with a mild-temperature photothermal therapeutic effect and multimodal imaging abilities was successfully synthesized. HMON@CuS/Gd2O3 exhibited an appropriate size distribution, L-glutathione (GSH)-responsive degradable properties, and high singlet oxygen generation characteristics. In this study, the nanoplatform specifically entered SKOV-3 cells and was entrapped in endo/lysosomes. With a mild near infrared (NIR) power density (.5 W/cm2), the HMON@CuS/Gd2O3 nanoplatform caused lysosome vacuolation, disrupted the lysosomal membrane integrity, and exerted antitumour effects in ovarian cancer. Additionally, our in vivo experiments indicated that HMON@CuS/Gd2O3 has enhanced T1 MR imaging, fluorescence (FL) imaging (wrapping fluorescent agent), and infrared thermal (IRT) imaging capacities. Using FL/MRI/IRT imaging, HMON@CuS/Gd2O3 selectively caused mild phototherapy in the cancer region, efficiently inhibiting the growth of ovarian cancer without systemic toxicity in vivo. Taken together, the results showed that these well-synthesized nanoplatforms are likely promising anticancer agents to treat ovarian cancer and show great potential for biomedical applications.

Evaluation of Phosphorus and Non-Phosphorus Neutral Oligonucleotide Backbones for Enhancing Therapeutic Index of Gapmer Antisense Oligonucleotides.

The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.

Suppression of amyloid-ß secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid.

Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-ß (Aß) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aß in mouse neurons. CLA treatment did not affect the level of ß-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aß protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aß generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aß in neurons.

Specific amino acid supplementation rescues the heart from lipid overload-induced insulin resistance and contractile dysfunction by targeting the endosomal mTOR-v-ATPase axis.

OBJECTIVE: The diabetic heart is characterized by extensive lipid accumulation which often leads to cardiac contractile dysfunction. The underlying mechanism involves a pivotal role for vacuolar-type H+-ATPase (v-ATPase, functioning as endosomal/lysosomal proton pump). Specifically, lipid oversupply to the heart causes disassembly of v-ATPase and endosomal deacidification. Endosomes are storage compartments for lipid transporter CD36. However, upon endosomal deacidification, CD36 is expelled to translocate to the sarcolemma, thereby inducing myocardial lipid accumulation, insulin resistance, and contractile dysfunction. Hence, the v-ATPase assembly may be a suitable target for ameliorating diabetic cardiomyopathy. Another function of v-ATPase involves the binding of anabolic master-regulator mTORC1 to endosomes, a prerequisite for the activation of mTORC1 by amino acids (AAs). We examined whether the relationship between v-ATPase and mTORC1 also operates reciprocally; specifically, whether AA induces v-ATPase reassembly in a mTORC1-dependent manner to prevent excess lipids from entering and damaging the heart. METHODS: Lipid overexposed rodent/human cardiomyocytes and high-fat diet-fed rats were treated with a specific cocktail of AAs (lysine/leucine/arginine). Then, v-ATPase assembly status/activity, cell surface CD36 content, myocellular lipid uptake/accumulation, insulin sensitivity, and contractile function were measured. To elucidate underlying mechanisms, specific gene knockdown was employed, followed by subcellular fractionation, and coimmunoprecipitation. RESULTS: In lipid-overexposed cardiomyocytes, lysine/leucine/arginine reinternalized CD36 to the endosomes, prevented/reversed lipid accumulation, preserved/restored insulin sensitivity, and contractile function. These beneficial AA actions required the mTORC1-v-ATPase axis, adaptor protein Ragulator, and endosomal/lysosomal AA transporter SLC38A9, indicating an endosome-centric inside-out AA sensing mechanism. In high-fat diet-fed rats, lysine/leucine/arginine had similar beneficial actions at the myocellular level as in vitro in lipid-overexposed cardiomyocytes and partially reversed cardiac hypertrophy. CONCLUSION: Specific AAs acting through v-ATPase reassembly reduce cardiac lipid uptake raising the possibility for treatment in situations of lipid overload and associated insulin resistance.

The Discovery of Naringenin as Endolysosomal Two-Pore Channel Inhibitor and Its Emerging Role in SARS-CoV-2 Infection.

The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.

Preclinical modeling of chronic inhibition of the Parkinson's disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo.

The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.

ß-Adrenergic control of sarcolemmal CaV1.2 abundance by small GTPase Rab proteins.

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. ß-Adrenergic receptor (ßAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. ßAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, ßAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented ßAR-activated Ca2+ current augmentation. Moreover, ßAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that ßAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for ßAR-regulation of cardiac CaV1.2.

Antiviral drugs targeting endosomal membrane proteins inhibit distant animal and human pathogenic viruses.

The endocytic pathway is a common strategy that several highly pathogenic viruses use to enter into the cell. To demonstrate the usefulness of this pathway as a common target for the development of broad-spectrum antivirals, the inhibitory effect of drug compounds targeting endosomal membrane proteins were investigated. This study entailed direct comparison of drug effectiveness against animal and human pathogenic viruses, namely Ebola (EBOV), African swine fever virus (ASFV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A panel of experimental and FDA-approved compounds targeting calcium channels and PIKfyve at the endosomal membrane caused potent reductions of entry up to 90% in SARS-CoV-2 S-protein pseudotyped retrovirus. Similar inhibition was observed against transduced EBOV glycoprotein pseudovirus and ASFV. SARS-CoV-2 infection was potently inhibited by selective estrogen receptor modulators in cells transduced with pseudovirus, among them Raloxifen inhibited ASFV with very low 50% inhibitory concentration. Finally, the mechanism of the inhibition caused by the latter in ASFV infection was analyzed. Overall, this work shows that cellular proteins related to the endocytic pathway can constitute suitable cellular targets for broad range antiviral compounds.

Inclusion of IR-820 into Soybean-Phosphatides-Based Nanoparticles for Near-Infrared-Triggered Release and Endolysosomal Escape in HaCaT Keratinocytes at Insignificant Cytotoxic Level.

PURPOSE: The degradation of drugs within endolysosomes has been widely addressed as a cause of poor bioavailability. One of the strategies to allow molecules to escape from a destructive fate is to introduce a photosensitizing moiety into a drug carrier enabling the permeabilization of endosomes and endolysosomes upon irradiation. This paper presents an alternative delivery nanosystem composed of cost-effective soybean phosphatides mixed with IR-820, a near-infrared (NIR) sensitizer, to load various active compounds and trigger an endolysosomal escape with a low cytotoxic effect. METHODS: IR-820-incorporated phosphatides-based nanoparticles were formulated using a thin-film hydration method to encapsulate different molecular probes and a drug model. The nanoparticles were characterized in vitro using dynamic light scattering, transmission electron microscopy, as well as ultraviolet-visible and fluorescence spectroscopy techniques. The NIR-corresponding generation of the photochemical products, the content release, and the cytotoxicity toward the HaCaT keratinocyte cell line were evaluated. The cellular internalization and endolysosomal escape were monitored using a cytochemical marker and fluorescent probes with a colocalization analysis. RESULTS: The IR-820-combined nanoparticles revealed the NIR-triggered changes in the singlet oxygen presence, nanoparticle architecture, and release rate without being cytotoxic. Additionally, the nanoplatform appeared to enhance cellular uptake of the macromolecules. The localization of the cytochemical marker and the colocalization analysis on the fluorescence signals of the encapsulated fluorophore and the lysosome-labeling reporter implied the transient endolysosomal escape of the cargo within the HaCaT cells after NIR irradiation. CONCLUSION: The inclusion of IR-820 into a soybean-phosphatides base ingredient provides NIR responsiveness, particularly the endolysosomal escape of the payload, to the formulated nanoparticles, while preserving the beneficial properties as a drug carrier. This alternative delivery nanomedicine system has future potential to provide high bioavailability of cytosolic drugs utilizing time- and spatial-controllable NIR triggerability as well as the synergistic therapeutic effects with NIR-biomodulation.

Portfolio Targeting Strategy To Realize the Assembly and Membrane Fusion-Mediated Delivery of Gold Nanoparticles to Mitochondria for Enhanced NIR Photothermal Therapies.

Targeting mitochondria has always been a challenging goal for therapeutic nanoparticle agents due to their heterotypic features and size, which usually lead to a lysosome/endosome endocytosis pathway. To overcome this limitation, in this work, a portfolio targeting strategy combining a small targeting molecule with a biomembrane was developed. Modification of small targeting molecule H2N-TPP on gold nanoparticles (GNPs) could not only facilitate the mitochondrial targeting but could also induce gold nanoparticle assembly. Therefore, the GNPs were endowed with good absorption and photothermal conversion abilities in the near-infrared (NIR) region. Meanwhile, a biomimetic strategy was adopted by wrapping the gold nanoparticle assembly (GNA) with cancer cell membranes (CCMs), which helped the GNA enter the prostatic cancer cell via a homotypic membrane-fusion process to avoid being trapped in endosomes/lysosomes. Thereafter, the GNA remaining in the cytoplasm could reach mitochondria more efficiently via guidance from H2N-TPP molecules. This "biomembrane-small molecule" combination targeting process was evidenced by fluorescence microscopy, and the highly efficient photothermal ablation of prostatic tumors in vivo was demonstrated. This portfolio targeting strategy could be extended to various nanodrugs/agents to realize an accurate subcellular targeting efficiency for cancer treatments or cell detections.

Ever-Evolving Identity of Magnetic Nanoparticles within Human Cells: The Interplay of Endosomal Confinement, Degradation, Storage, and Neocrystallization.

Considerable knowledge has been acquired in inorganic nanoparticles' synthesis and nanoparticles' potential use in biomedical applications. Among different materials, iron oxide nanoparticles remain unrivaled for several reasons. Not only do they respond to multiple physical stimuli (e.g., magnetism, light) and exert multifunctional therapeutic and diagnostic actions but also they are biocompatible and integrate endogenous iron-related metabolic pathways. With the aim to optimize the use of (magnetic) iron oxide nanoparticles in biomedicine, different biophysical phenomena have been recently identified and studied. Among them, the concept of a "nanoparticle's identity" is of particular importance. Nanoparticles' identities evolve in distinct biological environments and over different periods of time. In this Account, we focus on the remodeling of magnetic nanoparticles' identities following their journey inside cells. For instance, nanoparticles' functions, such as heat generation or magnetic resonance imaging, can be highly impacted by endosomal confinement. Structural degradation of nanoparticles was also evidenced and quantified in cellulo and correlates with the loss of magnetic nanoparticle properties. Remarkably, in human stem cells, the nonmagnetic products of nanoparticles' degradation could be subsequently reassembled into neosynthesized, endogenous magnetic nanoparticles. This stunning occurrence might account for the natural presence of magnetic particles in human organs, especially the brain. However, mechanistic details and the implication of such phenomena in homeostasis and disease have yet to be completely unraveled.This Account aims to assess the short- and long-term transformations of magnetic iron oxide nanoparticles in living cells, particularly focusing on human stem cells. Precisely, we herein overview the multiple and ever-evolving chemical, physical, and biological magnetic nanoparticles' identities and emphasize the remarkable intracellular fate of these nanoparticles.

Existing highly accumulating lysosomotropic drugs with potential for repurposing to target COVID-19.

Given the speed of viral infection spread, repurposing of existing drugs has been given the highest priority in combating the ongoing COVID-19 pandemic. Only drugs that are already registered or close to registration, and therefore have passed lengthy safety assessments, have a chance to be tested in clinical trials and reach patients quickly enough to help in the current disease outbreak. Here, we have reviewed available evidence and possible ways forward to identify already existing pharmaceuticals displaying modest broad-spectrum antiviral activity which is likely linked to their high accumulation in cells. Several well studied examples indicate that these drugs accumulate in lysosomes, endosomes and biological membranes in general, and thereby interfere with endosomal pathway and intracellular membrane trafficking crucial for viral infection. With the aim to identify other lysosomotropic drugs with possible inherent antiviral activity, we have applied a set of clear physicochemical, pharmacokinetic and molecular criteria on 530 existing drugs. In addition to publicly available data, we have also used our in silico model for the prediction of accumulation in lysosomes and endosomes. By this approach we have identified 36 compounds with possible antiviral effects, also against coronaviruses. For 14 of them evidence of broad-spectrum antiviral activity has already been reported, adding support to the value of this approach. Presented pros and cons, knowledge gaps and methods to identify lysosomotropic antivirals, can help in the evaluation of many drugs currently in clinical trials considered for repurposing to target COVID-19, as well as open doors to finding more potent and safer alternatives.

Antimicrobial Peptide Omiganan Enhances Interferon Responses to Endosomal Toll-Like Receptor Ligands in Human Peripheral Blood Mononuclear Cells.

LL-37 is a cationic antimicrobial peptide and the sole human member of cathelicidins. Besides its bactericidal properties, LL-37 is known to have direct immunomodulatory effects, among which enhancement of antiviral responses via endosomal toll-like receptors (TLRs). Omiganan pentahydrochloride is a synthetic cationic peptide in clinical development. Previously, omiganan was primarily known for its direct bactericidal and antifungal properties. We investigated whether omiganan enhances endosomal TLR responses, similar to LL-37. Human peripheral blood mononuclear cells were treated with endosomal TLR3, -7, -8, and -9 ligands in the presence of omiganan. Omiganan enhanced TLR-mediated interferon-α release. Subsequent experiments with TLR9 ligands showed that plasmacytoid dendritic cells were main contributors to omiganan-enhanced IFN production. Based on this type I interferon-enhancing effect, omiganan may qualify as potential treatment modality for virus-driven diseases. The molecular mechanism by which omiganan enhances endosomal TLR responses remains to be elucidated.

Analysis of Triterpenoid Saponins Reveals Insights into Structural Features Associated with Potent Protein Drug Enhancement Effects.

Plant-based saponins are amphipathic glycosides composed of a hydrophobic aglycone backbone covalently bound to one or more hydrophilic sugar moieties. Recently, the endosomal escape activity of triterpenoid saponins has been investigated as a potentially powerful tool for improved cytosolic penetration of protein drugs internalized by endocytic uptake, thereby greatly enhancing their pharmacological effects. However, only a few saponins have been studied, and the paucity in understanding the structure-activity relationship of saponins imposes significant limitations on their applications. To address this knowledge gap, 12 triterpenoid saponins with diverse structural side chains were screened for their utility as endosomolytic agents. These compounds were used in combination with a toxin (MAP30-HBP) comprising a type I ribosome-inactivating protein fused to a cell-penetrating peptide. Suitability of saponins as endosomolytic agents was assessed on the basis of cytotoxicity, endosomal escape promotion, and synergistic effects on toxins. Five saponins showed strong endosomal escape activity, enhancing MAP30-HBP cytotoxicity by more than 106 to 109 folds. These saponins also enhanced the apoptotic effect of MAP30-HBP in a pH-dependent manner. Additionally, growth inhibition of MAP30-HBP-treated SMMC-7721 cells was greater than that of similarly treated HeLa cells, suggesting that saponin-mediated endosomolytic effect is likely to be cell-specific. Furthermore, the structural features and hydrophobicity of the sugar side chains were analyzed to draw correlations with endosomal escape activity and derive predictive rules, thus providing new insights into structure-activity relationships of saponins. This study revealed new saponins that can potentially be exploited as efficient cytosolic delivery reagents for improved therapeutic drug effects.

The inducible amphisome isolates viral hemagglutinin and defends against influenza A virus infection.

The emergence of drug-resistant influenza type A viruses (IAVs) necessitates the development of novel anti-IAV agents. Here, we target the IAV hemagglutinin (HA) protein using multivalent peptide library screens and identify PVF-tet, a peptide-based HA inhibitor. PVF-tet inhibits IAV cytopathicity and propagation in cells by binding to newly synthesized HA, rather than to the HA of the parental virus, thus inducing the accumulation of HA within a unique structure, the inducible amphisome, whose production from the autophagosome is accelerated by PVF-tet. The amphisome is also produced in response to IAV infection in the absence of PVF-tet by cells overexpressing ABC transporter subfamily A3, which plays an essential role in the maturation of multivesicular endosomes into the lamellar body, a lipid-sorting organelle. Our results show that the inducible amphisomes can function as a type of organelle-based anti-viral machinery by sequestering HA. PVF-tet efficiently rescues mice from the lethality of IAV infection.

Arabidopsis FLYING SAUCER 2 Functions Redundantly with FLY1 to Establish Normal Seed Coat Mucilage.

Following exposure to water, mature Arabidopsis seeds are surrounded by a gelatinous capsule, termed mucilage. The mucilage consists of pectin-rich polysaccharides, which are produced in epidermal cells of the seed coat. Although pectin is a major component of plant cell walls, its biosynthesis and biological functions are not fully understood. Previously, we reported that a transmembrane RING E3 ubiquitin ligase, FLYING SAUCER 1 (FLY1) regulates the degree of pectin methyl esterification for mucilage capsule formation. The Arabidopsis thaliana genome has a single FLY1 homolog, FLY2. In this study, we show that the FLY2 protein functions in mucilage modification together with FLY1. FLY2 was expressed in seed coat epidermal cells during mucilage synthesis, but its expression level was much lower than that of FLY1. While fly2 showed no obvious difference in mucilage capsule formation from wild type, the fly1 fly2 double mutants showed more severe defects in mucilage than fly1 alone. FLY2-EYFP that was expressed under the control of the FLY1 promoter rescued fly1 mucilage, showing that FLY2 has the same molecular function as FLY1. FLY2-EYFP colocalized with marker proteins of Golgi apparatus (sialyltransferase-mRFP) and late endosome (mRFP-ARA7), indicating that as FLY1, FLY2 controls pectin modification by functioning in these endomembrane organelles. Furthermore, phylogenetic analysis suggests that FLY1 and FLY2 originated from a common ancestral gene by gene duplication prior to the emergence of Brassicaceae. Taken together, our findings suggest that FLY2 functions in the Golgi apparatus and/or the late endosome of seed coat epidermal cells in a manner similar to FLY1.