Protein aggregation drives major neurodegenerative diseases, yet most computational predictors collapse assembly into static risk scores and do not resolve the distinct structural determinants of nucleation and elongation. Here, we present SKALE 2.0, a phase-resolved geometric deep learning framework that represents proteins as multimodal structural graphs and learns mutation-induced aggregation phenotypes directly from three-dimensional topology. Across SOD1, TDP-43, MAPT, and PRNP, SKALE 2.0 recovered a conserved latent transition from nucleation to elongation while resolving distinct mutation-specific phase sensitivities. Representative protein language model, AlphaFold-derived feature, and non-phase-aware structural baselines failed to recover both phase-dependent mutation modulation and phase separability, indicating that explicit phase conditioning is essential. The learned geometry showed that nucleation is preferentially coupled to buried hydrophobic perturbations, whereas elongation is shaped by solvent-accessible interfaces that support fibril propagation. This framework explains how pathogenic variants can remain globally folded yet acquire aggregation competence through localized structural rewiring. Recombinant SOD1 experiments validated predicted suppressor, enhancer, and phase-switch mutations, demonstrating that initiation and propagation can be tuned independently. SKALE 2.0 links atomic topology to phase-specific assembly kinetics and enables a constraint-aware design of aggregation suppressors.

Prodrug strategies are widely used to improve the efficacy, selectivity, and safety of therapeutic agents. Among stimulus-responsive approaches, enzyme-triggered activation has emerged as a particularly attractive method because it can couple drug release to disease-associated biochemical activity. Cathepsins, a family of lysosomal proteases including 15 types, implicated in cancer, inflammation, and other pathological processes, have become important enzymatic triggers in this area. Most cathepsin-responsive systems focused on cathepsin B, whose elevated activity in tumors and favorable intracellular localization have supported the development of peptide-cleavable prodrugs, from small-molecule prodrugs to various drug conjugates. More recent studies have extended this concept beyond cathepsin B to other cathepsins such as L, E, and S, highlighting a shift toward substrate-selective design and improved biological precision. Meanwhile, comparative studies and unnatural amino acid engineering have emphasized that successful cathepsin targeting depends not only on enzyme overexpression but also on substrate specificity, circulation stability, intracellular trafficking, and productive payload release. This review summarizes the recent advances in cathepsin-activated prodrugs with a focus on the novel therapeutic applications and major challenges limiting translation, providing insight for the future development of cathepsin-activated prodrugs.

Protein aggregation drives proteinopathies ranging from ALS to systemic amyloidosis, yet the multiscale determinants bridging sequence, structure, and kinetics remain elusive. We present SKALE, an interpretable machine learning framework that integrates sequence motifs, AlphaFold-derived structural descriptors, and experimental kinetics to decode aggregation mechanisms. SKALE identifies latent hotspots that evade conventional tools and matches high-performing neural baselines while preserving computational efficiency. In ALS-linked SOD1 G86R, the model isolates a risk region at residues 72-91 where preserved beta-sheet geometry coincides with weakened hydrogen bonding to drive nucleation. Similarly, analysis of TDP-43 S332N reveals that a locally unwound helix increases surface exposure, a prediction validated by showing that targeted deletion of model-identified regions significantly reduces cellular aggregation. The framework generalizes to Tau P301L and PRNP variants where it uncovers distal aggregation-prone regions to discriminate pathogenic drivers from neutral mutations. Interpretability analysis further disentangles global from mutation-local mechanisms to reveal that beta-sheet propensity acts as a shared determinant while hydrogen bond dynamics define specific routes to nucleation. These findings establish SKALE as a scalable, disease-agnostic engine that combines high-fidelity prediction with biophysical resolution to decode the molecular logic of misfolding and guide therapeutic design.

Inflammation is a hallmark of amyotrophic lateral sclerosis (ALS), particularly in cases with SOD1 mutations. Using integrative transcriptomics, we analyzed gene expression changes in mouse models throughout progression, human induced-pluripotent stem cells (hiPSCs), and post-mortem spinal cord tissue from ALS patients. We identified a conserved upregulation of interferon (IFN) genes and IFN-stimulating genes (ISGs) in both mouse models and human ALS, with a predominance Type I IFNs (IFN-α/β) in mice and Type II IFNs (IFN-γ) in humans. In mouse models, we observed robust and sustained upregulation of Type I and II ISGs, including ATF3, beginning at disease onset stage and persisting throughout disease progression. Single-cell transcriptomics further pinpointed vascular endothelial cells as a major source of ISGs. Furthermore, we found that the STING-TBK1 axis is essential for the induction of Type II ISGs in ALS, as its deletion impaired their expression. Our study uncovers a conserved ISGs signature across ALS models and patients, highlighting the potential role of innate immune activation in ALS pathogenesis. These findings suggest that ISGs may serve as potential biomarkers and therapeutic targets for ALS. Integrative transcriptomics in amyotrophic lateral sclerosis models and patients reveals a conserved type I and type II interferon gene signature driven by STING-mediated innate immune activation.

Inspired by the "magic bullet" concept proposed over a century ago, antibody-drug conjugates (ADCs) are developed to enhance cancer therapy by linking monoclonal antibodies to a cytotoxic payload, aiming to overcome the limitations of conventional chemotherapy. To date, 17 ADCs have received regulatory approval for treating both hematologic and solid tumors. Despite their clinical success, developing ADCs with optimal therapeutic potential remains challenging. While selecting the appropriate antibody and cytotoxin is crucial, the linker plays a pivotal role in determining plasma stability and efficient payload release at the tumor site. Over the past decade, advances in linker technology have significantly improved the pharmacokinetics, efficacy, and toxicity profiles of ADCs. This review provides an overview of clinically validated linkers and recent innovations in linker design, focusing on drug release triggers, bioconjugation strategies, the impact of spacers on hydrophilicity, traceless drug release, and linker architecture, as well as a discussion of the bystander effect, offering insights for the rational design of next-generation ADCs.

Nucleobases serve as essential molecular frameworks present in both natural and synthetic compounds that exhibit notable antiviral activity. Through molecular modifications, novel nucleobase-containing drugs (NCDs) have been developed, exhibiting enhanced antiviral activity against a wide range of viruses, including the recently emerged SARS‑CoV‑2. This article provides a detailed examination of the significant advancements in NCDs from 2015 till current, encompassing various aspects concerning their mechanisms of action, pharmacology and antiviral properties. Additionally, the article discusses antiviral prodrugs relevant to the scope of this review. It fills in the knowledge gap by examining the structure–activity relationship and trend of NCDs as therapeutics against a diverse range of viral diseases, either as approved drugs, clinical candidates or as early-stage development prospects. Moreover, the article highlights on the status of this field of study and addresses the prevailing limitations encountered.

Neuroinflammation exacerbates the progression of SOD1-driven amyotrophic lateral sclerosis (ALS), although the underlying mechanisms remain largely unknown. Herein, we demonstrate that misfolded SOD1 (SOD1(Mut))-causing ALS results in mitochondrial damage, thus triggering the release of mtDNA and an RNA:DNA hybrid into the cytosol in an mPTP-independent manner to activate IRF3- and IFNAR-dependent type I interferon (IFN-I) and interferon-stimulating genes. The neuronal hyper-IFN-I and pro-inflammatory responses triggered in ALS-SOD1(Mut) were sufficiently robust to cause a strong physiological outcome in vitro and in vivo. cGAS/DDX41-STING-signaling is amplified in bystander cells through inter-neuronal gap junctions. Our results highlight the importance of a common DNA-sensing pathway between SOD1 and TDP-43 in influencing the progression of ALS.

During viral infection, the dynamic virus-host relationship is constantly in play. Many cellular proteins, such as RNA-binding proteins (RBPs), have been shown to mediate antiviral responses during viral infection. Here, we report that the RBP FUS/TLS (fused in sarcoma/translocated in liposarcoma) acts as a host-restricting factor against infection with coxsackievirus B3 (CVB3). Mechanistically, we found that deletion of FUS leads to increased viral RNA transcription and enhanced internal ribosome entry site (IRES)-driven translation, with no apparent impact on viral RNA stability. We further demonstrated that FUS physically interacts with the viral genome, which may contribute to direct inhibition of viral RNA transcription/translation. Moreover, we identified a novel function for FUS in regulating host innate immune response. We show that in the absence of FUS, gene expression of type I interferons and proinflammatory cytokines elicited by viral or bacterial infection is significantly impaired. Emerging evidence suggests a role for stress granules (SGs) in antiviral innate immunity. We further reveal that knockout of FUS abolishes the ability to form SGs upon CVB3 infection or poly(I·C) treatment. Finally, we show that, to avoid FUS-mediated antiviral response and innate immunity, CVB3 infection results in cytoplasmic mislocalization and cleavage of FUS through the enzymatic activity of viral proteases. Together, our findings in this study identify FUS as a novel host antiviral factor which restricts CVB3 replication through direct inhibition of viral RNA transcription and protein translation and through regulation of host antiviral innate immunity.IMPORTANCE Enteroviruses are common human pathogens, including those that cause myocarditis (coxsackievirus B3 [CVB3]), poliomyelitis (poliovirus), and hand, foot, and mouth disease (enterovirus 71). Understanding the virus-host interaction is crucial for developing means of treating and preventing diseases caused by these pathogens. In this study, we explored the interplay between the host RNA-binding protein FUS/TLS and CVB3 and found that FUS/TLS restricts CVB3 replication through direct inhibition of viral RNA transcription/translation and through regulation of cellular antiviral innate immunity. To impede the antiviral role of FUS, CVB3 targets FUS for mislocalization and cleavage. Findings from this study provide novel insights into interactions between CVB3 and FUS, which may lead to novel therapeutic interventions against enterovirus-induced diseases.

Mucosal-associated invariant T (MAIT) cells are a unique subset of innate-like T cells that bridge between innate and adaptive immunity. MAIT cells act like a 'biliary firewall' protecting the epithelial lining of the liver against pathogenic intruders. MAIT1 and MAIT17 subsets respond rapidly to pathogenic presence both in the liver as well as in the peripheral circulation. In addition to chronic hepatitis B virus (HBV) infection, MAIT cells also appear to serve as potential therapeutic targets in several other chronic ailments. Evidence indicates that MAIT cells have tissue repair functions also paving way for fibrotic changes during chronic HBV infection. Observations also suggest that HBV-hepatitis delta virus (HDV) co-infection disease progression is closely associated with loss of MAIT cells. Furthermore, reduction in the number of hepatic MAIT cells in patients with cirrhotic non-alcoholic fatty liver disease and HBV-associated primary liver cancer has also been reported. Given their concrete role against HBV disease progression, and has also become evident that the tumor microenvironment can cause functional impairment of MAIT cells. Here, we reviewed the protective and the pathological role of MAIT cells in chronic HBV infection and certain other related medical conditions based on the understanding that an optimal functioning of the MAIT cell arsenal is key to a "host-friendly" immune defense against HBV disease progression.

The ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understand betacoronaviruses as a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected over 50 million individuals' worldwide with more than ∼1 million fatalities. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks urge for better understanding of viral subversion of autophagy. Using MHV-A59 as a model betacoronavirus, time-course infections revealed significant loss in the protein level of ULK1, a canonical autophagy-regulating kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target ULK1, we conducted in-vitro and cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PLpro). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PLpro recognition sequence (LGGG) after G499, separating its N-terminal kinase domain from a C-terminal substrate recognition region. Over-expression of SARS-CoV-2 PLpro is sufficient to impair starvation-induced autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PLpro of betacoronaviruses induces viral pathogenesis by targeting cellular autophagy.

Severe acute respiratory syndrome coronavirus-2 is the etiological agent of the ongoing pandemic of coronavirus disease-2019, a multi-organ disease that has triggered an unprecedented global health and economic crisis. The virally encoded 3C-like protease (3CLpro ), which is named after picornaviral 3C protease (3Cpro ) due to their similarities in substrate recognition and enzymatic activity, is essential for viral replication and has been considered as the primary drug target. However, information regarding the cellular substrates of 3CLpro and its interaction with the host remains scarce, though recent work has begun to shape our understanding more clearly. Here we summarized and compared the mechanisms by which picornaviruses and coronaviruses have evolved to evade innate immune surveillance, with a focus on the established role of 3Cpro in this process. Through this comparison, we hope to highlight the potential action and mechanisms that are conserved and shared between 3Cpro and 3CLpro . In this review, we also briefly discussed current advances in the development of broad-spectrum antivirals targeting both 3Cpro and 3CLpro .

Enteroviruses (EVs) usurp the host autophagy pathway for pro-viral functions; however, the consequence of EV-induced diversion of autophagy on organelle quality control is poorly defined. Using coxsackievirus B3 (CVB3) as a model EV, we explored the interplay between EV infection and selective autophagy receptors, i.e., Tax1-binding protein 1/TRAF6-binding protein (T6BP), optineurin (OPTN), and nuclear dot 10 protein 52 (NDP52), known to be involved in regulating the clearance of damaged mitochondria, a process termed as mitophagy. Following CVB3 infection, we showed significant perturbations of the mitochondrial network coincident with degradation of the autophagy receptor protein T6BP, similar phenomenon to what we previously observed on NDP52. Notably, protein levels of OPTN are not altered during early infection and slightly reduced upon late infection. Cell culture studies revealed that T6BP degradation occurs independent of the function of host caspases and viral proteinase 3C, but requires the proteolytic activity of viral proteinase 2A. Further investigation identified the cleavage site on T6BP after the amino acid 621 that separates the C-terminal ubiquitin-binding domain from the other functional domains at the N-terminus. Genetic silencing of T6BP and OPTN results in the attenuation of CVB3 replication, suggesting a pro-viral activity for these two proteins. Finally, functional assessment of cleaved fragments from NDP52 and T6BP revealed abnormal binding affinity and impaired capacity to be recruited to depolarized mitochondria. Collectively, these results suggest that CVB3 targets autophagy receptors to impair selective autophagy.

Coxsackievirus B3 (CVB3) is a prevalent etiological agent for viral myocarditis and neurological disorders, particularly in infants and young children. Virus-encoded proteinases have emerged as cytopathic factors that contribute to disease pathogenesis in part through targeting the cellular recycling machinery of autophagy. Although it is appreciated that CVB3 can usurp cellular macroautophagy/autophagy for pro-viral functions, the precise mechanisms by which viral proteinases disrupt autophagy remain incompletely understood. Here we identified TFEB (transcription factor EB), a master regulator of autophagy and lysosome biogenesis, as a novel target of CVB3 proteinase 3 C. Time-course infections uncovered a significant loss of full-length TFEB and the emergence of a lower-molecular mass (similar to 63 kDa) fragment. Cellular and in vitro cleavage assays revealed the involvement of viral proteinase 3 C in the proteolytic processing of TFEB, while site-directed mutagenesis identified the site of cleavage after glutamine 60. Assessment of TFEB transcriptional activity using a reporter construct discovered a loss of function of the cleavage fragment despite nuclear localization and retaining of the ability of DNA and protein binding. Furthermore, we showed that CVB3 infection was also able to trigger cleavage-independent nuclear translocation of TFEB that relied on the serine-threonine phosphatase PPP3/calcineurin. Finally, we demonstrated that both TFEB and TFEB [Delta 60] serve roles in viral egress albeit through differing mechanisms. Collectively, this study reveals that CVB3 targets TFEB for proteolytic processing to disrupt host lysosomal function and enhance viral infection.

Genetic analyses of patients with amyotrophic lateral sclerosis (ALS) have revealed a strong association between mutations in genes encoding many RNA-binding proteins (RBPs), includingTARDBP,FUS,hnRNPA1,hnRNPA2B1,MATR3,ATXN2,TAF15,TIA-1, andEWSR1, and disease onset/progression. RBPs are a group of evolutionally conserved proteins that participate in multiple steps of RNA metabolism, including splicing, polyadenylation, mRNA stability, localization, and translation. Dysregulation of RBPs, as a consequence of gene mutations, impaired nucleocytoplasmic trafficking, posttranslational modification (PTM), aggregation, and sequestration by abnormal RNA foci, has been shown to be involved in neurodegeneration and the development of ALS. While the exact mechanism by which dysregulated RBPs contribute to ALS remains elusive, emerging evidence supports the notion that both a loss of function and/or a gain of toxic function of these ALS-linked RBPs play a significant role in disease pathogenesis through facilitating abnormal protein interaction, causing aberrant RNA metabolism, and by disturbing ribonucleoprotein granule dynamics and phase transition. In this review article, we summarize the current knowledge on the molecular mechanism by which RBPs are dysregulated and the influence of defective RBPs on cellular homeostasis during the development of ALS. The strategies of ongoing clinical trials targeting RBPs and/or relevant processes are also discussed in the present review.

Coxsackievirus B3 (CVB3) is a single-stranded positive RNA virus that usurps cellular machinery, including the evolutionarily anti-viral autophagy pathway, for productive infections. Despite the emergence of double-membraned autophagosome-like vesicles during CVB3 infection, very little is known about the mechanism of autophagy initiation. In this study, we investigated the role of established autophagy factors in the initiation of CVB3-induced autophagy. Using siRNA-mediated gene-silencing and CRISPR-Cas9-based gene-editing in culture cells, we discovered that CVB3 bypasses the ULK1/2 and PI3K complexes to trigger autophagy. Moreover, we found that CVB3-induced LC3 lipidation occurred independent of WIPI2 and the transmembrane protein ATG9 but required components of the late-stage ubiquitin-like ATG conjugation system including ATG5 and ATG16L1. Remarkably, we showed the canonical autophagy factor ULK1 was cleaved through the catalytic activity of the viral proteinase 3C. Mutagenesis experiments identified the cleavage site of ULK1 after Q524, which separates its N-terminal kinase domain from C-terminal substrate binding domain. Finally, we uncovered PI4KIIIβ (a PI4P kinase), but not PI3P or PI5P kinases as requisites for CVB3-induced LC3 lipidation. Taken together, our studies reveal that CVB3 initiates a non-canonical form of autophagy that bypasses ULK1/2 and PI3K signaling pathways to ultimately converge on PI4KIIIβ- and ATG5–ATG12–ATG16L1 machinery.

Host nucleases are implicated in antiviral response through the processing of pathogen-derived nucleic acids. Among many host RNases, decapping enzymes DCP1 and 2, and 5′→3′ exonuclease XRN1, which are components of the RNA decay machinery, have been extensively studied in prokaryotes, plants, and invertebrates but less so in mammalian systems. As a result, the implication of XRN1 and DCPs in viral replication, in particular, the spatio-temporal dynamics during RNA viral infections remains elusive. Here, we highlight that XRN1 and DCPs play a critical role in limiting several groups of RNA viral infections. This antiviral activity was not obvious in wild-type cells but clearly observed in type I interferon (IFN-I)-deficient cells. Mechanistically, infection with RNA viruses induced the enrichment of XRN1 and DCPs in viral replication complexes (vRCs), hence forming distinct cytoplasmic aggregates. These aggregates served as sites for direct interaction between XRN1, DCP1/2, and viral ribonucleoprotein that contains viral RNA (vRNA). Although these XRN1-DCP1/2-vRC-containing foci resemble antiviral stress granules (SGs) or P-body (PB), they did not colocalize with known SG markers and did not correlate with critical PB functions. Furthermore, the presence of 5′ mono- and 5′ triphosphate structures on vRNA was not required for the formation of XRN1-DCP1/2-vRC-containing foci. On the other hand, single-, double-stranded, and higher-ordered vRNA species play a role but are not deterministic for efficient formation of XRN1-DCP1/2 foci and consequent antiviral activity in a manner proportional to RNA length. These results highlight the mechanism behind the antiviral function of XRN1-DCP1/2 in RNA viral infections independent of IFN-I response, protein kinase R and PB function.

PerspectivesThe Pathophysiology of COVID-19 and SARS-CoV-2 InfectionConsideration of Pannexin 1 channels in COVID-19 pathology and treatmentLeigh Anne Swayne, Scott R. Johnstone, Chen Seng Ng, Juan C. Sanchez-Arias, Miranda E. Good, Silvia Penuela, Alexander W. Lohman, Abigail G. Wolpe, Victor E. Laubach, Michael Koval, and Brant E. IsaksonLeigh Anne SwayneDivision of Medical Sciences, University of Victoria, Victoria, British Columbia, Canada, Scott R. JohnstoneFralin Biomedical Research Institute at Virginia Tech Carilion Center for Heart and Reparative Medicine Research, Virginia Tech, Roanoke, VirginiaDepartment of Biological Sciences, Virginia Tech, Roanoke, Virginia, Chen Seng NgDepartment of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, CanadaCentre for Heart Lung Innovation, St. Paul's Hospital, University of British Columbia, Vancouver, British Columbia, Canada, Juan C. Sanchez-AriasDivision of Medical Sciences, University of Victoria, Victoria, British Columbia, Canada, Miranda E. GoodMolecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts, Silvia PenuelaDepartment of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada, Alexander W. LohmanDepartment of Cell Biology and Anatomy, University of Calgary, Calgary, Alberta, CanadaHotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, Abigail G. WolpeRobert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VirginiaDepartment of Cell Biology, University of Virginia School of Medicine, Charlottesville, Virginia, Victor E. LaubachDepartment of Surgery, University of Virginia School of Medicine, Charlottesville, VirginiaDepartment of Molecular Physiology and Biophysics, University of Virginia School of Medicine, Charlottesville, Virginia, Michael KovalDepartment of Medicine, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Emory University School of Medicine, Atlanta, Georgia, and Brant E. IsaksonRobert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VirginiaDepartment of Molecular Physiology and Biophysics, University of Virginia School of Medicine, Charlottesville, VirginiaPublished Online:30 Jun 2020https://doi.org/10.1152/ajplung.00146.2020This is the final version - click for previous versionMoreSectionsPDF (299 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat INTRODUCTIONPannexin 1 (PANX1) is a ubiquitously expressed, channel-forming protein found in a number of tissues throughout the body (e.g., lung, vasculature, liver, central nervous system, immune system) that is important in many key physiological and immune responses (18, 55). PANX1 channels passively flux ATP (predominantly), multiple metabolites, and likely other small anions (37, 39). PANX1 channels regulate inflammation and host responses to several pathogens, including viruses (36, 42, 53). While there is currently no evidence suggesting novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and PANX1 directly interact, there is an urgent need for therapeutic strategies, especially those targeting the hyperinflammation and cytokine storm that occurs in severe cases of COVID-19 (27, 41). Here we argue that PANX1, and drugs known to target PANX1 (including the FDA-approved drug probenecid), should be the focus of further investigation in the context of SARS-CoV-2 infection and its associated pathology in COVID-19 patients.REGULATION OF INFLAMMATION BY PANX1 IN THE CONTEXT OF COVID-19COVID-19 patients frequently present with hypoxemia and dyspnea requiring supportive oxygen therapy before reaching a more severe hyperinflammatory phase of the disease and acute respiratory distress syndrome (ARDS) (4, 11, 26). Control of the early phase of innate immunity, enabling a productive adaptive immune response, is critical to ensure patient recovery (56). It has been suggested that immunosuppression treatment for COVID-19 patients displaying hyperinflammation could limit disease progression as well as limit viral entry (38, 65). There are several lines of evidence demonstrating that PANX1 channel opening (and release of ATP) enhances inflammatory responses, including in the systemic endothelium (lung microvasculature), lung epithelium, olfactory epithelium, and the parenchyma of several tissues throughout the body (13, 17, 22, 29, 31, 35, 51). Multiple studies have shown that PANX1 signaling exacerbates inflammatory responses through: being activated and enhanced by TNFα-receptor signaling, being implicated in the inflammasome, involvement in leukocyte recruitment, and playing a role in the production and secretion of proinflammatory cytokines such as IL-1β and IL-6 by endothelial cells and other cell types (12, 22, 24, 35, 41, 51, 63). Given that disruption (both deletion and inhibition) of PANX1 in endothelial cells significantly reduces inflammation in several injury models (22, 28, 51, 59), PANX1 represents a potential target in reducing inflammatory burden and the damaging effects of the cytokine storm in COVID-19 patients. With intense vascular inflammation observed in severe cases of SARS-CoV-2 infection (14, 66), effective treatments to dampen hyperinflammation represent an urgent treatment need.Of particular interest to SARS-CoV-2 infection, endothelial PANX1 has been shown to play a key role in regulating lung vascular inflammation and edema in response to ischemia/reperfusion injury (51). Control of pulmonary edema is crucial in limiting the severity of ARDS (58). In this light, TRPV4 channels in the alveolo-capillary unit were recently proposed to be a pharmacologically tractable target for treatment of ARDS associated with COVID-19 (30). In addition to direct control of lung barrier function by TRPV4 inhibitors, there is evidence that TRPV4 channels could induce PANX1 channel activity (45, 49, 50), suggesting that PANX1 inhibitors might improve the efficacy of TRPV4 channel inhibitors. Defining the molecular basis for coordinated regulation of TRPV4 and PANX1 channels should facilitate the design of therapeutic approaches attenuating this signaling axis in treatment of COVID-19.In addition to its general role in inflammatory signaling, PANX1 (either directly or pharmacologically) has been implicated in host responses to viral infection and regulation of virus life cycle [e.g., human immunodeficiency virus (HIV), hepatitis B, influenza, vesicular stomatitis virus (VSV), etc.]. PANX1 is expressed in key cells and tissues targeted by SARS-CoV-2, including airway epithelium (46), lung endothelium (34, 51), and neurons (7) (as well as many cell types and tissues throughout the body). In the case of HIV (36, 42, 53), ATP release via PANX1 channels following viral binding stimulates purinergic signaling pathways that enhance viral binding, uptake, and replication. Extracellular ATP has also been linked to viral infection and sequelae: it triggers HIV-1 release from cells (23), is released from cells following VSV infection (64), and is linked to ARDS associated with adenoviral infection (32). How extracellular ATP and purinergic receptors generally regulate entry of viruses has not yet been elucidated, and no links have yet been made between extracellular ATP and coronaviruses. Coronavirus membrane fusion can occur at the plasma membrane or at endosomes (25, 54, 62). Coronaviruses have been shown to enter cells via macropinocytosis, a clathrin- and caveolin-independent process (20, 57). Notably, elevated extracellular ATP can also trigger interactions between PANX1 and P2X7 receptors and their internalization to endosomes through a clathrin- and caveolin-independent process reminiscent of micropinocytosis (6, 8). If direct links between PANX1 and the SARS-CoV-2 life cycle are identified, targeting PANX1 could help mitigate the significant viral titers observed with COVID-19 that result in endothelial damage and neuronal tissue accumulation (33, 44, 68).The above snapshot of PANX1 regulation of inflammatory cascades and viral pathologies supports the need for further study to explore potential direct links to COVID-19. From a therapeutics standpoint for COVID-19, PANX1 also has some intriguing preexisting ties and potential. For instance, nucleotide antiviral drugs, of which remdesivir has shown some effectiveness, may exhibit anti-PANX1 activity. Moreover, probenecid is an FDA-approved drug that blocks PANX1, often recapitulating the effects of PANX1 deletion. Given the evidence described below, we argue PANX1 blockers should be considered in COVID-19 preclinical drug repurposing studies.PANX1 AND NUCLEOTIDE ANALOG ANTIVIRALSNucleotide analog antivirals are designed to compete for incorporation into newly synthesized viral nucleic acid chains thereby disrupting virus life cycles. In preliminary analyses of ongoing clinical trials (1, 10), remdesivir, first described in the treatment of Ebola, has shown modest effects in reducing time to recovery (2). With respect to PANX1, tenofovir (not under consideration for COVID-19 but a nucleotide analog antiviral), used in the treatment of hepatitis B and HIV, inhibited PANX1-mediated ATP release in a mouse macrophage cell line (RAW264.7 cells) and a human liver cell line (HepG2 cells) (19). It is possible the inhibitory action of tenofovir on PANX1 could be via an intracellular mechanism, due to the fact that the drug is metabolized into a nucleotide analog inside the cell. However, high concentrations of extracellular ATP inhibit PANX1 and lead to PANX1 internalization (8), raising another possibility: that the active form of the drug might somehow be released into the extracellular space and block the channel from an external site, like ATP. In light of these prior findings, it could be valuable to determine whether, like tenofovir, remdesivir also impacts both PANX1 channel activity and possibly PANX1-associated inflammatory signaling in COVID-19. This work would be facilitated by the recent advances in our understanding of the PANX1 structure, including identification of key extracellular regulatory residues (16, 39). Remdesivir's effects on PANX1 channels are not known but would merit investigation in light of the blocking effect of the related drug, tenofovir, and the established role of PANX1 in inflammation and regulation of virus life cycles.REPURPOSED FDA-APPROVED DRUGS THAT BLOCK PANX1 FOR COVID-19 TREATMENT?Probenecid (commercially known as Probalan, Benemid, or Benuryl) is an FDA-approved treatment for gout that is also a well-established PANX1 inhibitor (61). Influenza A viral infection and lung viral load were attenuated following probenecid treatment both in vitro and in vivo (43). Probenecid also decreased inflammasome-dependent IL-1β secretion from macrophages in vitro (15), reduced the inflammatory response in sepsis (55), and suppressed hyperinflammation resulting from severe influenza A infection in mice (48). Additionally, probenecid treatment lowered the required dose of another antiviral medication, oseltamivir (43), likely due to probenecid's ability to increase plasma levels of the antiviral drug (47). Note that probenecid also inhibits P2X7 receptors (3), which is also likely to contribute to the antiviral and anti-inflammatory activity. Probenecid has also been shown to have a protective effect in ischemia/reperfusion injury by inhibiting secretion of the lysosomal cathepsin proteases (60). This suggests a mechanism of protection from SARS-CoV-2 infection, since cathepsins promote coronavirus infection through proteolytic cleavage of the spike protein (67). Thus, considering probenecid is relatively well tolerated, has demonstrated action on viral infection-associated inflammation, and decreases required doses of other drugs, it could be of interest to investigate its potential use for COVID-19.Another PANX1 channel-inhibiting FDA-approved drug with potential for the treatment of COVID-19 is spironolactone, an aldosterone antagonist used initially as a diuretic for the treatment of high blood pressure (21). In addition to its potential benefit as a PANX1 blocker in the context of SARS-CoV-2 infection, it was recently suggested that spironolactone may be useful in COVID-19-associated ARDS patients with hypertension (9). It was postulated that spironolactone might selectively increase plasma levels of the spike protein receptor ACE2 (27, 52), increasing the proportion of circulating to lung-endothelial cell-membrane-associated ACE2 levels (thereby minimizing lung infection), as a safer mechanism of action than ACE inhibitors that target cell-bound ACE2 (9). Further investigation is needed to determine whether this is the case and to determine whether there are benefits of the PANX1-inhibiting action of spironolactone in the context of COVID-19.A POTENTIAL FOR THE DIRECT TARGETING OF PANX1 CHANNELSFinally, if PANX1 is found to be involved in the primary regulation of SARS-CoV-2 infectivity and inflammatory responses, it may also be worth considering direct targeting of the channel functions. Currently there are no FDA-approved PANX1-specific blockers, although a PANX1-specific inhibitor peptide, pannexin intracellular loop 2 peptide (PxIL2P), has shown promise in reducing inflammatory responses in vitro and in vivo (35, 63). PxIL2P contains a short mimetic sequence for the IL2 region of PANX1 attached to an HIV-TAT transactivation protein (5) that binds to the second intracellular loop of PANX1 and blocks channel release of ATP, altering intracellular Ca2+ flux (35, 63). Using PxIL2P in cultured endothelial cells blocks PANX1-regulated expression and release of cytokines including IL-1β and CxCL10 and limits monocyte adhesion in the vasculature (35, 63). Thus direct targeting of the PANX1 channel may have functionality in reducing SARS-CoV-2 infectivity and vascular inflammatory responses in COVID19 patients.CONCLUSIONAlthough there are currently no direct lines of evidence linking PANX1 to COVID-19, the central role of PANX1 in regulating inflammation, and more generally viral infection, provides a rationale supporting preclinical investigation of PANX1 and repurposing of approved PANX1-targeting drugs like probenecid as potential treatments. A summary of our perspective is illustrated in Fig. 1. We postulate that evaluating current COVID-19 treatment protocols for their effects on PANX1 may lead to improved combination therapeutic approaches by including specific PANX1 inhibitors as part of a treatment regimen.Fig. 1.Possible roles for Pannexin 1 (PANX1) in COVID-19 pathology. Pannexin intracellular loop 2 peptide (PxIL2P) could potentially be commercialized for specific PANX1 inhibition of inflammatory cues (e.g., IL-1β). Probenecid, an FDA-approved drug used in the treatment of gout, could help dampen the hyperinflammation observed in COVID-19 and could also have an impact on the life cycle of the virus. In addition to affecting the life cycle of the virus, remdesivir could potentially impact inflammation through blocking PANX1.Download figureDownload PowerPointGRANTSSupport for researching and writing this Perspective was from the Canadian Institutes of Health Research (MOP142215) and from the Natural Sciences and Engineering Research Council (RGPIN-2017-03889) (L.A.S.), American Heart Association Career Development Award 19CDA34630036 (S.R.J.), as well as NIH R00-HL143165 (M.E.G.), NIH R01-HL137112 (M.K. and B.E.I.), NIH R01-HL120840 (B.E.I.), and NIH R01-AA025854 (M.K.).DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the authors.AUTHOR CONTRIBUTIONSL.A.S. and B.E.I. conceived and designed research; L.A.S., S.R.J., C.S.N., J.C.S.-A., M.E.G., S.P., A.W.L., A.G.W., V.E.L., M.K., and B.E.I. analyzed data; L.A.S. and B.E.I. interpreted results of experiments; L.A.S. and B.E.I. drafted manuscript; L.A.S., S.R.J., C.S.N., J.C.S.-A., M.E.G., S.P., A.W.L., A.G.W., V.E.L., M.K., and B.E.I. edited and revised manuscript; L.A.S., S.R.J., C.S.N., J.C.S.-A., M.E.G., S.P., A.W.L., A.G.W., V.E.L., M.K., and B.E.I. approved final version of manuscript.REFERENCES1. 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We recently discovered that coxsackievirus B3 (CVB3) is a potent oncolytic virus against KRAS mutant lung adenocarcinoma. Nevertheless, the evident toxicity restricts the use of wild-type (WT)-CVB3 for cancer therapy. The current study aims to engineer the CVB3 to decrease its toxicity and to extend our previous research to determine its safety and efficacy in treating TP53/RB1 mutant small-cell lung cancer (SCLC). A microRNA-modified CVB3 (miR-CVB3) was generated via inserting multiple copies of tumor-suppressive miR-145/miR-143 target sequences into the viral genome. In vitro experiments revealed that miR-CVB3 retained the ability to infect and lyse KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC cells, but with a markedly reduced cytotoxicity toward cardiomyocytes. In vivo study using a TP53/RB1-mutant SCLC xenograft model demonstrated that a single dose of miR-CVB3 via systemic administration resulted in a significant tumor regression. Most strikingly, mice treated with miR-CVB3 exhibited greatly attenuated cardiotoxicities and decreased viral titers compared to WT-CVB3-treated mice. Collectively, we generated a recombinant CVB3 that is powerful in destroying both KRAS mutant lung adenocarcinoma and TP53/RB1-mutant SCLC, with a negligible toxicity toward normal tissues. Future investigation is needed to address the issue of genome instability of miR-CVB3, which was observed in ~40% of mice after a prolonged treatment.

In conjunction with the development of genome-wide technology, numerous studies have revealed the importance of regulatory mechanisms to avoid the onset of autoimmunity. In this, protein regulators and the newly identified low-abundant RNA species participate in the regulation of type I interferon (IFN-I) and proinflammatory genes induced by innate immune sensors. In this review, we briefly look into some of the autoimmune diseases profiled by dysregulations of IFN-I signaling and the regulatory mechanisms critical for immunological homeostasis.