Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Kunitz-type protease inhibitor TFPI2 remodels stemness and immunosuppressive tumor microenvironment in glioblastoma.

Glioblastoma (GBM) tumors consist of multiple cell populations, including self-renewing glioblastoma stem cells (GSCs) and immunosuppressive microglia. Here we identified Kunitz-type protease inhibitor TFPI2 as a critical factor connecting these cell populations and their associated GBM hallmarks of stemness and immunosuppression. TFPI2 promotes GSC self-renewal and tumor growth via activation of the c-Jun N-terminal kinase-signal transducer and activator of transcription (STAT)3 pathway. Secreted TFPI2 interacts with its functional receptor CD51 on microglia to trigger the infiltration and immunosuppressive polarization of microglia through activation of STAT6 signaling. Inhibition of the TFPI2-CD51-STAT6 signaling axis activates T cells and synergizes with anti-PD1 therapy in GBM mouse models. In human GBM, TFPI2 correlates positively with stemness, microglia abundance, immunosuppression and poor prognosis. Our study identifies a function for TFPI2 and supports therapeutic targeting of TFPI2 as an effective strategy for GBM.

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications.

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

Microglia-organized scar-free spinal cord repair in neonatal mice.

Spinal cord injury in mammals is thought to trigger scar formation with little regeneration of axons1-4. Here we show that a crush injury to the spinal cord in neonatal mice leads to scar-free healing that permits the growth of long projecting axons through the lesion. Depletion of microglia in neonatal mice disrupts this healing process and stalls the regrowth of axons, suggesting that microglia are critical for orchestrating the injury response. Using single-cell RNA sequencing and functional analyses, we find that neonatal microglia are transiently activated and have at least two key roles in scar-free healing. First, they transiently secrete fibronectin and its binding proteins to form bridges of extracellular matrix that ligate the severed ends of the spinal cord. Second, neonatal-but not adult-microglia express several extracellular and intracellular peptidase inhibitors, as well as other molecules that are involved in resolving inflammation. We transplanted either neonatal microglia or adult microglia treated with peptidase inhibitors into spinal cord lesions of adult mice, and found that both types of microglia significantly improved healing and axon regrowth. Together, our results reveal the cellular and molecular basis of the nearly complete recovery of neonatal mice after spinal cord injury, and suggest strategies that could be used to facilitate scar-free healing in the adult mammalian nervous system.

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor.

Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV-resident cysteine protease called SERA6, enabling host RBC rupture through SERA6-mediated degradation of the RBC cytoskeleton protein ß-spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV-located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in ß-spectrin cleavage and RBC rupture. Drug-like inhibitors of SERA6 autoprocessing similarly prevent ß-spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.

Exploring the therapeutic potential of DV-B-120 as an inhibitor of dengue virus infection.

Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.

Inhibition of autocrine HGF maturation overcomes cetuximab resistance in colorectal cancer.

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.

An extended conformation of SARS-CoV-2 main protease reveals allosteric targets.

SignificanceThe coronavirus main protease (Mpro) is required for viral replication. Here, we obtained the extended conformation of the native monomer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro by trapping it with nanobodies and found that the catalytic domain and the helix domain dissociate, revealing allosteric targets. Another monomeric state is termed compact conformation and is similar to one protomer of the dimeric form. We designed a Nanoluc Binary Techonology (NanoBiT)-based high-throughput allosteric inhibitor assay based on structural conformational change. Our results provide insight into the maturation, dimerization, and catalysis of the coronavirus Mpro and pave a way to develop an anticoronaviral drug through targeting the maturation process to inhibit the autocleavage of Mpro.

Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

Structural basis of nirmatrelvir and ensitrelvir activity against naturally occurring polymorphisms of the SARS-CoV-2 main protease.

SARS-CoV-2 is the causative agent of COVID-19. The main viral protease (Mpro) is an attractive target for antivirals. The clinically approved drug nirmatrelvir and the clinical candidate ensitrelvir have so far showed great potential for treatment of viral infection. However, the broad use of antivirals is often associated with resistance generation. Herein, we enzymatically characterized 14 naturally occurring Mpro polymorphisms that are close to the binding site of these antivirals. Nirmatrelvir retained its potency against most polymorphisms tested, while mutants G143S and Q189K were associated with diminished inhibition constants. For ensitrelvir, diminished inhibition constants were observed for polymorphisms M49I, G143S, and R188S, but not for Q189K, suggesting a distinct resistance profile between inhibitors. In addition, the crystal structures of selected polymorphisms revealed interactions that were critical for loss of potency. In conclusion, our data will assist the monitoring of potential resistant strains, support the design of combined therapy, as well as assist the development of the next generation of Mpro inhibitors.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

Structural basis for the inhibition of the HCoV-NL63 main protease Mpro by X77.

Human coronaviruses are a group of pathogens that primarily cause respiratory and intestinal diseases. Infection can easily cause respiratory symptoms, as well as a variety of serious complications. There are several types of human coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and SARS-CoV-2. The prevalence of COVID-19 has led to a growing focus on drug research against human coronaviruses. The main protease (Mpro) from human coronaviruses is a relatively conserved that controls viral replication. X77 was discovered to have extremely high inhibitory activity against SARS-CoV-2 Mpro through the use of computer-simulated docking. In this paper, we have resolved the crystal structure of the HCoV-NL63 Mpro complexed with X77 and analyzed their interaction in detail. This data provides essential information for solving their binding modes and their structural determinants. Then, we compared the binding modes of X77 with SARS-CoV-2 Mpro and HCoV-NL63 Mpro in detail. This study illustrates the structural basis of HCoV-NL63 Mpro binding to the inhibitor X77. The structural insights derived from this study will inform the development of new drugs with broad-spectrum resistance to human coronaviruses.

Structural basis of rosmarinic acid inhibitory mechanism on SARS-CoV-2 main protease.

The SARS-CoV-2 coronavirus is characterized by high mutation rates and significant infectivity, posing ongoing challenges for therapeutic intervention. To address potential challenges in the future, the continued development of effective drugs targeting SARS-CoV-2 remains an important task for the scientific as well as the pharmaceutical community. The main protease (Mpro) of SARS-CoV-2 is an ideal therapeutic target for COVID-19 drug development, leading to the introduction of various inhibitors, both covalent and non-covalent, each characterized by unique mechanisms of action and possessing inherent strengths and limitations. Natural products, being compounds naturally present in the environment, offer advantages such as low toxicity and diverse activities, presenting a viable source for antiviral drug development. Here, we identified a natural compound, rosmarinic acid, which exhibits significant inhibitory effects on the Mpro of the SARS-CoV-2. Through detailed structural biology analysis, we elucidated the precise crystal structure of the complex formed between rosmarinic acid and SARS-CoV-2 Mpro, revealing the molecular basis of its inhibitory mechanism. These findings not only enhance our understanding of the antiviral action of rosmarinic acid, but also provide valuable structural information and mechanistic insights for the further development of therapeutic strategies against SARS-CoV-2.

Applying the bioisosterism strategy to obtain lead compounds against SARS-CoV-2 cysteine proteases: An in-silico approach.

SARS-CoV-2 cysteine proteases are essential nonstructural proteins due to their role in the formation of the virus multiple enzyme replication-transcription complex. As a result, those functional proteins are extremely relevant targets in the development of a new drug candidate to fight COVID-19. Based on this fact and guided by the bioisosterism strategy, the present work has selected 126 out of 1050 ligands from DrugBank website. Subsequently, 831 chemical analogs containing bioisosteres, some of which became structurally simplified, were created using the MB-Isoster software, and molecular docking simulations were performed using AutoDock Vina. Finally, a study of physicochemical properties, along with pharmacokinetic profiles, was carried out through SwissADME and ADMETlab 2.0 platforms. The promising results obtained with the molecules encoded as DB00549_BI_005, DB04868_BI_003, DB11984_BI_002, DB12364_BI_006 and DB12805_BI_004 must be confirmed by molecular dynamics studies, followed by in vitro and in vivo empirical tests that ratify the advocated in-silico results.

A low-background, fluorescent assay to evaluate inhibitors of diverse viral proteases.

Multiple coronaviruses (CoVs) can cause respiratory diseases in humans. While prophylactic vaccines designed to prevent infection are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), incomplete vaccine efficacy, vaccine hesitancy, and the threat of other pathogenic CoVs for which vaccines do not exist have highlighted the need for effective antiviral therapies. While antiviral compounds targeting the viral polymerase and protease are already in clinical use, their sensitivity to potential resistance mutations as well as their breadth against the full range of human and preemergent CoVs remain incompletely defined. To begin to fill that gap in knowledge, we report here the development of an improved, noninfectious, cell-based fluorescent assay with high sensitivity and low background that reports on the activity of viral proteases, which are key drug targets. We demonstrate that the assay is compatible with not only the SARS-CoV-2 Mpro protein but also orthologues from a range of human and nonhuman CoVs as well as clinically reported SARS-CoV-2 drug-resistant Mpro variants. We then use this assay to define the breadth of activity of two clinically used protease inhibitors, nirmatrelvir and ensitrelvir. Continued use of this assay will help define the strengths and limitations of current therapies and may also facilitate the development of next-generation protease inhibitors that are broadly active against both currently circulating and preemergent CoVs. IMPORTANCE Coronaviruses (CoVs) are important human pathogens with the ability to cause global pandemics. Working in concert with vaccines, antivirals specifically limit viral disease in people who are actively infected. Antiviral compounds that target CoV proteases are already in clinical use; their efficacy against variant proteases and preemergent zoonotic CoVs, however, remains incompletely defined. Here, we report an improved, noninfectious, and highly sensitive fluorescent method of defining the sensitivity of CoV proteases to small molecule inhibitors. We use this approach to assay the activity of current antiviral therapies against clinically reported SARS-CoV-2 protease mutants and a panel of highly diverse CoV proteases. Additionally, we show this system is adaptable to other structurally nonrelated viral proteases. In the future, this assay can be used to not only better define the strengths and limitations of current therapies but also help develop new, broadly acting inhibitors that more broadly target viral families.

Enterovirus D68 Infection Induces TDP-43 Cleavage, Aggregation, and Neurotoxicity.

Enterovirus D68 (EV-D68), which causes severe respiratory diseases and irreversible central nervous system damage, has become a serious public health problem worldwide. However, the mechanisms by which EV-D68 exerts neurotoxicity remain unclear. Thus, we aimed to analyze the effects of EV-D68 infection on the cleavage, subcellular translocation, and pathogenic aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in respiratory or neural cells. The results showed that EV-D68-encoded proteases 2A and 3C induced TDP-43 translocation and cleavage, respectively. Specifically, 3C cleaved residue 327Q of TDP-43. The 3C-mediated cleaved TDP-43 fragments had substantially decreased protein solubility compared with the wild-type TDP-43. Hence, 3C activity promoted TDP-43 aggregation, which exerted cytotoxicity to diverse human cells, including glioblastoma T98G cells. The effects of commercially available antiviral drugs on 3C-mediated TDP-43 cleavage were screened, and the results revealed lopinavir as a potent inhibitor of EV-D68 3C protease. Overall, these results suggested TDP-43 as a conserved host target of EV-D68 3C. This study is the first to provide evidence on the involvement of TDP-43 dysregulation in EV-D68 pathogenesis. IMPORTANCE Over the past decade, the incidence of enterovirus D68 (EV-D68) infection has increased worldwide. EV-D68 infection can cause different respiratory symptoms and severe neurological complications, including acute flaccid myelitis. Thus, elucidating the mechanisms underlying EV-D68 toxicity is important to develop novel methods to prevent EV-D68 infection-associated diseases. This study shows that EV-D68 infection triggers the translocalization, cleavage, and aggregation of TDP-43, an intracellular protein closely related to degenerative neurological disorders. The viral protease 3C decreased TDP-43 solubility, thereby exerting cytotoxicity to host cells, including human glioblastoma cells. Thus, counteracting 3C activity is an effective strategy to relieve EV-D68-triggered cell death. Cytoplasmic aggregation of TDP-43 is a hallmark of degenerative diseases, contributing to neural cell damage and central nervous system (CNS) disorders. The findings of this study on EV-D68-induced TDP-43 formation extend our understanding of virus-mediated cytotoxicity and the potential risks of TDP-43 dysfunction-related cognitive impairment and neurological symptoms in infected patients.

Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies.

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.