Antiviral Potency Research Articles

Comparative Analyses of Antiviral Potencies of Second-Generation Integrase Strand Transfer Inhibitors (INSTIs) and the Developmental Compound 4d Against a Panel of Integrase Quadruple Mutants

Second-generation integrase strand transfer inhibitors (INSTIs) are strongly recommended for people living with HIV-1 (PLWH). The emergence of resistance to second-generation INSTIs has been infrequent and has not yet been a major issue in high-income countries. However, the delayed rollouts of these INSTIs in low- to middle-income countries during the COVID-19 pandemic combined with increased transmission of drug-resistant mutants worldwide are leading to an increase in INSTI resistance. Herein, we evaluated the antiviral potencies of our lead developmental INSTI 4d and the second-generation INSTIs dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) against a panel of IN quadruple mutants. The mutations are centered around G140S/Q148H, including positions L74, E92, and T97 combined with E138A/K/G140S/Q148H. All of the tested INSTIs lose potency against these IN quadruple mutants compared with the wild-type IN. In single-round infection assays, compound 4d retained higher antiviral potencies (EC50 values) than second-generation INSTIs against a subset of quadruple mutants. These findings may advance understanding of mechanisms that contribute to resistance and, in so doing, facilitate development of new INSTIs with improved antiviral profiles.

Alternative splicing expands the antiviral IFITM repertoire in Chinese rufous horseshoe bats.

Species-specific interferon responses are shaped by the virus-host arms race. The human interferon-induced transmembrane protein (IFITM) family consists of three antiviral IFITM genes that arose by gene duplication. These genes restrict virus entry and are key players in antiviral interferon responses. The unique IFITM repertoires in different species influence their resistance to viral infections, but the role of IFITMs in shaping the enhanced antiviral immunity of reservoir bat species is unclear. Here, we identified an IFITM gene in Chinese rufous horseshoe bat, a natural host of severe acute respiratory syndrome (SARS)-related coronaviruses, that is alternatively spliced to produce two IFITM isoforms in native cells as shown by transcriptomics. These bat IFITMs have conserved structures in vitro as demonstrated by circular dichroism spectroscopy, yet they exhibit distinct antiviral specificities against influenza A virus, Nipah virus and coronaviruses including SARS-CoV, SARS-CoV-2 and MERS-CoV. In parallel with human IFITM1-3, bat IFITM isoforms localize to distinct sites of virus entry which influences their antiviral potency. Further bioinformatic analysis of IFITM repertoires in 206 mammals reveals that alternative splicing is a recurring strategy for IFITM diversification, albeit less widely adopted than gene duplication. These findings demonstrate that alternative splicing is a key strategy for evolutionary diversification in the IFITM family. Our study also highlights an example of convergent evolution where species-specific selection pressures led to expansion of the IFITM family through multiple means, underscoring the importance of IFITM diversity as a component of innate immunity.

The isolation, bioactivity, and synthesis of natural products from Litsea verticillate with anti-HIV activities

Natural products isolated from Litsea verticillata have attracted considerable attention from the chemical community due to their unique structures and promising anti-HIV activities. Recent progresses in the isolation and bioactivity studies for these natural molecules were summarized comprehensively. From the 23 previously uncharacterized compounds isolated from the plant Litsea verticillata, litseaverticillol B demonstrated the most potent anti-HIV activity in vitro, with IC50 ranging from 2 to 3 μg/mL. Meanwhile, litseaverticillol E displayed the highest selectivity index (SI = 3.1), indicating a favorable balance between antiviral potency and cellular toxicity. The plausible biosynthetic pathways and the total synthetic approaches for the representative members (litseaverticillols) were introduced in detail.

The effects of Remdesivir’s functional groups on its antiviral potency and resistance against the SARS-CoV-2 polymerase

Remdesivir (RDV, Veklury®) is the first FDA-approved antiviral treatment for COVID-19. It is a nucleotide analogue (NA) carrying a 1'-cyano (1'-CN) group on the ribose and a pseudo-adenine nucleobase whose contributions to the mode of action (MoA) are not clear. Here, we dissect these independent contributions by employing RDV-TP analogues. We show that while the 1'-CN group is directly responsible for transient stalling of the SARS-CoV-2 replication/transcription complex (RTC), the nucleobase plays a role in the strength of this stalling. Conversely, RNA extension assays show that the 1'-CN group plays a role in fidelity and that RDV-TP can be incorporated as a GTP analogue, albeit with lower efficiency. However, a mutagenic effect by the viral polymerase is not ascertained by deep sequencing of viral RNA from cells treated with RDV. We observe that once added to the 3' end of RNA, RDV-MP is sensitive to excision and its 1'-CN group does not impact its nsp14-mediated removal. A >14-fold RDV-resistant SARS-CoV-2 isolate can be selected carrying two mutations in the nsp12 sequence, S759A and A777S. They confer both RDV-TP discrimination over ATP by nsp12 and stalling during RNA synthesis, leaving more time for excision-repair and potentially dampening RDV efficiency. We conclude that RDV presents a multi-faced MoA. It slows down or stalls overall RNA synthesis but is efficiently repaired from the primer strand, whereas once in the template, read-through inhibition adds to this effect. Its efficient incorporation may corrupt proviral RNA, likely disturbing downstream functions in the virus life cycle.

Exploring 4,7-Disubstituted Pyrimido[4,5-d]pyrimidines as Antiviral and Anticancer Agents.

Thirteen new 4,7-disubstituted pyrimido[4,5-d]pyrimidines were synthesized via a straightforward methodology starting from thiourea. The anti-proliferative activity of these compounds was evaluated across a diverse panel of eight cancer cell lines, with derivatives 7d and 7h showing efficacy against several hematological cancer types. Furthermore, all compounds were assessed for their antiviral potency against a panel of viruses. Compounds featuring a cyclopropylamino group and an aminoindane moiety exhibited remarkable efficacy against human coronavirus 229E (HCoV-229E). These findings highlight the pyrimidino[4,5-d]pyrimidine scaffold as an interesting framework for the design of novel antiviral agents against HCoVs, with compounds 7a, 7b, and 7f emerging as strong candidates for further investigation.

Synthesis and Structure-Activity Relationship of Covalent Inhibitors of SARS-CoV-2 Papain-Like Protease with Antiviral Potency

The papain-like protease (PLpro) is a highly conserved domain encoded by the coronavirus (CoV) genome and it plays an essential role in the replication and maturation of the virus in addition to weakening host immune response. Due to the virus’s reliance on PLpro for survival and propagation, small-molecule inhibitors of PLpro serve as an attractive model for direct-acting antiviral therapeutic agents against SARS-CoV-2. Building upon existing work aimed at designing covalent inhibitors against PLpro, we report the synthesis and structure–activity relationship of analogs based on the known covalent inhibitor 1 (Sanders, et al.2023). To evaluate the efficacy of synthesized derivatives, we conducted enzymatic inhibition assays, SARS-CoV-2/HeLa-ACE2 cellular potency and toxicity assays, and profiled the most promising analogs via in vitro ADME and in vivo pharmacokinetic studies. Additionally, we describe computational docking of profiled compounds bound to PLpro to elucidate the structure–activity relationship of compounds based on 1 and offer suggestions for optimizing the potency and selectivity of the electrophilic warhead and improving ADME and PK properties for this chemotype. Relative to the parent compound, new designs demonstrate comparable potency and target selectivity for PLpro. The accomplished SAR campaign provides novel insight for future development of antivirals against SARS-CoV-2.

A novel HIV triple broadly neutralizing antibody (bNAb) combination-based passive immunization of infant rhesus macaques achieves durable protective plasma neutralization levels and mediates anti-viral effector functions.

To eliminate vertical HIV transmission and achieve therapy-free viral suppression among children living with HIV, novel strategies beyond antiretroviral therapy (ART) are necessary. Our group previously identified a triple broadly neutralizing antibody (bNAb) combination comprising of 3BNC117, PGDM1400 and PGT151 that mediates robust in vitro neutralization and non-neutralizing effector functions against a cross-clade panel of simian human immunodeficiency viruses (SHIVs). In this study, we evaluated the safety, pharmacokinetics, and antiviral potency of this bNAb combination in infant rhesus macaques (RMs). We demonstrate that subcutaneous infusion of the triple bNAb regimen was well tolerated in pediatric monkeys and resulted in durable systemic and mucosal distribution. Plasma obtained from passively-immunized RMs demonstrated potent HIV-neutralizing and Fc-mediated antiviral effector functions. Finally, using the predicted serum neutralization 80% inhibitory dilution titer (PT80) biomarker threshold of >200, which was recently identified as a surrogate endpoint for evaluation of the preventative efficacy of bNAbs against mucosal viral acquisition in human clinical trials, we demonstrated that our regimen has PT80>200 against a large panel of plasma and breast milk-derived HIV strains and cross-clade SHIV variants. This data will guide the development of combination bNAbs for eliminating vertical HIV transmission and for achieving ART-free viral suppression among children living with HIV.

Adaptive multi-epitope targeting and avidity-enhanced nanobody platform for ultrapotent, durable antiviral therapy

Pathogens constantly evolve and can develop mutations that evade host immunity and treatment. Addressing these escape mechanisms requires targeting evolutionarily conserved vulnerabilities, as mutations in these regions often impose fitness costs. We introduce adaptive multi-epitope targeting with enhanced avidity (AMETA), a modular and multivalent nanobody platform that conjugates potent bispecific nanobodies to a human immunoglobulin M (IgM) scaffold. AMETA can display 20+ nanobodies, enabling superior avidity binding to multiple conserved and neutralizing epitopes. By leveraging multi-epitope SARS-CoV-2 nanobodies and structure-guided design, AMETA constructs exponentially enhance antiviral potency, surpassing monomeric nanobodies by over a million-fold. These constructs demonstrate ultrapotent, broad, and durable efficacy against pathogenic sarbecoviruses, including Omicron sublineages, with robust preclinical results. Structural analysis through cryoelectron microscopy and modeling has uncovered multiple antiviral mechanisms within a single construct. At picomolar to nanomolar concentrations, AMETA efficiently induces inter-spike and inter-virus cross-linking, promoting spike post-fusion and striking viral disarmament. AMETA's modularity enables rapid, cost-effective production and adaptation to evolving pathogens.

High-Throughput Synthesis and Evaluation of Antiviral Copolymers for Enveloped Respiratory Viruses.

COVID-19 made apparent the devastating impact viral pandemics have had on global health and order. Development of broad-spectrum antivirals to provide early protection upon the inevitable emergence of new viral pandemics is critical. In this work, antiviral polymers are discovered using a combination of high-throughput polymer synthesis and antiviral screening, enabling diverse polymer compositions to be explored. Amphipathic polymers, with ionizable tertiary amine groups, are the most potent antivirals, effective against influenza virus and SARS-CoV-2, with minimal cytotoxicity. It is hypothesized that these polymers interact with the viral membrane as they showed no activity against a nonenveloped virus (rhinovirus). The switchable chemistry of the polymers during endosomal acidification was evaluated using lipid monolayers, indicating that a complex synergy between hydrophobicity and ionization drives polymer-membrane interactions. This new high-throughput methodology can be adapted to continue to engineer the potency of the lead candidates or develop antiviral polymers against other emerging viral classes.

Study on the Recombinant Human Interferon α1b, α2b, and Gamma Transient Expression and in Vitro Activities in Tobacco.

Interferons (IFNs) are universally acknowledged for their pivotal role in antiviral and anticancer responses. Thus, the primary aim of our study was to explore the expressions of IFN-α1b, α2b, and gamma in tobacco leaves via agrobacterium-mediated transient transformation and investigate their possible activities. Briefly, fusion with green fluorescent protein tags aided in detecting the expressed IFN proteins in the foliar tissues. The genetic constructs encoding these fusion proteins were inserted into the MagnICON plant transient expression vector, followed by transformation into the Agrobacterium strain GV3101. The transformed bacteria were then used to infiltrate tobacco leaves. After post-infiltration, protein expression was confirmed within 72 h via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the fusion proteins were subsequently purified using high-performance liquid chromatography for identification. Both the antiviral and anticancer potencies of these IFN fusion proteins were evaluated using the WISH/VSV (WISH cells/Vesicular stomatitis virus) microneutralization and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively. Results indicated robust expression of the targeted IFN genes in plant tissues and significant biological activities against pathogens and cancer cells. Consequently, this study substantiated the viability of producing these therapeutic proteins in plants, potentially revolutionizing the manufacture of interferons biologically.

Preclinical and clinical antiviral characterization of AB-836, a potent capsid assembly modulator against hepatitis B virus

HBV capsid assembly modulators (CAMs) target the core protein and inhibit pregenomic RNA encapsidation and viral replication. HBV CAMs also interfere with cccDNA formation during de novo infection, which in turn suppresses transcription and production of HBV antigens. In this report, we describe the antiviral activities of AB-836, a potent and highly selective HBV CAM. AB-836 inhibited viral replication (EC50 = 0.010 μM) in HepDE19 cells, and cccDNA formation (EC50 = 0.18 μM) and HBsAg production (EC50 = 0.20 μM) in HepG2-NTCP cells during de novo infection. AB-836 showed broad genotype coverage, remained active against variants resistant to nucleos(t)ide analogs, and demonstrated improved antiviral potency against core variants resistant to other CAMs. AB-836 also mediated potent inhibition of HBV replication in a hydrodynamic injection mouse model, reducing both serum and liver HBV DNA. In a Phase 1 clinical study, 28 days of once-daily AB-836 oral dosing at 50, 100, and 200 mg resulted in mean serum HBV DNA declines of 2.57, 3.04, and 3.55 log10 IU/mL from baseline, respectively. Neither on-treatment viral rebound nor the emergence of viral resistance was observed during the 28-day treatment period. Furthermore, HBV DNA sequence analysis of baseline samples from the Phase 1 study revealed that 51.4% of the chronic hepatitis B participants contained at least one core polymorphism within the CAM-binding pocket, suggesting that genetic variations exist at this site. While AB-836 was discontinued due to clinical safety findings, data from the preclinical and clinical studies could help inform future optimization of HBV CAMs.

From the "One-Molecule, One-Target, One-Disease" Concept towards Looking for Multi-Target Therapeutics for Treating Non-Polio Enterovirus (NPEV) Infections.

Non-polio enteroviruses (NPEVs), namely coxsackieviruses (CV), echoviruses (E), enteroviruses (EV), and rhinoviruses (RV), are responsible for a wide variety of illnesses. Some infections can progress to life-threatening conditions in children or immunocompromised patients. To date, no treatments have been approved. Several molecules have been evaluated through clinical trials without success. To overcome these failures, the multi-target directed ligand (MTDL) strategy could be applied to tackle enterovirus infections. This work analyzes registered clinical trials involving antiviral drugs to highlight the best candidates and develops filters to apply to a selection for MTDL synthesis. We explicitly stated the methods used to answer the question: which solution can fight NPEVs effectively? We note the originality and relevance of this proposal in relation to the state of the art in the enterovirus-inhibitors field. Several combinations are possible to broaden the antiviral spectrum and potency. We discuss data related to the virus and data related to each LEAD compound identified so far. Overall, this study proposes a perspective on different strategies to overcome issues identified in clinical trials and evaluate the "MTDL" potential to improve the efficacy of drugs, broaden the antiviral targets, possibly reduce the adverse effects, drug design costs and limit the selection of drug-resistant virus variants.

7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

There are a limited number of effective vaccines against dengue virus (DENV) and significant efforts are being made to develop potent anti-virals. Previously, we described that platelet-chemokine CXCL4 negatively regulates interferon (IFN)-α/β synthesis and promotes DENV2 replication. An antagonist to CXCR3 (CXCL4 receptor) reversed it and inhibited viral replication. In a concurrent search, we identified CXCR3-antagonist from our compound library, namely 7D, which inhibited all serotypes of DENV in vitro. With a half-life of ~2.85 h in plasma and no significant toxicity, 7D supplementation (8 mg/kg-body-weight) to DENV2-infected IFNα/β/γR−/−AG129 or wild-type C57BL6 mice increased synthesis of IFN-α/β and IFN-λ, and rescued disease symptoms like thrombocytopenia, leukopenia and vascular-leakage, with improved survival. 7D, having the property to inhibit Sirt-1 deacetylase, promoted acetylation and phosphorylation of STAT3, which in-turn increased plasmablast proliferation, germinal-center maturation and synthesis of neutralizing-antibodies against DENV2 in mice. A STAT3-inhibitor successfully inhibited these effects of 7D. Together, these observations identify compound 7D as a stimulator of IFN-α/β/λ synthesis via CXCL4:CXCR3:p38:IRF3 signaling, and a booster for neutralizing-antibody generation by promoting STAT3-acetylation in plasmablasts, capable of protecting dengue infection.

GROWTH RESPONSE AND ANTIVIRAL POTENTIALS OF NEEM (AZADIRACHTA INDICA J.) AND SCENT (OCIMUM SANCTUM L.) LEAF MEALS AGAINST NEWCASTLE AND INFECTIOUS BURSAL DISEASES IN BROILER CHICKS

Newcastle disease (ND) and infectious bursal disease (IBD) are the most feared viral disease of poultry. Viral reversion, vaccine failure, risk of autoimmune diseases and allergic disorder has led to the search for alternatives for vaccination. The effects of neem (Azadirachta indica) and scent (Ocimum santum) leaf meals fed singly or in combination on the growth performance and their antiviral potencies in broiler chicks were investigated. Two hundred 1-day old Ross 308 unsexed broiler chicks were randomly allotted to 5 treatments, consisting of 5 replicates with 8 birds each. Treatments 1 and 2 were basal diets with no leaf meals, treatments 3, 4 and 5 had the basal diets plus 3% neem leaf meal (NLM), 3% scent leaf meal (SLM) and the blend of 2% NLM + 2% SLM, respectively. Birds in treatments 2-5 were unvaccinated against Newcastle disease and Infectious bursal disease while birds in treatment 1 were vaccinated. Performance indices were measured on a weekly basis. At day-21, blood samples were randomly collected from 2 birds per replicate for haemagglutination inhibition and agar gel precipitation tests. Data were subjected to descriptive statistics and ANOVA at P<0.05. Birds fed diets supplemented with a combination of 2% neem + 2% scent leaves had the highest weight gain (819.49g/b) and lowest feed conversion ratio (1.83). In conclusion, birds fed diets supplemented with 3% neem and 3% scent leaf meals singly had haemagglutination inhibition titre values and conferred immunity above protective threshold against Newcastle disease and produced antibodies against Infectious bursal disease.

Dynamic features of virus protein 1 and substitutions in the 3-phenyl ring determine the potency and broad-spectrum activity of capsid-binding pyrazolo[3,4-d]pyrimidines against rhinoviruses

Pyrazolo[3,4-d]pyrimidines represent one potent class of well tolerated and highly active rhinovirus (RV) inhibitors that act as capsid binders. The lead compound OBR-5-340 inhibits a broad-spectrum of RVs. Aiming to improve lead activity, we evaluated the impact of structural modifications in the 3-phenyl ring of OBR-5-340 on its potency and spectrum of anti-RV activity vitro. Our results demonstrate the crucial role of substitution at position 4 for strong, broad-spectrum anti-RV activity. The 4-methyl (RCB23137) and 4-chloro (RCB23138) derivatives outperformed OBR-5-340 in terms of potency and anti-RV activity spectrum. Based on these findings, the compounds were selected for computational binding studies. Molecular dynamic simulations with six RVs differing in OBR-5-340, RCB23137, and RCB23138 sensitivity proved the impact of dynamic features of two VP1 loops enveloping these inhibitors on antiviral potency.

Strategic Fluorination to Achieve a Potent, Selective, Metabolically Stable, and Orally Bioavailable Inhibitor of CSNK2.

The host kinase casein kinase 2 (CSNK2) has been proposed to be an antiviral target against β-coronaviral infection. To pharmacologically validate CSNK2 as a drug target in vivo, potent and selective CSNK2 inhibitors with good pharmacokinetic properties are required. Inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold possess outstanding potency and selectivity for CSNK2, but bioavailability and metabolic stability are often challenging. By strategically installing a fluorine atom on an electron-rich phenyl ring of a previously characterized inhibitor 1, we discovered compound 2 as a promising lead compound with improved in vivo metabolic stability. Compound 2 maintained excellent cellular potency against CSNK2, submicromolar antiviral potency, and favorable solubility, and was remarkably selective for CSNK2 when screened against 192 kinases across the human kinome. We additionally present a co-crystal structure to support its on-target binding mode. In vivo, compound 2 was orally bioavailable, and demonstrated modest and transient inhibition of CSNK2, although antiviral activity was not observed, possibly attributed to its lack of prolonged CSNK2 inhibition.

Discovery of potential inhibitors targeting SARS-CoV-2 Mpro.

The coronavirus disease (COVID-19) pandemic, resulting from human-to-human transmission of a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has caused a global health emergency. The lack of a specific drug or treatment strategy against this disease makes it devastating. Given that the main protease (Mpro) of SARS-CoV-2 plays an indispensable role in viral polyprotein processing, its successful inhibition prevents viral replication and constrains virus spread. Therefore, developing an effective SARS-CoV-2 Mpro inhibitor to treat COVID-19 is imperative. We employed a high-throughput screening (HTS) method based on fluorescence polarization (FP) assay and further confirmed by the fluorescence resonance energy transfer (FRET) method for the discovery of Mpro inhibitors. Then multiple approaches were taken to investigate the inhibition profiles of the hit compounds against Mpro, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay, surface plasmon resonance analysis (SPR), high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS), cytopathic effect (CPE) assay, molecule docking, and the drug-likeness analysis. In this study, four Mpro inhibitors with low toxicity were selected from HTS. According to SPR, all the hit compounds had medium binding affinities toward SARS-CoV-2 Mpro. HPLC-Q-TOF-MS results revealed the non-covalent linkage of each compound with SARS-CoV-2 Mpro. Molecule docking simulated the molecule interactions between each compound and the substrate binding pocket of SARS-CoV-2 Mpro. CPE assay was used to detect their inhibitory activities against coronaviruses HCoV-OC43 and HCoV-229E. In particular, the IMB63-8G compound demonstrated the highest antiviral potency [50% effective concentration (IC50) value of 1.71 μg/mL] and selectivity against HCoV-OC43 (SI = 39), which was more than 4-fold higher than that of ribavirin (RBV). Besides, the IMB63-8G compound possessed favorable drug-likeness characteristics. Our results will highlight the therapeutic potential of these compounds for the treatment of SARS-CoV-2 infection.

The activation cascade of the broad-spectrum antiviral bemnifosbuvir characterized at atomic resolution.

Bemnifosbuvir (AT-527) and AT-752 are guanosine analogues currently in clinical trials against several RNA viruses. Here, we show that these drugs require a minimal set of 5 cellular enzymes for activation to their common 5'-triphosphate AT-9010, with an obligate order of reactions. AT-9010 selectively inhibits essential viral enzymes, accounting for antiviral potency. Functional and structural data at atomic resolution decipher N6-purine deamination compatible with its metabolic activation. Crystal structures of human histidine triad nucleotide binding protein 1, adenosine deaminase-like protein 1, guanylate kinase 1, and nucleoside diphosphate kinase at 2.09, 2.44, 1.76, and 1.9 Å resolution, respectively, with cognate precursors of AT-9010 illuminate the activation pathway from the orally available bemnifosbuvir to AT-9010, pointing to key drug-protein contacts along the activation pathway. Our work provides a framework to integrate the design of antiviral nucleotide analogues, confronting requirements and constraints associated with activation enzymes along the 5'-triphosphate assembly line.

Anti-enterovirus 71 activity of native fucosylated chondroitin sulfates and their derivatives

Enterovirus 71 (EV71) is recognized as a major causative agent of hand, foot, and mouth disease (HFMD), posing a significant global public health concern due to its widespread impact and resulting in a major public health issue worldwide. Despite its prevalence, current clinical therapy lacks effective antiviral agents. Fucosylated chondroitin sulfates (FCS) derived from sea cucumber exhibits a range of biological activities including potent antiviral effects. This study provides compelling evidence of the potent antiviral efficacy of FCS against EV71. To further elucidate the impact of structural variations on the anti-EV71 activity, native FCSs with diverse sulfation patterns and a varity of FCS derivatives were prepared and analyzed. Notably, this study presents the detailed structural characterization of FCSs from the sea cucumbers Holothuria scabra Jaege and Holothuria fuscopunctata. Analysis of the structure-activity relationships revealed that molecular weight, sulfated fucose branches, and sulfation pattern were all crucial factors contributing to the potent inhibitory effects of FCS against EV71. Interestingly, molecular weight emerged as the most significant structural determinant of the antiviral potency. These findings suggest the promising potential of utilizing FCS as an innovative EV71 entry inhibitor for the treatment of HFMD.

Antiviral Mx proteins have an ancient origin and widespread distribution among eukaryotes.

First identified in mammals, Mx proteins are potent antivirals against a broad swathe of viruses. Mx proteins arose within the Dynamin superfamily of proteins (DSP), mediating critical cellular processes, such as endocytosis and mitochondrial, plastid, and peroxisomal dynamics. And yet, the evolutionary origins of Mx proteins are poorly understood. Using a series of phylogenomic analyses with stepwise increments in taxonomic coverage, we show that Mx proteins predate the interferon signaling system in vertebrates. Our analyses find an ancient monophyletic DSP lineage in eukaryotes that groups vertebrate and invertebrate Mx proteins with previously undescribed fungal MxF proteins, the relatively uncharacterized plant and algal Dynamin 4A/4C proteins, and representatives from several early-branching eukaryotic lineages. Thus, Mx-like proteins date back close to the origin of Eukarya. Our phylogenetic analyses also reveal that host-encoded and NCLDV (nucleocytoplasmic large DNA viruses)-encoded DSPs are interspersed in four distinct DSP lineages, indicating recurrent viral theft of host DSPs. Our analyses thus reveal an ancient history of viral and antiviral functions encoded by the Dynamin superfamily in eukaryotes.