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The endemic nature of the Ebola virus disease in Africa underscores the need for prophylactic and therapeutic drugs that are affordable and easy to administer. Through a phenotypic screening employing viral pseudotypes and our in-house chemical library, we identified a promising hit featuring a thiophene scaffold, exhibiting antiviral activity in the micromolar range. Following up on this thiophene hit, a new series of compounds that retain the five-membered heterocyclic scaffold while modifying several substituents was synthesized. Initial screening using a pseudotype viral system and validation assays employing authentic Ebola virus demonstrated the potential of this new chemical class as viral entry inhibitors. Subsequent investigations elucidated the mechanism of action through site-directed mutagenesis. Furthermore, we conducted studies to assess the pharmacokinetic profile of selected compounds to confirm its pharmacological and therapeutic potential.

The life cycle of Ebola and Marburg viruses includes a step of the virion envelope fusion with the cell membrane. Here, we analyzed whether the fusion of liposome membranes under the action of fragments of fusion peptides of Ebola and Marburg viruses depends on the composition of lipid vesicles. A fluorescence assay and electron microscopy were used to quantify the fusogenic activity of the virus fusion peptides and to identify the lipid determinants affecting membrane merging. Differential scanning calorimetry of lipid phase transitions revealed alterations in the physical properties of the lipid matrix produced by virus fusion peptides. Additionally, we found that plant polyphenols, quercetin, and myricetin inhibited vesicle fusion induced by the Marburg virus fusion peptide.

The increasing frequency of filovirus outbreaks in African countries has led to a pressing need for the development of effective antifilovirus agents. In continuation of our previous research on the antifilovirus activity of monoterpenoid derivatives, we synthesized a series of (+)-fenchol and (-)-isopinocampheol derivatives by varying the type of heterocycle and linker length. Derivatives with an N-alkylpiperazine cycle proved to be the most potent antiviral compounds, with half-maximal inhibitory concentration (IC 50 ) 1.4-20 μМ against Lenti-EboV-GP infection and 11.3-47 μМ against Lenti-MarV-GP infection. Mechanism-of-action experiments revealed that the compounds may exert their action by binding to surface glycoproteins (GPs). It was demonstrated that the binding of the synthesized compounds to the Marburg virus GP is less efficient as compared to the Ebola virus GP. Furthermore, it was shown that the compounds possess lysosomotropic properties. Thus, the antiviral activity may be due to dual effects. This study offers new antiviral agents that are worthy of further exploration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Diphyllin is a naturally occurring lignan comprised of an aryl naphthalene lactone scaffold that demonstrates beneficial biological activities in disease models of cancer, obesity, and viral infection. A target of diphyllin and naturally occurring derivatives is the vacuolar ATPase (V-ATPase) complex. Although diphyllin-related natural products are active with in vitro models for viral entry, the potencies and unknown pharmacokinetic properties limit well-designed in vivo evaluations. Previous studies demonstrated that diphyllin derivatives have the utility of blocking the Ebola virus cell entry pathway. However, diphyllin shows limited potency and poor oral bioavailability in mice. An avenue to improve the potency was used in a new library of synthetic derivatives of diphyllin. Diphyllin derivatives exploiting ether linkages at the 4-position with one-to-three carbon spacers to an oxygen or nitrogen atom provided compounds with EC 50 values ranging from 7 to 600 nM potency and selectivity up to >500 against Ebola virus in infection assays. These relative potencies are reflected in the Ebola virus infection of primary macrophages, a cell type involved in early pathogenesis. A target engagement study reveals that reducing the ATPV0a2 protein expression enhanced the potency of diphyllin derivatives to block EBOV entry, consistent with effects on the endosomal V-ATPase function. Despite the substantial enhancement of antiviral potencies, limitations were identified, including rapid clearance predicted by in vitro microsome stability assays. However, compounds with similar or improved half-lives relative to diphyllin demonstrated improved pharmacokinetic profiles in vivo. Importantly, these derivatives displayed suitable plasma levels using oral administration, establishing the feasibility of in vivo antiviral testing., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. The corresponding author is a founder and CSO of Amplified Sciences, Inc and Aromarc Therapeutics, Inc. These companies hold no competing or common interests in the work being submitted for publication. All other authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

High-consequence pathogens such as the Ebola, Marburg, and Lassa viruses are handled in maximum-containment biosafety level 4 (BSL-4) laboratories. Genetic material is often isolated from such viruses and subsequently removed from BSL-4 laboratories for a multitude of downstream analyses using readily accessible technologies and equipment available at lower-biosafety level laboratories. However, it is essential to ensure that these materials are free of viable viruses before removal from BSL-4 laboratories to guarantee sample safety. This study details the in-house procedure used for validating the inactivation of Ebola, Marburg, and Lassa virus cultures after incubation with AVL lysis buffer (Qiagen) and ethanol. This study's findings show that no viable virus was detectable when high-titer cultures of Ebola, Marburg, and Lassa viruses were incubated with AVL lysis buffer for 10 min, followed by an equal volume of 95% ethanol for 3 min, using a method with a sensitivity of ≤0.8 log 10 TCID 50 as the limit of detection.

Since the largest and most fatal Ebola virus epidemic during 2014-2016, there have been several consecutive filoviral outbreaks in recent years, including those in 2021, 2022, and 2023. Ongoing outbreak prevalence and limited FDA-approved filoviral therapeutics emphasize the need for novel small molecule treatments. Here, we showcase the structure-activity relationship development of N-substituted pyrrole-based heterocycles and their potent, submicromolar entry inhibition against diverse filoviruses in a target-based pseudovirus assay. Inhibitor antiviral activity was validated using replication-competent Ebola, Sudan, and Marburg viruses. Mutational analysis was used to map the targeted region within the Ebola virus glycoprotein. Antiviral counter-screen and phospholipidosis assays were performed to demonstrate the reduced off-target activity of these filoviral entry inhibitors. Favorable antiviral potency, selectivity, and drug-like properties of the N-substituted pyrrole-based heterocycles support their potential as broad-spectrum antifiloviral treatments.

The recent outbreak of the Ebola virus (EBOV) has marked it as one of the most severe health threats globally. Among various anti-EBOV inhibitors studied, galidesivir (BCX4430) has shown remarkable efficacy. This study aims to identify novel potential anti-EBOV drugs among galidesivir analogs, focusing on the Zaire ebolavirus (Z-EBOV), which exhibits a mortality rate of 90%. We subjected 200 candidate compounds to molecular docking calculations, followed by an evaluation of the bioactivity of the top 25 compounds using the OSIRIS Property Explorer. Initial 50 ns molecular dynamics (MD) simulations were then performed. According to our findings, only six compounds exhibited positive drug scores. We further performed molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) calculations of binding energy over 50 ns, selecting the two top-performing compounds for extended 150 ns MD simulations. CID 117698807 and CID 117712809 showed higher binding stability compared to galidesivir, with Δ G binding values of -36.7 and -53.4 kcal/mol, respectively. Both compounds demonstrated high stability within the Z-EBOV-V24 active site over the 150 ns MD simulations. Hence, our study proposes CID 117698807 and CID 117712809 as potential anti-Z-EBOV-V24 drug candidates, warranting further investigation.Communicated by Ramaswamy H. Sarma.

VP30 and VP40 proteins of Ebola and Marburg viruses have been recognized as potential targets for antiviral drug development due to their essential roles in the viral lifecycle. Targeting these proteins could disrupt key stages of the viral replication process, inhibiting the viruses' ability to propagate and cause disease. The current study aims to perform molecular docking and virtual screening on deep-sea fungal metabolites targeting Marburg virus VP40 Dimer, matrix protein VP40 from Ebola virus Sudan, Ebola VP35 Interferon Inhibitory Domain, and VP35 from Marburg virus. The top ten compounds for each protein target were chosen using the glide score. All the compounds obtained indicate a positive binding interaction. Furthermore, AdmetSAR was utilized to investigate the pharmacokinetics of the inhibitors chosen. Gliotoxin was used as a ligand with Marburg virus VP40 Dimer, Austinol with matrix protein VP40 from Ebola virus Sudan, Ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) with Ebola VP35 Interferon Inhibitory Domain, and Dehydroaustinol with VP35 from Marburg virus. MD modeling and MMPBSA studies were used to provide a better understanding of binding behaviors. Pre-clinical experiments can assist validate our in-silico studies and assess whether the molecule can be employed as an anti-viral drug., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Alanzi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Favipiravir is a ribonucleoside analogue that has been explored as a therapeutic for the treatment of Ebola Virus Disease (EVD). Promising data from rodent models has informed nonhuman primate trials, as well as evaluation in patients during the 2013-2016 West African EVD outbreak of favipiravir treatment. However, mixed results from these studies hindered regulatory approval of favipiravir for the indication of EVD. This study examined the influence of route of administration, duration of treatment, and treatment schedule of favipiravir in immune competent mouse and guinea pig models using rodent-adapted Zaire ebolavirus (EBOV). A dose of 300 mg/kg/day of favipiravir with an 8-day treatment was found to be fully effective at preventing lethal EVD-like disease in BALB/c mice regardless of route of administration (oral, intraperitoneal, or subcutaneous) or whether it was provided as a once-daily dose or a twice-daily split dose. Preclinical data generated in guinea pigs demonstrates that an 8-day treatment of 300 mg/kg/day of favipiravir reduces mortality following EBOV challenge regardless of route of treatment or duration of treatments for 8, 11, or 15 days. This work supports the future translational development of favipiravir as an EVD therapeutic.

The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.

In the 1990s, monoclonal antibodies (mAbs) progressed from scientific tools to advanced therapeutics, particularly for the treatment of cancers and autoimmune and inflammatory disorders. In the arena of infectious disease, the inauguration of mAbs as a post-exposure treatment in humans against Ebola virus (EBOV) occurred in response to the 2013-2016 West Africa outbreak. This review recounts the history of a candidate mAb treatment, ZMapp, beginning with its emergency use in the 2013-2016 outbreak and advancing to randomized controlled trials into the 2018-2020 African outbreak. We end with a brief discussion of the hurdles and promise toward mAb therapeutic use against infectious disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

For those exposed to filovirus, such as Sudan virus and Ebola virus, a new study offers hope.

Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.

Background: Ebola virus (EBOV) is a genus of negative-strand RNA viruses belonging to the family Filoviradae that was first described in 1976 in the present-day Democratic Republic of the Congo. It has intermittently affected substantial human populations in West Africa and presents itself as a global health menace due to the high mortality rate of patients, high transmission rate, difficult patient management, and the emergence of complicated autoimmune disease-like conditions post-infection., Objective: EBOV or other EBOV-like species as a biochemical weapon pose a significant risk; hence, the need to develop both prophylactic and therapeutic medications to combat the virus is unquestionable., Methods: In this review work, we have compiled the literature pertaining to transmission, pathogenesis, immune response, and diagnosis of EBOV infection. We included detailed structural details of EBOV along with all the available therapeutics against EBOV disease. We have also highlighted current developments and recent advances in therapeutic approaches against Ebola virus disease (EVD)., Discussion: The development of preventive vaccines against the virus is proving to be a successful effort as of now; however, problems concerning logistics, product stability, multi- dosing, and patient tracking are prominent in West Africa. Monoclonal antibodies that target EBOV proteins have also been developed and approved in the clinic; however, no small drug molecules that target these viral proteins have cleared clinical trials. An understanding of clinically approved vaccines and their shortcomings also serves an important purpose for researchers in vaccine design in choosing the right vector, antigen, and particular physicochemical properties that are critical for the vaccine's success against the virus across the world., Conclusion: Our work brings together a comprehensive review of all available prophylactic and therapeutic medications developed and under development against the EBOV, which will serve as a guide for researchers in pursuing the most promising drug discovery strategies against the EBOV and also explore novel mechanisms of fighting against EBOV infection., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ebola virus disease (EVD) is a severe and frequently lethal disease caused by Ebola virus (EBOV). The latest occasional EVD outbreak (2013-2016) in Western African, which was accompanied by a high fatality rate, showed the great potential of epidemic and pandemic spread. Antiviral therapies against EBOV are very limited, strain-dependent (only antibody therapies are available) and mostly restricted to symptomatic treatment, illustrating the urgent need for novel antiviral strategies. Thus, we evaluated the effect of the clinically widely used antifungal itraconazole and the antidepressant fluoxetine for a repurposing against EBOV infection. While itraconazole, similar to U18666A, directly binds to and inhibits the endosomal membrane protein Niemann-Pick C1 (NPC1), fluoxetine, which belongs to the structurally unrelated group of weakly basic, amphiphile so-called "functional inhibitors of acid sphingomyelinase" (FIASMA) indirectly acts on the lysosome-residing acid sphingomyelinase via enzyme detachment leading to subsequent lysosomal degradation. Both, the drug-induced endolysosomal cholesterol accumulation and the altered endolysosomal pH, might interfere with the fusion of viral and endolysosomal membrane, preventing infection with EBOV. We further provide evidence that cholesterol imbalance is a conserved cross-species mechanism to hamper EBOV infection. Thus, exploring the endolysosomal host-pathogen interface as a suitable antiviral treatment may offer a general strategy to combat EBOV infection.

Viral infections have a great impact on human health. The urgent need to find a cure against different viruses led us to investigations in a vast range of drugs. Azithromycin (AZT), classified as a macrolide, showed various effects on different known viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV), Zika, Ebola, Enterovirus (EVs) and Rhinoviruses (RVs), and Influenza A previously; namely, these viruses, which caused global concerns, are considered as targets for AZT different actions. Due to AZT background in the treatment of known viral infections mentioned above (which is described in this study), in the early stages of COVID-19 (a new zoonotic disease caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) development, AZT drew attention to itself due to its antiviral and immunomodulatory effects as a valuable candidate for COVID-19 treatment. AZT usage instructions for treating different viral infections have always been under observation, and COVID-19 is no exception. There are still debates about the use of AZT in COVID-19 treatment. However, eventually, novel researches convinced WHO to announce the discontinuation of AZT use (alone or in combination with hydroxychloroquine) in treating SARS-CoV-2 infection. This research aims to study the structure of all of the viruses mentioned above and the molecular and clinical effects of AZT against the virus., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

In the current context of the COVID-19 pandemic, it appears that our scientific resources and the medical community are not sufficiently developed to combat rapid viral spread all over the world. A number of viruses causing epidemics have already disseminated across the world in the last few years, such as the dengue or chinkungunya virus, the Ebola virus, and other coronavirus families such as Middle East respiratory syndrome (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV). The outbreaks of these infectious diseases have demonstrated the difficulty of treating an epidemic before the creation of vaccine. Different antiviral drugs already exist. However, several of them cause side effects or have lost their efficiency because of virus mutations. It is essential to develop new antiviral strategies, but ones that rely on more natural compounds to decrease the secondary effects. Polysaccharides, which have come to be known in recent years for their medicinal properties, including antiviral activities, are an excellent alternative. They are essential for the metabolism of plants, microorganisms, and animals, and are directly extractible. Polysaccharides have attracted more and more attention due to their therapeutic properties, low toxicity, and availability, and seem to be attractive candidates as antiviral drugs of tomorrow.

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013-2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement., Competing Interests: The authors have declared that no competing interests exist.

Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses., Competing Interests: JC has served as a consultant for Luna Biologics and Merck Sharp & Dohme Corp., is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics and AstraZeneca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Murin, Gilchuk, Crowe and Ward.)

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC 50(VeroE6)  = 2.3 μM; IC 50(Calu3)  = 0.24 μM), 3'-O-4-oxatetradodecanoyl (4b) (IC 50(VeroE6)  = 2.0 μM; IC 50(Calu3)  = 0.18 μM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC 50(VeroE6)  = 2.4 μM; IC 50(Calu3)  = 0.25 μM) derivatives exhibited less activity than RDV (IC 50(VeroE6)  = 0.85 μM; IC 50(Calu3)  = 0.06 μM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC 50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV., Competing Interests: Declaration of competing interest The increasing prevalence of COVID-19 is a severe public health problem affecting people globally. COVID-19 is taking a devastating toll on human lives. Several existing drugs and potential drug candidates such as remdesivir (RDV) have been considered for repurposing as COVID-19 treatments. Our group has previously studied the impact of fatty acylation on the antiviral activity of different anti-HIV (human immunodeficiency virus) nucleoside drugs. The conjugates were more potent and less toxic than their parent nucleoside analogs, providing a much higher selectivity index. Furthermore, the conjugates significantly enhanced the cellular uptake versus the parent analogs and corresponding physical mixtures of fatty acids and nucleosides. Herein, we report the synthesis of fatty acyl derivatives of RDV and structural characterization of their antiviral activity against SARS-CoV-2 in VeroE6 cells and Calu3 cells. Five fatty acids, myristic acid, 12-azidododecanoic acid, 12-thioethyldodecanoic acid, 4-oxatetradecanoic acid, and palmitic acid were conjugated to RDV at its 2′-O- and 3′-O-position. Most monofatty acyl derivatives of RDV demonstrated IC(50) values against SARS-CoV-2 that were only slightly decreased relative to RDV. Against the EBOV trVLP and infectious EBOV, any of the monofatty acyl conjugates had IC(50)s similar to that of RDV. Overall, these data together indicate that RDV can be modified with fatty acids at positions 2′ or 3′, without demonstrating a significant loss of antiviral activity in cell culture against SARS-CoV-2 and EBOV. Future studies will determine if these modifications result in greater long-acting effect, bioavailability, and stability in animal models., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Ebola virus (EBOV) belongs to the Filoviridae family and causes severe illnesses such as hemorrhagic fever with a high mortality rate up to 90%. Now two antibody drugs termed Inmazeb and Ebanga have been approved for treating EBOV infection. However, clinical studies have demonstrated that the mortality rate of the patients who received these two antibody drugs remains above 30%. Therefore, novel therapeutics with better efficacy is still desired. The isolated human IgG1 constant domain 2 (CH2 domain) has been proposed as a scaffold for the development of C-based single domain antibodies (C-sdAbs) as therapeutic candidates against viral infections and other diseases. Here, we screened and identified a novel C-sdAb termed M24 that targets EBOV glycoprotein (GP) from a C-sdAb phage display library. M24 neutralizes the pseudotype EBOV with IC 50 of 0.8 nmol/L (12 ng/mL) and has modest neutralizing activity against authentic EBOV. Epitope determination, including molecular docking and site mutation analysis, discloses that M24 binds to the internal fusion loop (IFL) within GP2, a transmembrane subunit of GP. Interestingly, we found that the binding of M24 to GP at pH 5.5 has dramatically decreased compared to the binding at pH 7.5, which may lead to weak efficacy in the neutralization of authentic EBOV. Since no sdAb against EBOV infection has been reported to date, our results not only give a proof of concept that sdAbs could be utilized for the development of potential therapeutic candidates against EBOV infection, but also provide useful information for the discovery and improvement of anti-EBOV agents., (© 2021. Wuhan Institute of Virology, CAS.)

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability-the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses., Competing Interests: KC is a member of the scientific advisory boards of Integrum Scientific, LLC, Biovaxys Technology Corp, and the Pandemic Security Initiative of Celdara Medical, LLC, and he has consulted for Axon Advisors, LLC. JL is a consultant for Celdara Medical, LLC. Author JT was employed by company Mapp Biopharmaceutical. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wirchnianski, Wec, Nyakatura, Herbert, Slough, Kuehne, Mittler, Jangra, Teruya, Dye, Lai and Chandran.)

Ebola virus (EBOV) has emerged as a significant public health concern since the 2013-2016 outbreak in West Africa. Currently, no effective antiviral treatments have been approved for clinical use. Compound 1 RYL-634 is a quinolone-derived compound that can inhibit dihydroorotate dehydrogenase, a rate-limiting enzyme in the de novo pyrimidine synthesis pathway and it exhibited antiviral activity against multiple RNA virus infection. In this study, we evaluated the efficacy of a panel of newly developed compounds based on RYL-634 against EBOV infection. Our data showed that RYL-634 as well as its derivatives are effective against EBOV transcription- and replication-competent virus-like particle (trVLP) infection and authentic EBOV infection in vitro at low nanomolar IC 50 values and relatively high CC 50 . Of note, the new derivative RYL-687 had the lowest IC 50 at approximately 7 nM and was almost 6 times more potent than remdesivir (GS-5734). Exogenous addition of different metabolites in the pyrimidine de novo synthesis pathway confirmed DHODH as the target of RYL-687. These data provide evidence that such quinolone-derived compounds are promising therapeutic candidates against EBOV infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease., (© 2021. The Author(s).)

Ebola virus (EBOV), one of the most infectious human viruses and a leading cause of viral hemorrhagic fever, imposes a potential public health threat with several recent outbreaks. Despite the difficulties associated with working with this pathogen in biosafety level-4 containment, a protective vaccine and antiviral therapeutic were recently approved. However, the high mortality rate of EBOV infection underscores the necessity to continuously identify novel antiviral strategies to help expand the scope of prophylaxis/therapeutic management against future outbreaks. This includes identifying antiviral agents that target EBOV entry, which could improve the management of EBOV infection. Herein, using EBOV glycoprotein (GP)-pseudotyped particles, we screened a panel of natural medicinal extracts, and identified the methanolic extract of Perilla frutescens (PFME) as a robust inhibitor of EBOV entry. We show that PFME dose-dependently impeded EBOV GP-mediated infection at non-cytotoxic concentrations, and exerted the most significant antiviral activity when both the extract and the pseudoparticles are concurrently present on the host cells. Specifically, we demonstrate that PFME could block viral attachment and neutralize the cell-free viral particles. Our results, therefore, identified PFME as a potent inhibitor of EBOV entry, which merits further evaluation for development as a therapeutic strategy against EBOV infection.

The unprecedented 2013-2016 West Africa Ebola outbreak accelerated several medical countermeasures (MCMs) against Ebola virus disease (EVD). Several investigational products (IPs) were used throughout the outbreak but were not conclusive for efficacy results. Only the Randomized Controlled Trial (RCT) on ZMapp was promising but inconclusive. More recently, during the second-largest Ebola outbreak in North Kivu and Ituri provinces, Democratic Republic of the Congo (DRC), four IPs, including one small molecule (Remdesivir), two monoclonal antibody (mAb) cocktails (ZMapp and REGN-EB3) and a single mAb (mAb114), were evaluated in an RCT, the Pamoja Tulinde Maisha (PALM) study. Two products (REGN-EB3 and mAb114) demonstrated efficacy as compared to the control arm, ZMapp. There were remarkably few side effects recorded in the trial. The FDA approved both medications in this scientifically sound study, marking a watershed moment in the field of EVD therapy. These products can be produced relatively inexpensively and can be stockpiled. The administration of mAbs in EVD patients appears to be safe and effective, while several critical knowledge gaps remain; the impact of early administration of Ebola-specific mAbs on developing a robust immune response for future Ebola virus exposure is unknown. The viral mutation escape, leading to resistance, presents a potential limitation for single mAb therapy; further improvements need to be explored. Understanding the contribution of Fc-mediated antibody functions such as antibody-dependent cellular cytotoxicity (ADCC) of those approved mAbs is still critical. The potential merit of combination therapy and post-exposure prophylaxis (PEP) need to be demonstrated. Furthermore, the PALM trial has accounted for 30% of mortality despite the administration of specific treatments. The putative role of EBOV soluble Glycoprotein (sGP) as a decoy to the immune system, the virus persistence, and relapses might be investigated for treatment failure. The development of pan-filovirus or pan-species mAbs remains essential for protection. The interaction between FDA-approved mAbs and vaccines remains unclear and needs to be investigated. In this review, we summarize the efficacy and safety results of the PALM study and review current research questions for the further development of mAbs in pre-exposure or emergency post-exposure use., Competing Interests: OTM is employed by Leidos Biomedical Research. SM is employed by Ridgeback Biotherapeutics, and is listed as inventor on the patent application for mAb 114, US Application No.62/087, 087 (PCT Application No. PCT/US2015/060733) related to anti-Ebola virus antibodies and their use. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tshiani Mbaya, Mukumbayi and Mulangu.)

In recent years, a number of novel filoviruses (e.g. Lloviu virus (LLOV) and Bombali virus (BOMV)) have been discovered. While antibody-based therapeutics have recently been approved for treatment of infections with the filovirus Ebola virus (EBOV), no treatment options for novel filoviruses currently exist. Further, the development of antivirals against them is complicated by the fact that only sequence information, but no actual virus isolates, are available. To address this issue, we developed a reverse genetics-based minigenome system for BOMV, which allows us to assess the activity of the BOMV polymerase. Together with similar systems that we have developed for other filoviruses in the past (i.e. LLOV and Reston virus (RESTV)), we then assessed the efficiency of remdesivir, a known inhibitor of the EBOV polymerase that has recently been tested in a clinical trial for efficacy against Ebola disease. We show that remdesivir is indeed also active against the polymerases of BOMV, LLOV, and RESTV, with comparable IC 50 values to its activity against EBOV. This suggests that treatment with remdesivir might represent a viable option in case of infections with novel filoviruses., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014-2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 10 1 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Ebola Virus (EBOV) is one of the deadliest pathogenic virus which causes hemorrhagic fever. Though many Ebola-human interaction studies and databases are already reported, the unavailability of an adequate model and lack of publically accessible resources requires a comprehensive study to curate the Ebola-Human-Drug interactions. In total, 270 human proteins interacted with EBOV are collected from published experimental evidence. Then the protein-protein interaction networks are generated as EBOV-human and EBOV-Human-Drugs interaction. These results can help the researcher to find the effective repurposed drug for EBOV treatment. Further, the illustration of gene enrichment and pathway analysis would provide knowledge and insight of EBOV-human interaction describes the importance of the study. Investigating the networks may help to identify a suitable human-based drug target for ebola research community. The inclusion of an emerging concept, a human-based drug targeted therapy plays a very significant role in drug repurposing which reduces the time and effort is the highlight of the current research. An integrated database namely, Ebolabase has been developed and linked with other repositories such as Epitopes, Structures, Literature, Genomics and Proteomics. All generated networks are made to be viewed in a customized manner and the required data can be downloaded freely. The Ebolabase is available at http://ebola.bicpu.edu.in., (Copyright © 2021. Published by Elsevier B.V.)

Remdesivir is an antiviral drug initially designed against the Ebola virus. The results obtained with it both in biochemical studies in vitro and in cell line assays in vivo were very promising, but it proved to be ineffective in clinical trials. Remdesivir exhibited far better efficacy when repurposed against SARS-CoV-2. The chemistry that accounts for this difference is the subject of this study. Here, we examine the hypothesis that remdesivir monophosphate (RMP)-containing RNA functions as a template at the polymerase site for the second run of RNA synthesis, and as mRNA at the decoding center for protein synthesis. Our hypothesis is supported by the observation that RMP can be incorporated into RNA by the RNA-dependent RNA polymerases (RdRps) of both viruses, although some of the incorporated RMPs are subsequently removed by exoribonucleases. Furthermore, our hypothesis is consistent with the fact that RdRp of SARS-CoV-2 selects RMP for incorporation over AMP by 3-fold in vitro , and that RMP-added RNA can be rapidly extended, even though primer extension is often paused when the added RMP is translocated at the i + 3 position (with i the nascent base pair at an initial insertion site of RMP) or when the concentrations of the subsequent nucleoside triphosphates (NTPs) are below their physiological concentrations. These observations have led to the hypothesis that remdesivir might be a delayed chain terminator. However, that hypothesis is challenged under physiological concentrations of NTPs by the observation that approximately three-quarters of RNA products efficiently overrun the pause.

In seeking to increase the library of fluorine containing adenine-derived carbocyclic nucleoside antiviral candidates, d-like and l-like 6'-fluoro-3-deazaneplanocin and its 3-bromo derivative lacking the 4'-hydroxylmethylene substituent (2/3 and 4/5, respectively) are presented. Their synthesis was accomplished from d-ribose by developing a more facile precursor route than suggested by the literature. The 2/4d-like pair displayed significant anti-filo virial properties while the enantiomeric l-like congeners 3/5 were inactive. Target compounds 2/4 also were active towards measles and norovirus. The effect of 2/4 is further evidence of the role fluoro-derived adenine carbocyclic nucleoside can play in antiviral drug discovery. Furthermore, the simplicity of their synthesis lends them to more efficacious analogs and to scale-up optimization. There were no other relevant antiviral properties for 2/3 and 4/5 (except BK polyomavirus for 3/5)., (Copyright © 2021. Published by Elsevier Ltd.)

Filoviruses, mainly consisting of Ebola viruses (EBOV) and Marburg viruses (MARV), are enveloped negative-strand RNA viruses which can infect humans to cause severe hemorrhagic fevers and outbreaks with high mortality rates. The filovirus infection is mediated by the interaction of viral envelope glycoprotein (GP) and the human endosomal receptor Niemann-Pick C1 (NPC1). Blocking this interaction will prevent the infection. Therefore, we utilized an In silico screening approach to conduct virtual compound screening against the NPC1 receptor-binding site (RBS). Twenty-six top-hit compounds were purchased and evaluated by in vitro cell based inhibition assays against pseudotyped or replication-competent filoviruses. Two classes (A and U) of compounds were identified to have potent inhibitory activity against both Ebola and Marburg viruses. The IC 50 values are in the lower level of micromolar concentrations. One compound (compd-A) was found to have a sub-micromolar IC 50 value (0.86 μM) against pseudotyped Marburg virus. The cytotoxicity assay (MTT) indicates that compd-A has a moderate cytotoxicity level but the compd-U has much less toxicity and the CC 50 value was about 100 μM. Structure-activity relationship (SAR) study has found some analogs of compd-A and -U have reduced the toxicity and enhanced the inhibitory activity. In conclusion, this work has identified several qualified lead-compounds for further drug development against filovirus infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Human pathogenic RNA viruses are threats to public health because they are prone to escaping the human immune system through mutations of genomic RNA, thereby causing local outbreaks and global pandemics of emerging or re-emerging viral diseases. While specific therapeutics and vaccines are being developed, a broad-spectrum therapeutic agent for RNA viruses would be beneficial for targeting newly emerging and mutated RNA viruses. In this study, we conducted a screen of repurposed drugs using Sendai virus (an RNA virus of the family Paramyxoviridae), with human-induced pluripotent stem cells (iPSCs) to explore existing drugs that may present anti-RNA viral activity. Selected hit compounds were evaluated for their efficacy against two important human pathogens: Ebola virus (EBOV) using Huh7 cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using Vero E6 cells. Selective estrogen receptor modulators (SERMs), including raloxifene, exhibited antiviral activities against EBOV and SARS-CoV-2. Pioglitazone, a PPARγ agonist, also exhibited antiviral activities against SARS-CoV-2, and both raloxifene and pioglitazone presented a synergistic antiviral effect. Finally, we demonstrated that SERMs blocked entry steps of SARS-CoV-2 into host cells. These findings suggest that the identified FDA-approved drugs can modulate host cell susceptibility against RNA viruses., (© 2021 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

To date, the 'one bug-one drug' approach to antiviral drug development cannot effectively respond to the constant threat posed by an increasing diversity of viruses causing outbreaks of viral infections that turn out to be pathogenic for humans. Evidently, there is an urgent need for new strategies to develop efficient antiviral agents with broad-spectrum activities. In this paper, we identified camphene derivatives that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses, including influenza virus A/PR/8/34 (H1N1), Ebola virus (EBOV), and the Hantaan virus. The lead-compound 2a , with pyrrolidine cycle in its structure, displayed antiviral activity against influenza virus (IC 50 = 45.3 µM), Ebola pseudotype viruses (IC 50 = 0.12 µM), and authentic EBOV (IC 50 = 18.3 µM), as well as against pseudoviruses with Hantaan virus Gn-Gc glycoprotein (IC 50 = 9.1 µM). The results of antiviral activity studies using pseudotype viruses and molecular modeling suggest that surface proteins of the viruses required for the fusion process between viral and cellular membranes are the likely target of compound 2a . The key structural fragments responsible for efficient binding are the bicyclic natural framework and the nitrogen atom. These data encourage us to conduct further investigations using bicyclic monoterpenoids as a scaffold for the rational design of membrane-fusion targeting inhibitors.

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies., Competing Interests: Declaration of interests A.L.B., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is a shareholder of Integral Molecular. J.E.C. has served as a consultant for Lilly and Luna Biologics, is a member of the Scientific Advisory Boards of CompuVax and Meissa Vaccines, and is the founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from and IDBiologics and AstraZeneca. Vanderbilt University has applied for a patent that is related to antibodies discussed in this work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ansuvimab (ansuvimab-zykl; EBANGA™) is a human monoclonal antibody developed by Ridgeback Biotherapeutics, which binds to the glycoprotein on Zaire ebolavirus (Ebola virus) to block its entry into host cells. Ansuvimab has been recently approved in the USA for the treatment of infection caused by Z. ebolavirus in adult and paediatric patients, including in neonates born to a mother who is RT-PCR positive for Z. ebolavirus infection, following the results of the PALM phase II/III trial. This article summarizes the milestones in the development of ansuvimab leading to this first approval for the treatment of infections caused by Ebola virus in adults and paediatric patients.

Ansuvimab-zykl (Ebanga), a human monoclonal antibody, has been approved to treat Zaire ebolavirus (Ebolavirus) infection.The most common adverse effects of the drug are fever, tachycardia, diarrhea, vomiting, hypotension, tachypnea, and chills; these are also common symptoms of Ebolavirus infection., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: To support the rapid development of an antibody cocktail against Ebola virus and avoid unnecessary exposure to infectious environments, an automatic and fast turnover triplex assay was developed using Simoa ®  (Quanterix Corporation, MA, USA). Materials & methods: A robust triplex assay was developed and validated for simultaneous quantification of the antibody cocktail against Ebola virus in cynomolgus serum. Results: The assay had a quantitation range of 78.1-5000 ng/ml. The intra- and interassay precisions (%CV) were within 11.4 and 13.9%, and the accuracies (%RE) were within -10.8 to 6.8%, respectively. Cross-reactivity was evaluated, and the results met the acceptance criteria. Conclusion: The assay was successfully applied to a pharmacokinetics study following a single-dose intravenous administration of 10 mg/kg the antibody cocktail against Ebola virus to cynomolgus monkeys.

Ebola virus has been responsible for two major epidemics over the last several years and there has been a strong effort to find potential treatments that can improve the disease outcome. Antiviral favipiravir was thus tested on non-human primates infected with Ebola virus. Half of the treated animals survived the Ebola virus challenge, whereas the infection was fully lethal for the untreated ones. Moreover, the treated animals that did not survive died later than the controls. We evaluated the hematological, virological, biochemical, and immunological parameters of the animals and performed proteomic analysis at various timepoints of the disease. The viral load strongly correlated with dysregulation of the biological functions involved in pathogenesis, notably the inflammatory response, hemostatic functions, and response to stress. Thus, the management of viral replication in Ebola virus disease is of crucial importance in preventing the immunopathogenic disorders and septic-like shock syndrome generally observed in Ebola virus-infected patients., Competing Interests: The authors have declared that no competing interests exist.

Introduction . The consistent emergence/reemergence of filoviruses into a world that previously lacked an approved pharmaceutical intervention parallels an experience repeatedly played-out for most other emerging pathogenic zoonotic viruses. Investment to preemptively develop effective and low-cost prophylactic and therapeutic interventions against viruses that have high potential for emergence and societal impact should be a priority. Areas covered . Candidate drugs can be characterized into those that interfere with cellular processes required for Ebola virus (EBOV) replication (host-directed), and those that directly target virally encoded functions (direct-acting). We discuss strategies to identify pharmaceutical interventions for EBOV infections. PubMed/Web of Science databases were searched to establish a detailed catalog of these interventions. Expert opinion. Many drug candidates show promising in vitro inhibitory activity, but experience with EBOV shows the general lack of translation to in vivo efficacy for host-directed repurposed drugs. Better translation is seen for direct-acting antivirals, in particular monoclonal antibodies. The FDA-approved monoclonal antibody treatment, Inmazeb™ is a success story that could be improved in terms of impact on EBOV-associated disease and mortality, possibly by combination with other direct-acting agents targeting distinct aspects of the viral replication cycle. Costs need to be addressed given EBOV emergence primarily in under-resourced countries.

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development., Competing Interests: The authors have declared that no competing interests exist.

The endocytic pathway is a common strategy that several highly pathogenic viruses use to enter into the cell. To demonstrate the usefulness of this pathway as a common target for the development of broad-spectrum antivirals, the inhibitory effect of drug compounds targeting endosomal membrane proteins were investigated. This study entailed direct comparison of drug effectiveness against animal and human pathogenic viruses, namely Ebola (EBOV), African swine fever virus (ASFV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A panel of experimental and FDA-approved compounds targeting calcium channels and PIKfyve at the endosomal membrane caused potent reductions of entry up to 90% in SARS-CoV-2 S-protein pseudotyped retrovirus. Similar inhibition was observed against transduced EBOV glycoprotein pseudovirus and ASFV. SARS-CoV-2 infection was potently inhibited by selective estrogen receptor modulators in cells transduced with pseudovirus, among them Raloxifen inhibited ASFV with very low 50% inhibitory concentration. Finally, the mechanism of the inhibition caused by the latter in ASFV infection was analyzed. Overall, this work shows that cellular proteins related to the endocytic pathway can constitute suitable cellular targets for broad range antiviral compounds., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Following vaccination with the live attenuated, recombinant vesicular stomatitis virus Indiana serotype Ebola virus (rVSV-EBOV) vaccine, persons may exhibit a transient vaccine-associated viremia. To investigate the potential for Old World sand flies to transmit this vaccine following feeding on a viremic person, we fed laboratory-reared Phlebotomus papatasi an artificial blood meal containing 7.2 log10 plaque-forming units of rVSV-EBOV. Replication or dissemination was not detected in the body or legs of any P. papatasi collected at seven (n = 75) or 15 (n = 75) days post-feed. These results indicate a low potential for rVSV-EBOV to replicate and disseminate in P. papatasi, a species whose geographic distribution ranges from Morocco to southwest Asia and as far north as southern Europe.

Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi-pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi-pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi-pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi-pi stacking without disrupting the overall lectin structure. These findings show that pi-pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes.

Ebola virus (EBOV), a member of the mononegaviral family Filoviridae, causes severe disease associated with high lethality in humans. Despite enormous progress in development of EBOV medical countermeasures, no anti-EBOV treatment has been approved. We designed an immunotoxin in which a single-chain variable region fragment of the EBOV glycoprotein-specific monoclonal antibody 6D8 was fused to the effector domains of Pseudomonas aeruginosa exotoxin A (PE38). This immunotoxin, 6D8-PE38, bound specifically to cells expressing EBOV glycoproteins. Importantly, 6D8-PE38 targeted EBOV-infected cells, as evidenced by inhibition of infectious EBOV production from infected cells, including primary human macrophages. The data presented here provide a proof of concept for immunotoxin-based targeted killing of infected cells as a potential antiviral intervention for Ebola virus disease., Competing Interests: This work was supported in part through Battelle Memorial Institute’s former prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I and Laulima Government Solutions, LLC current prime contract with NIAID under Contract No. HHSN272201800013C (S.Y.). Y.C. and J.H.K. performed this work as former employees of Battelle Memorial Institute and current employees of Tunnel Government Services (TGS), a subcontractor of Laulima Government Solutions, LLC under Contract No. HHSN272201800013C. Battelle Memorial Institute/Laulima Government Solutions/Tunnell Government Services serve(d) as employers for government contractors only and had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials

REGN-EB3 (INMAZEB ® , Regeneron Pharmaceuticals) is a combination of three fully-human monoclonal antibodies-atoltivimab (REGN3470), maftivimab (REGN3479), and odesivimab (REGN3471) -that target Ebola virus glycoprotein. Based on the results of the PALM study conducted during an Ebola outbreak in the Democratic Republic of Congo, REGN-EB3 was recently approved by the US FDA as a treatment for Ebola virus infection. This article summarizes the milestones in the development of REGN-EB3 leading to this first approval for the treatment of infection caused by Zaire ebolavirus (Ebola virus) in adult and paediatric patients.

Filoviruses, such as Ebola virus and Marburg virus, are of significant human health concern. From 2013 to 2016, Ebola virus caused 11,323 fatalities in Western Africa. Since 2018, two Ebola virus disease outbreaks in the Democratic Republic of the Congo resulted in 2354 fatalities. Although there is progress in medical countermeasure (MCM) development (in particular, vaccines and antibody-based therapeutics), the need for efficacious small-molecule therapeutics remains unmet. Here we describe a novel high-throughput screening assay to identify inhibitors of Ebola virus VP40 matrix protein association with viral particle assembly sites on the interior of the host cell plasma membrane. Using this assay, we screened nearly 3000 small molecules and identified several molecules with the desired inhibitory properties. In secondary assays, one identified compound, sangivamycin, inhibited not only Ebola viral infectivity but also that of other viruses. This finding indicates that it is possible for this new VP40-based screening method to identify highly potent MCMs against Ebola virus and its relatives.

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.

In this study, we screened a large library of (+)-camphor and (-)-borneol derivatives to assess their filovirus entry inhibition activities using pseudotype systems. Structure-activity relationship studies revealed several compounds exhibiting submicromolar IC 50 values. These compounds were evaluated for their effect against natural Ebola virus (EBOV) and Marburg virus. Compound 3b (As-358) exhibited the good antiviral potency (IC 50  = 3.7 μM, SI = 118) against Marburg virus, while the hydrochloride salt of this compound 3b·HCl had a strong inhibitory effect against Ebola virus (IC 50  = 9.1 μM, SI = 31) and good in vivo safety (LD 50  > 1000 mg/kg). The results of molecular docking and in vitro mutagenesis analyses suggest that the synthesized compounds bind to the active binding site of EBOV glycoprotein similar to the known inhibitor toremifene., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

At a time when the world faces an emotional breakdown, crushing our dreams, if not, taking our lives, we realize that together we must fight the war against the COVID-19 outbreak even if almost the majority of the scientific community finds itself confined at home. Every day, we, scientists, listen to the latest news with its promises and announcements. Across the world, a surge of clinical trials trying to cure or slow down the coronavirus pandemic has been launched to bring hope instead of fear and despair. One first proposed clinical trial has drawn worldwide hype to the benefit of chloroquine (CQ), in the treatment of patients infected by the recently emerged deadly coronavirus (SARS-CoV-2). We should consider this information in light of the long-standing anti-inflammatory and anti-viral properties of CQ-related drugs. Yet, none of the articles promoting the use of CQ in the current pandemic evoked a possible molecular or cellular mechanism of action that could account for any efficacy. Here, given the interaction of viruses with macroautophagy (hereafter referred to as autophagy), a CQ-sensitive anti-viral safeguard pathway, we would like to discuss the pros, but also the cons concerning the current therapeutic options targeting this process.