High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

Mannose-binding lectin (MBL) targets diverse microorganisms for phagocytosis and complement-mediated lysis by binding specific surface glycans. Although recombinant human MBL (rhMBL) trials have focused on reconstitution therapy, safety studies have identified no barriers to its use at higher levels. Ebola viruses cause fatal hemorrhagic fevers for which no treatment exists and that are feared as potential biothreat agents. We found that mice whose rhMBL serum concentrations were increased ≥7-fold above average human levels survived otherwise fatal Ebola virus infections and became immune to virus rechallenge. Because Ebola glycoproteins potentially model other glycosylated viruses, rhMBL may offer a novel broad-spectrum antiviral approach

Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine

Synthesis and structure–activity relationships of 3,5-disubstituted-pyrrolo[2,3-b]pyridines as inhibitors of adaptor-associated kinase 1 with antiviral activity

There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine–threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3-b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy621258105831FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP106169/ZZ14/ZThis work was supported by award number W81XWH-16-1− 0691 from the Department of Defense (DoD), Congressionally Directed Medical Research Programs (CDMRP) to S.E., P.H.; Grant 12393481 from the Defense Threat Reduction Agency (DTRA), Fundamental Research to Counter Weapons of Mass Destruction to S.E., P.H.; and seed grant from the Stanford SPARK program. S.V. is the recipient of a doctoral fellowship from the Research FoundationFlanders (1S00116N). S.P. was supported by the Child Health Research Institute, Lucile Packard Foundation for Children’s Health and the Stanford CSTA (grant number UL1 TR000093). The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, Sao Paulo Research Foundation-FAPESP, Takeda, and ̃ Wellcome [106169/ZZ14/Z

Synthesis and Structure-Activity Relationships of 3,5-Disubstituted-pyrrolo[2,3-b]pyridines as Inhibitors of Adaptor-Associated Kinase 1 with Antiviral Activity

There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine-threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3- b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy.status: publishe

Optimization of Isothiazolo[4,3‑<i>b</i>]­pyridine-Based Inhibitors of Cyclin G Associated Kinase (GAK) with Broad-Spectrum Antiviral Activity

There is an urgent need for strategies to combat dengue and other emerging viral infections. We reported that cyclin G-associated kinase (GAK), a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, regulates intracellular trafficking of multiple unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the discovery of potent, selective GAK inhibitors based on an isothiazolo­[4,3-<i>b</i>]­pyridine scaffold, albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery of novel isothiazolo­[4,3-<i>b</i>]­pyridines that maintain high GAK affinity and selectivity. These compounds demonstrate improved in vitro activity against dengue virus, including in human primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses. Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral action of these compounds. These findings demonstrate the potential utility of a GAK-targeted broad-spectrum approach for combating currently untreatable emerging viral infections

Calcium Regulation of Hemorrhagic Fever Virus Budding: Mechanistic Implications for Host-Oriented Therapeutic Intervention

<div><p>Hemorrhagic fever viruses, including the filoviruses (Ebola and Marburg) and arenaviruses (Lassa and Junín viruses), are serious human pathogens for which there are currently no FDA approved therapeutics or vaccines. Importantly, transmission of these viruses, and specifically late steps of budding, critically depend upon host cell machinery. Consequently, strategies which target these mechanisms represent potential targets for broad spectrum host oriented therapeutics. An important cellular signal implicated previously in EBOV budding is calcium. Indeed, host cell calcium signals are increasingly being recognized to play a role in steps of entry, replication, and transmission for a range of viruses, but if and how filoviruses and arenaviruses mobilize calcium and the precise stage of virus transmission regulated by calcium have not been defined. Here we demonstrate that expression of matrix proteins from both filoviruses and arenaviruses triggers an increase in host cytoplasmic Ca²⁺ concentration by a mechanism that requires host Orai1 channels. Furthermore, we demonstrate that Orai1 regulates both VLP and infectious filovirus and arenavirus production and spread. Notably, suppression of the protein that triggers Orai activation (Stromal Interaction Molecule 1, STIM1) and genetic inactivation or pharmacological blockade of Orai1 channels inhibits VLP and infectious virus egress. These findings are highly significant as they expand our understanding of host mechanisms that may broadly control enveloped RNA virus budding, and they establish Orai and STIM1 as novel targets for broad-spectrum host-oriented therapeutics to combat these emerging BSL-4 pathogens and potentially other enveloped RNA viruses that bud via similar mechanisms.</p></div

Synta66 decreases the number of viral particles in supernatants.

<p>A virus plaque assay used to quantify infectious virions in supernatants of virus infected cells demonstrates that Synta66 (30 μM) significantly inhibits the budding and spread of live LASV, JUNV, MARV, and EBOV. Error bars indicate standard error of mean (SEM) and statistical significance was determined by two way ANOVA with Bonferroni multiple comparisons (**p<0.01, ***p < 0.001, ****p < 0.0001).</p

Viral matrix protein expression mobilizes cytoplasmic Ca²⁺.

<p>Viral matrix proteins or vector controls and the genetically encoded Ca²⁺ indicator R-GECO-1 were co-expressed in WT HEK293T cells, HEK293T cells stably expressing the dominant negative permeation defective Orai (E106A) mutant, or WT HEK293T cells treated with the Orai inhibitor Synta66 (50 μM, lower right). Cytoplasmic R-GECO-1 fluorescence emission (580nM) was monitored between 6 and 24 hours post transfection from cells cultured in an environmentally controlled chamber on the microscope stage. Each trace represents the average +/- SEM of at least two experiments of at least 20 cells.</p