Claudia S. Sepúlveda

Imidazo[2,1-b]thiazole carbohydrate derivatives: Synthesis and antiviral activity against Junin virus, agent of Argentine hemorrhagic fever

Herein, we describe the syntheses of 3,5-disubstituted imidazo[2,1-b]thiazole. The cyclization step was performed in two different conditions by using either thermal or microwave heating. Comparing the results of both methodologies, we found that the microwave assistance is an improved alternative to obtain this family of heterocyclic compound. Compounds were first evaluated for cytotoxicity in Vero cells by MTT method and then, the antiviral activity was assayed by a virus yield inhibition assay in the range of concentrations lower than the corresponding CC(50), using JUNV strain IV4454 as the model system. The most active compounds (3 and 4), showed a level of antiviral activity against JUNV in monkey Vero cells better than the reference substance ribavirin. Then, they are promising lead compound for further analysis and characterization to establish their therapeutic potential against hemorrhagic fever viruses.

Inhibition of Junin virus RNA synthesis by an antiviral acridone derivative

There are no specific approved drugs for the treatment of agents of viral hemorrhagic fevers (HF) and antiviral therapies against these viruses are urgently needed. The present study characterizes the potent and selective antiviral activity against the HF causing arenavirus Junin virus (JUNV) of the compound 10-allyl-6-chloro-4-methoxy-9(10H)-acridone, designated 3f. The effectiveness of 3f to inhibit JUNV multiplication was not importantly affected by the initial multiplicity of infection, with similar effective concentration 50% (EC(50)) values in virus yield inhibition assays performed in Vero cells in the range of 0.2-40 plaque forming units (PFU)/cell. Mechanistic studies demonstrated that 3f did not affect the initial steps of adsorption and internalization. The subsequent process of viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR in compound-treated cells relative to non-treated cells. The addition of exogenous guanosine rescued the infectivity and RNA synthesis of JUNV in 3f-treated cells in a dose-dependent manner, but the reversal was partial, suggesting that the reduction of the GTP pool contributed to the antiviral activity of 3f, but it was not the main operative mechanism. The comparison of 3f with two other viral RNA inhibitors, ribavirin and mycophenolic acid, showed that ribavirin did not act against JUNV through the cellular enzyme inosine monophosphate dehydrogenase (IMPDH) inhibition whereas the anti-JUNV activity of mycophenolic acid was mainly targeted at this enzyme.

Lichen secondary metabolites from the cultured lichen mycobionts of Teloschistes chrysophthalmus and Ramalina celastri and their antiviral activities.

Lichens and spore-derived cultured mycobionts of Teloschistes chrysophthalmus and Ramalina celastri were studied chemically, and results indicated that they produced, respectively, parietin and usnic acid as major secondary metabolites, which were purified and identified. Identification of the compounds was performed by high performance liquid chromatography and structural elucidation by nuclear magnetic resonance (1H) and electron impact mass spectrometry. Usnic acid exhibited antiviral activity whereas parietin had a virucidal effect against the arenaviruses Junín and Tacaribe

Synthesis and antiviral activity of azoles obtained from carbohydrates

Herein we describe the synthesis of 1,2,4-triazolyl-3-thione;1,3,4-oxadiazole, and imidazo[2,1-b]thiazole derivatives from carbohydrates. The antiviral activity of these compounds was tested against Dengue and Junin virus (the etiological agent of Argentine hemorrhagic fever). The 3-(p-bromobenzoyl)-5-(1,2-O-isopropylidene-3-O-methyl-alpha-d-xylofuranos-5-ulos-5-yl)imidazo[2,1-b]thiazole was able to inhibit the replication of both viruses in Vero cells at concentration significantly lower than the CC(50).

Synthesis and evaluation of N-substituted acridones as antiviral agents against haemorrhagic fever viruses.

Background: In the present study, a series of N-substituted acridone derivatives was synthesized and evaluated against two haemorrhagic fever viruses (HFV). Methods: Compounds were tested against Junin virus (JUNV), an arenavirus agent of Argentine haemorrhagic fever, and dengue virus (DENV), a flavivirus agent of the most prevalent arthropod-borne viral disease in humans. Results: Among tested compounds, two N-allyl acridones (derivatives 3c and 3f) elicited a potent and selective antiviral activity against JUNV (strain IV4454) and DENV-2 (strain NGC) with 50% effective concentration values between 2.5 and 5.5 µM, as determined by virus yield inhibition. No cytotoxicity was detected at concentrations up to 1,000 µM, resulting in selectivity indices > 181.8–400.0. Both acridones were effective against a wide spectrum of arenaviruses and the four serotypes of DENV. Furthermore, 3c and 3f failed to inactivate virus before cell infection as well as to induce a refractory state by cell pretreatment, indicating that the inhibitory effect was exerted through a blockade in virus multiplication during the infectious process. Conclusion: These data are the first demonstration that acridone derivatives have a potent antiviral activity that block in vitro multiplication of HFV belonging to Arenaviridae and Flaviviridae, such as JUNV and DENV.

Acridones As Antiviral Agents: Synthesis, Chemical and Biological Properties

Acridones are a class of compounds that have attracted attention in recent years for their wide range of biological properties, including selective inhibition of diverse human pathogenic viruses. The wide spectrum of antiviral activity includes DNA and RNA viruses, such as herpes simplex virus, cytomegalovirus, adenovirus, hepatitis C virus, dengue virus, and Junin virus, among others, indicative of the involvement of cellular factors as potential targets of acridone derivatives. At the present, their precise mode of action is not clearly determined, although the predominant action seems to be centered on the synthesis of nucleic acids. Regarding this point, inhibitory activity against cellular and viral enzymes and the ability to intercalate into nucleic acid molecules was demonstrated for some acridone compounds. Then, the possibility of a multiple effect on different targets renewed interest in these agents for virus chemotherapy allowing a potent inhibitory effectiveness associated to less feasibility of generating antiviral resistance. This review summarizes the current knowledge regarding the methods of synthesis, the antiviral properties of acridone derivatives, their mechanism of action, and structural characteristics related to antiviral activity as well as the perspectives of this class of compounds for clinical application against human viral infections.

Inhibition of arenavirus infection by thiuram and aromatic disulfides

A selected group of aromatic disulfides, thiuram disulfides and thiosulfones, provided by the National Cancer Institute, were evaluated in vitro for their inhibitory activity against Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever. The aromatic disulfides NSC4492 and NSC71033 and the thiuram disulfide NSC14560 were, respectively, the more potent virucidal and antiviral agents against JUNV, with inactivating concentration 50% (IC(50)) values of 0.2-0.5 microM for virucidal compounds and antiviral effective concentration 50% (EC(50)) of 8.5 microM for NSC14560. Both types of compounds exhibited inhibitory activity against three arenaviruses. Additionally, a comparable efficacy in the antiviral action of NSC14560 was observed in monkey, hamster or human cells with selectivity indices in the range 55.9-85.7. Time of addition experiments showed that the main antiviral activity of NSC14560 was situated before 5h of infection, but a significant inhibition was still observed when the compound was added up 9h p.i. This compound did not induce a refractory state to infection by cell pretreatment. Nor did it prevent viral entry, but the cytoplasmic and membrane expression of the main viral proteins was inhibited. The possible involvement of the RING finger motif of arenavirus Z protein as target for the thiuram disulfide is discussed.

AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication

We have recently demonstrated that AR‐12 (OSU‐03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR‐12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR‐12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α—dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over‐expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR‐12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR‐12—stimulated the co‐localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug‐induced autophagosome formation and reduced the anti‐viral protection afforded by AR‐12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR‐12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR‐12 acts as a broad‐specificity anti‐viral drug in vitro and in vivo. We argue future patient studies with AR‐12 are warranted. J. Cell. Physiol. 231: 2286–2302, 2016.

Targeting of arenavirus RNA synthesis by a carboxamide-derivatized aromatic disulfide with virucidal activity.

Several arenaviruses can cause severe hemorrhagic fever (HF) in humans, representing a public health threat in endemic areas of Africa and South America. The present study characterizes the potent virucidal activity of the carboxamide-derivatized aromatic disulfide NSC4492, an antiretroviral zinc finger-reactive compound, against Junín virus (JUNV), the causative agent of Argentine HF. The compound was able to inactivate JUNV in a time and temperature-dependent manner, producing more than 99 % reduction in virus titer upon incubation with virions at 37°C for 90 min. The ability of NSC4492-treated JUNV to go through different steps of the multiplication cycle was then evaluated. Inactivated virions were able to bind and enter into the host cell with similar efficiency as control infectious particles. In contrast, treatment with NSC4492 impaired the capacity of JUNV to drive viral RNA synthesis, as measured by quantitative RT-PCR, and blocked viral protein expression, as determined by indirect immunofluorescence. These results suggest that the disulfide NSC4492 targets on the arenavirus replication complex leading to impairment in viral RNA synthesis. Additionally, analysis of VLP produced in NSC4492-treated cells expressing JUNV matrix Z protein revealed that the compound may interact with Z resulting in an altered aggregation behavior of this protein, but without affecting its intrinsic self-budding properties. The potential perspectives of NSC4492 as an inactivating vaccinal compound for pathogenic arenaviruses are discussed.

Antiviral activity of A771726, the active metabolite of leflunomide, against Junín virus

The aim of this study was to investigate the effect of A771726, the active metabolite of leflunomide, (CONICET‐UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad against the infection with Junín virus (JUNV), agent of Argentine hemorrhagic fever (AHF). The treatment with non‐cytotoxic concentrations of A771726 of Vero and A549 cells infected with JUNV inhibited virus replication in a dose‐dependent manner, as determined by virus yield reduction assay. The antiviral effectiveness of A771726 was not importantly affected by the multiplicity of infection and the virus strain. Moreover, the combination of A771726 and ribavirin had a significantly more potent antiviral activity than each single drug treatment. Mechanistic studies showed that the main action of A771726 is exerted before 6u2009h of JUNV infection. Accordingly, inhibition of viral RNA synthesis was detected in treated infected cells by real time RT‐PCR. The exogenous addition of uridine or orotic acid produced a partial reversal of the inhibitory effect of A771726 on infective virus production whereas a total reversion was detected on JUNV RNA synthesis, probably by restoration of the enzymatic activity of dihydroorotate dehydrogenase (DHODH) and the intracellular pyrimidine pools. In conclusion, these results suggest that the antiviral target would be viral RNA synthesis through pyrimidine depletion, but any other effect of the compound on JUNV infection cannot be excluded. This study opens the possibility of the therapeutic application of a wide spectrum host‐targeted compound alone or in combination with ribavirin to combat AHF as well as other human pathogenic arenaviruses.