Host Cell Targets for Unconventional Antivirals against RNA Viruses.

The recent COVID-19 crisis has highlighted the importance of RNA-based viruses. The most prominent members of this group are SARS-CoV-2 (coronavirus), HIV (human immunodeficiency virus), EBOV (Ebola virus), DENV (dengue virus), HCV (hepatitis C virus), ZIKV (Zika virus), CHIKV (chikungunya virus), and influenza A virus. With the exception of retroviruses which produce reverse transcriptase, the majority of RNA viruses encode RNA-dependent RNA polymerases which do not include molecular proofreading tools, underlying the high mutation capacity of these viruses as they multiply in the host cells. Together with their ability to manipulate the immune system of the host in different ways, their high mutation frequency poses a challenge to develop effective and durable vaccination and/or treatments. Consequently, the use of antiviral targeting agents, while an important part of the therapeutic strategy against infection, may lead to the selection of drug-resistant variants. The crucial role of the host cell replicative and processing machinery is essential for the replicative cycle of the viruses and has driven attention to the potential use of drugs directed to the host machinery as therapeutic alternatives to treat viral infections. In this review, we discuss small molecules with antiviral effects that target cellular factors in different steps of the infectious cycle of many RNA viruses. We emphasize the repurposing of FDA-approved drugs with broad-spectrum antiviral activity. Finally, we postulate that the ferruginol analog (18-(phthalimide-2-yl) ferruginol) is a potential host-targeted antiviral.

A new host-targeted antiviral cyclolignan (SAU-22.107) for Dengue Virus infection in cell cultures. Potential action mechanisms based on cell imaging.

Dengue virus (DENV) infection is the most arbovirosis in the world. However, medications have not been approved for its treatment. Drug discovery based on the host-targeted antiviral (HTA) constitutes a new promising strategy, considering their high genetic barrier to resistance and the low probability of selecting drug resistance strains. In this study, we have tested fifty-seven podophyllotoxin-related cyclolignans on DENV-2 infected cells and found the most promising compound was S.71. Using cellular and molecular biology experiments, we have discovered that the new lignan altered the distribution of microtubules, induced changes in cell morphology, and caused retraction of the rough endoplasmic reticulum. In addition, the compound alters the viral envelope protein and the double-stranded RNA, while there is a decrease in negative-strand RNA synthesis; especially when the compound was added between 6- and 12-hours post-infection. Altogether, S.71 decreases the viral yield through an HTA-related mechanism of action, possibly altering the DENV genome replication and/or polyprotein translation, through the alteration of microtubule distribution and endoplasmic reticulum deterioration. Finally, pharmacokinetic predictors show that S.71 falls within the standard ranges established for drugs.

Antifungal Activity of Amphiphilic Perylene Bisimides.

Perylene-based compounds, either naturally occurring or synthetic, have shown interesting biological activities. In this study, we report on the broad-spectrum antifungal properties of two lead amphiphilic perylene bisimides, compounds 4 and 5, which were synthesized from perylene-3,4,9,10-tetracarboxylic dianhydride by condensation with spermine and an ammonium salt formation. The antifungal activity was evaluated using a collection of fungal strains and clinical isolates from patients with onychomycosis or sporotrichosis. Both molecules displayed an interesting antifungal profile with MIC values in the range of 2-25 µM, being as active as several reference drugs, even more potent in some particular strains. The ammonium trifluoroacetate salt 5 showed the highest activity with a MIC value of 2.1 µM for all tested Candida spp., two Cryptococcus spp., two Fusarium spp., and one Neoscytalidium spp. strain. Therefore, these amphiphilic molecules with the perylene moiety and cationic ammonium side chains represent important structural features for the development of novel antifungals.

A rapid and simple quantitative method for specific detection of smaller coterminal RNA by PCR (DeSCo-PCR): application to the detection of viral subgenomic RNAs.

RNAs that are 5'-truncated versions of a longer RNA but share the same 3' terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to-and amplify-the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost, and specific detection of these truncated RNAs, we report detection of smaller coterminal RNA by PCR (DeSCo-PCR). DeSCo-PCR uses a nonextendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP, and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5'-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA.

LA PÉRDIDA DE FUNCIÓN DE LA QUINASA DEPENDIENTE DE CICLINA 5 (CDK5) ALTERA EL CITOESQUELETO Y REDUCE LA INFECCIÓN in vitro POR EL VIRUS DEL DENGUE 2 / The loss of function of Cyclin-Dependent Kinase 5 (CDK5) alters the Cytoskeleton and decrease the in vitro Dengue Virus-2 infection

RESUMEN La quinasa dependiente de ciclina 5 (CDK5) regula diversas funciones en neuronas, células endoteliales y epiteliales, entre ellas la dinámica del citoesqueleto. Así mismo, se ha reportado que componentes del citoesqueleto, tales como, filamentos de actina y microtúbulos juegan un rol importante durante la infección por el virus dengue (DENV). El objetivo del presente trabajo fue evaluar por dos métodos, inhibición química y silenciamiento génico, la participación de CDK5 durante la infección por DENV-2. La actividad antiviral de roscovitina fue evaluada usando ensayos de Unidades Formadoras de Placa (PFU). La eficiencia de transfección y el silenciamiento de CDK5, empleando miARNs artificiales, se determinó por citometría de flujo. El efecto sobre la proteína de envoltura viral y elementos del citoesqueleto se evidenció mediante microscopia avanzada de fluorescencia y análisis de imágenes. Roscovitina mostró actividad antiviral en etapas pre y post-infectivas en una forma dependiente de la dosis. El tratamiento con roscovitina y miRCDK5 mostró ser efectivo reduciendo la cantidad de CDK5 en células no infectadas. En células infectadas y transfectadas con miRCDK5, así como tratadas con el inhibidor, se observó una reducción significativa de la proteína de envoltura viral; sin embargo, no se encontró reducción significativa de CDK5. Además, el tratamiento con roscovitina indujo cambios celulares morfológicos evidentes en células infectadas. Los resultados indican la potencial participación de CDK5 durante la infección por DENV-2, posiblemente mediando la traducción proteica o la replicación del genoma viral a través de la regulación de la dinámica del citoesqueleto. Se requieren datos adicionales para esclarecer la mecanística del fenómeno usando métodos alternativos.

ABSTRACT Cyclin-Dependent Kinase 5 (CDK5) regulates several functions in neurons, endothelial, and epithelial cells, including the cytoskeleton dynamics. Likewise, it has been reported that some cytoskeleton elements, such as actin filaments and microtubules, play an essential role during Dengue virus (DENV) infection. This work aimed to evaluate the role of CDK5 during DENV-2 infection by two methods, chemical inhibition, and gene silencing. The antiviral activity of roscovitine was evaluated using Plaque Forming Units (PFU) assay. The transfection efficiency and knockdown of CDK5, using artificial miRNAs, was carried out by flow cytometry. The effect on the viral envelope protein and cytoskeleton elements was evidenced by advanced fluorescence microscopy and image analysis. Roscovitine showed antiviral activity in pre and post-infection stages in a dose-dependent manner. Treatment with roscovitine and miRCDK5 decrease the amount of CDK5 in uninfected cells. In cells infected and transfected with miRCDK5, as well as treated with the inhibitor, a significant reduction of the viral envelope protein was observed; however, no significant reduction of CDK5 was found. Also, evident morphological cellular changes were observed during the treatment with roscovitine in infected cells. The results indicate the potential participation of CDK5 during DENV-2 infection, possibly mediating protein translation or replication of the viral genome through the cytoskeletal dynamics regulation. Additional data are required to clarify the mechanistic of these phenomena using alternative methods.

Anti-Herpetic, Anti-Dengue and Antineoplastic Activities of Simple and Heterocycle-Fused Derivatives of Terpenyl-1,4-Naphthoquinone and 1,4-Anthraquinone.

Quinones are secondary metabolites of higher plants associated with many biological activities, including antiviral effects and cytotoxicity. In this study, the anti-herpetic and anti-dengue evaluation of 27 terpenyl-1,4-naphthoquinone (NQ), 1,4-anthraquinone (AQ) and heterocycle-fused quinone (HetQ) derivatives was done in vitro against Human Herpesvirus (HHV) type 1 and 2, and Dengue virus serotype 2 (DENV-2). The cytotoxicity on HeLa and Jurkat tumor cell lines was also tested. Using plaque forming unit assays, cell viability assays and molecular docking, we found that NQ 4 was the best antiviral compound, while AQ 11 was the most active and selective molecule on the tested tumor cells. NQ 4 showed a fair antiviral activity against Herpesviruses (EC50: <0.4 µg/mL, <1.28 µM) and DENV-2 (1.6 µg/mL, 5.1 µM) on pre-infective stages. Additionally, NQ 4 disrupted the viral attachment of HHV-1 to Vero cells (EC50: 0.12 µg/mL, 0.38 µM) with a very high selectivity index (SI = 1728). The in silico analysis predicted that this quinone could bind to the prefusion form of the E glycoprotein of DENV-2. These findings demonstrate that NQ 4 is a potent and highly selective antiviral compound, while suggesting its ability to prevent Herpes and Dengue infections. Additionally, AQ 11 can be considered of interest as a leader for the design of new anticancer agents.

Anti-herpetic and anti-dengue activity of abietane ferruginol analogues synthesized from (+)-dehydroabietylamine.

The abietane-type diterpenoid (+)-ferruginol (1), a bioactive compound isolated from several plants, has attracted much attention as consequence of its pharmacological properties, which includes antibacterial, antifungal, antimicrobial, cardioprotective, anti-oxidative, anti-plasmodial, leishmanicidal, anti-ulcerogenic, anti-inflammatory and antitumor actions. In this study, we report on the antiviral evaluation of ferruginol (1) and several analogues synthesized from commercial (+)-dehydroabietylamine. Thus, the activity against Human Herpesvirus type 1, Human Herpesvirus type 2 and Dengue Virus type 2, was studied. Two ferruginol analogues showed high antiviral selectivity index and reduced viral plaque-size in post-infection stages against both Herpes and Dengue viruses. A promising lead, compound 8, was ten-fold more potent (EC50 = 1.4 µM) than the control ribavirin against Dengue Virus type 2. Our findings suggest that the 12-hydroxyabieta-8,11,13-triene skeleton, which is characteristic of the diterpenoid ferruginol (1), is an interesting molecular scaffold for development of novel antivirals. In addition, the cytotoxic and antifungal activities of the synthesized ferruginol analogues have also been investigated. (©)20155 Elsevier Science. All rights reserved.