Sofosbuvir Selects for Drug-Resistant Amino Acid Variants in the Zika Virus RNA-Dependent RNA-Polymerase Complex In Vitro.

The nucleotide analog sofosbuvir, licensed for the treatment of hepatitis C, recently revealed activity against the Zika virus (ZIKV) in vitro and in animal models. However, the ZIKV genetic barrier to sofosbuvir has not yet been characterized. In this study, in vitro selection experiments were performed in infected human hepatoma cell lines. Increasing drug pressure significantly delayed viral breakthrough (p = 0.029). A double mutant in the NS5 gene (V360L/V607I) emerged in 3 independent experiments at 40-80 µM sofosbuvir resulting in a 3.9 ± 0.9-fold half- maximal inhibitory concentration (IC50) shift with respect to the wild type (WT) virus. A triple mutant (C269Y/V360L/V607I), detected in one experiment at 80 µM, conferred a 6.8-fold IC50 shift with respect to the WT. Molecular dynamics simulations confirmed that the double mutant V360L/V607I impacts the binding mode of sofosbuvir, supporting its role in sofosbuvir resistance. Due to the distance from the catalytic site and to the lack of reliable structural data, the contribution of C269Y was not investigated in silico. By a combination of sequence analysis, phenotypic susceptibility testing, and molecular modeling, we characterized a double ZIKV NS5 mutant with decreased sofosbuvir susceptibility. These data add important information to the profile of sofosbuvir as a possible lead for anti-ZIKV drug development.

SARS-CoV-2 RNA-dependent RNA polymerase as a therapeutic target for COVID-19.

Introduction: The current SARS-CoV-2 pandemic urgently demands for both prevention and treatment strategies. RNA-dependent RNA-polymerase (RdRp), which has no counterpart in human cells, is an excellent target for drug development. Given the time-consuming process of drug development, repurposing drugs approved for other indications or at least successfully tested in terms of safety and tolerability, is an attractive strategy to rapidly provide an effective medication for severe COVID-19 cases.Areas covered: The currently available data and upcominSg studies on RdRp which can be repurposed to halt SARS-CoV-2 replication, are reviewed.Expert opinion: Drug repurposing and design of novel compounds are proceeding in parallel to provide a quick response and new specific drugs, respectively. Notably, the proofreading SARS-CoV-2 exonuclease activity could limit the potential for drugs designed as immediate chain terminators and favor the development of compounds acting through delayed termination. While vaccination is awaited to curb the SARS-CoV-2 epidemic, even partially effective drugs from repurposing strategies can be of help to treat severe cases of disease. Considering the high conservation of RdRp among coronaviruses, an improved knowledge of its activity in vitro can provide useful information for drug development or drug repurposing to combat SARS-CoV-2 as well as future pandemics.

Unique Domain for a Unique Target: Selective Inhibitors of Host Cell DDX3X to Fight Emerging Viruses.

Emerging viruses like dengue, West Nile, chikungunya, and Zika can cause widespread viral epidemics. Developing novel drugs or vaccines against specific targets for each virus is a difficult task. As obligate parasites, all viruses exploit common cellular pathways, providing the possibility to develop broad-spectrum antiviral agents targeting host factors. The human DEAD-box RNA helicase DDX3X is an essential cofactor for viral replication but dispensable for cell viability. Herein, we exploited the presence of a unique structural motif of DDX3X not shared by other cellular enzymes to develop a theoretical model to aid in the design of a novel class of highly selective inhibitors acting against such specific targets, thus limiting off-targeting effects. High-throughput virtual screening led us to identify hit compound 5, endowed with promising antienzymatic activity. To improve its aqueous solubility, 5 and its two enantiomers were synthesized and converted into their corresponding acetate salts (compounds 11, 12, and 13). In vitro mutagenesis and biochemical and cellular assays further confirmed that the developed molecules were selective for DDX3X and were able to suppress replication of West Nile and dengue viruses in infected cells in the micromolar range while showing no toxicity for uninfected cells. These results provide proof of principle for a novel strategy in developing highly selective and broad-spectrum antiviral molecules active against emerging and dangerous viral pathogens. This study paves the way for the development of larger focused libraries targeting such domain to expand SAR studies and fully characterize their mode of interaction.

Synthesis and Antiviral Activity of Novel 1,3,4-Thiadiazole Inhibitors of DDX3X.

The human ATPase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X) emerged as a novel therapeutic target in the fight against both infectious diseases and cancer. Herein, a new family of DDX3X inhibitors was designed, synthesized, and tested for its inhibitory action on the ATPase activity of the enzyme. The potential use of the most promising derivatives it has been investigated by evaluating their anti-HIV-1 effects, revealing inhibitory activities in the low micromolar range. A preliminary ADME analysis demonstrated high metabolic stability and good aqueous solubility. The promising biological profile, together with the suitable in vitro pharmacokinetic properties, make these novel compounds a very good starting point for further development.

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein.

Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro.

A strong correlation between Zika virus (ZIKV) infection and severe neurological disease in newborns and occasionally adults has emerged in the Brazilian outbreak. Efficient human cell-based assays are required to test candidate inhibitors of ZIKV replication. The aim of this work was to investigate ZIKV propagation and quantification in different cell lines. The human (U87, A549, Huh7), mosquito (C6/36) and monkey (VERO E6) cell lines tested were all permissive to ZIKV infection. When assessed by plaque forming units (PFU) in three different target cell lines, the maximal production of ZIKV was achieved in Huh7 at day 3 post-infection (6.38±0.44 log10PFU/ml). The C6/36 cell line showed a low and slow production of virus when compared with other cell lines. A549 readout cells generated a larger number of plaques compared to Huh7 but not to VERO E6 cells. ZIKV PFU and RNA titers showed the highest correlation when Huh7 and A549 were used as the producer and readout cells, respectively. Also, U87 cells produced ZIKV RNA titers which were highly correlated with PFU independently from the readout cell line. Using the best virus-cell system, sofosbuvir and ribavirin EC50 were 1.2µM and 1.1µM when measured through plaque assay, and 4.2µM and 5.2µM when measured by quantitative real time PCR (qRT-PCR), respectively. In summary, ZIKV can efficiently infect different human cell lines and rapidly reach peak viral titers. Overall, A549 cells appear to be as efficient as the VERO E6 gold standard for plaque assay allowing the use of human, rather than simian, cells for evaluating candidate anti-ZIKV compounds by the reference assay. The possibility to replace the labor-intensive plaque assay with the more rapid and easy-to-perform qRT-PCR is appealing and warrants further investigation.

Discovery and Structure-Based Optimization of 2-Ureidothiophene-3-carboxylic Acids as Dual Bacterial RNA Polymerase and Viral Reverse Transcriptase Inhibitors.

We are concerned with the development of novel anti-infectives with dual antibacterial and antiretroviral activities for MRSA/HIV-1 co-infection. To achieve this goal, we exploited for the first time the mechanistic function similarity between the bacterial RNA polymerase (RNAP) "switch region" and the viral non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Starting from our previously discovered RNAP inhibitors, we managed to develop potent RT inhibitors effective against several resistant HIV-1 strains with maintained or enhanced RNAP inhibitory properties following a structure-based design approach. A quantitative structure-activity relationship (QSAR) analysis revealed distinct molecular features necessary for RT inhibition. Furthermore, mode of action (MoA) studies revealed that these compounds inhibit RT noncompetitively, through a new mechanism via closing of the RT clamp. In addition, the novel RNAP/RT inhibitors are characterized by a potent antibacterial activity against S. aureus and in cellulo antiretroviral activity against NNRTI-resistant strains. In HeLa and HEK 293 cells, the compounds showed only marginal cytotoxicity.

Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

A novel methodology for large-scale phylogeny partition.

Understanding the determinants of virus transmission is a fundamental step for effective design of screening and intervention strategies to control viral epidemics. Phylogenetic analysis can be a valid approach for the identification of transmission chains, and very-large data sets can be analysed through parallel computation. Here we propose and validate a new methodology for the partition of large-scale phylogenies and the inference of transmission clusters. This approach, on the basis of a depth-first search algorithm, conjugates the evaluation of node reliability, tree topology and patristic distance analysis. The method has been applied to identify transmission clusters of a phylogeny of 11,541 human immunodeficiency virus-1 subtype B pol gene sequences from a large Italian cohort. Molecular transmission chains were characterized by means of different clinical/demographic factors, such as the interaction between male homosexuals and male heterosexuals. Our method takes an advantage of a flexible notion of transmission cluster and can become a general framework to analyse other epidemics.

Recombination analysis and structure prediction show correlation between breakpoint clusters and RNA hairpins in the pol gene of human immunodeficiency virus type 1 unique recombinant forms.

Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly throughout the genome, leading to viral recombination. Some recombination hotspots have been identified and found to correlate with RNA structure or sequence features. The aim of this study was to evaluate the presence of recombination hotspots in the pol gene of HIV-1 and to assess their correlation with the underlying RNA structure. Analysis of the recombination pattern and breakpoint distribution in a group of unique recombinant forms (URFs) detected two recombination hotspots in the pol region. Two stable and conserved hairpins were consistently predicted corresponding to the identified hotspots using six different RNA-folding algorithms on the URF parental strains. These findings suggest that such hairpins may play a role in the higher recombination rates detected at these positions.

Use of peripheral blood DNA for genotype antiretroviral resistance testing in drug-naive HIV-infected subjects.

Parallel analysis of peripheral blood mononuclear cell DNA and plasma RNA from 169 drug-naive human immunodeficiency virus-infected subjects revealed that evaluation of both compartments increases the sensitivity of detection of drug resistance-related mutations, compared with examination of either source alone. Peripheral blood mononuclear cell DNA may play a role in the surveillance of transmitted antiretroviral resistance.