The emergence or re-emergence of viruses with epidemic and/or pandemic potential, such as Ebola, Zika, Middle East Respiratory Syndrome (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus 1 and 2 (SARS and SARS-CoV-2) viruses, or new strains of influenza represents significant human health threats due to the absence of available treatments. Vaccines represent a key answer to control these viruses. However, in the case of a public health emergency, vaccine development, safety, and partial efficacy concerns may hinder their prompt deployment. Thus, developing broad-spectrum antiviral molecules for a fast response is essential to face an outbreak crisis as well as for bioweapon countermeasures. So far, broad-spectrum antivirals include two main categories: the family of drugs targeting the host-cell machinery essential for virus infection and replication, and the family of drugs directly targeting viruses. Among the molecules directly targeting viruses, nucleoside analogues form an essential class of broad-spectrum antiviral drugs. In this review, we will discuss the interest for broad-spectrum antiviral strategies and their limitations, with an emphasis on virus-targeted, broad-spectrum, antiviral nucleoside analogues and their mechanisms of action.

Dengue virus affects millions of people worldwide each year. To date, there is no drug for the treatment of dengue-associated disease. Nucleosides are effective antivirals and work by inhibiting the accurate replication of the viral genome. Nucleobases offer a cheaper alternative to nucleosides for broad antiviral applications. Metabolic activation of nucleobases involves condensation with 5-phosphoribosyl-1-pyrophosphate to give the corresponding nucleoside-5’-monophosphate. This could provide an alternative to phosphorylation of a nucleoside, a step that is often rate limiting and inefficient in activation of nucleosides. We evaluated more than 30 nucleobases and corresponding nucleosides for their antiviral activity against dengue virus. Five nucleobases and two nucleosides were found to induce potent antiviral effects not previously described. Our studies further revealed that nucleobases were usually more active with a better tissue culture therapeutic index than their corresponding nucleosides. The development of viral lethal mutagenesis, an antiviral approach that takes into account the quasispecies behavior of RNA viruses, represents an exciting prospect not yet studied in the context of dengue replication. Passage of the virus in the presence of the nucleobase 3a (T-1105) and corresponding nucleoside 3b (T-1106), favipiravir derivatives, induced an increase in apparent mutations, indicating lethal mutagenesis as a possible antiviral mechanism. A more concerted and widespread screening of nucleobase libraries is a very promising approach to identify dengue virus inhibitors including those that may act as viral mutagens. Dengue virus is a world-wide public health menace estimated to infect hundreds of millions of people per year. Vaccines to prevent dengue virus infection have had limited success due in part to the requirement to elicit effective immune responses against the four dengue serotypes. There is an urgent unmet need for anti-dengue virus therapies. Nucleosides are effective antiviral small molecules which usually work by inhibiting the accurate replication of the viral genome. Typically, nucleosides must be converted within the cell to their triphosphate form to inhibit virus replication, thus inefficient phosphorylation often leads to suboptimal activity. We screened a small library of nucleobases that require an activation pathway different from nucleosides to achieve the same active form. We identified some known and previously undescribed dengue virus nucleobase inhibitors and their corresponding nucleosides. Our investigation of the mechanism of action of one nucleobase and its corresponding nucleoside found evidence for enhanced mutagenesis of the dengue virus genome in the presence of the compounds in cell culture. A wide screening of nucleobases libraries is a promising strategy to discover dengue virus inhibitors including potential viral mutagens.

Every year, dengue virus affects hundreds of millions of individuals worldwide. To date, there is no specific medication to treat dengue virus infections. Nucleobases, the base of a nucleoside without ribose, are understudied as potential treatments for viral infections. Antiviral nucleobases are converted in infected cells to their corresponding nucleoside triphosphate active form. Importantly, the conversion of nucleobases to their active nucleotide form and their antiviral effect can be enhanced when combined with de novo nucleotide biosynthesis inhibitors. In this work, we evaluated seven purine and pyrimidine nucleobases alone or combined with six purine or pyrimidine de novo nucleotide biosynthesis inhibitors, including novel prodrugs. Our study revealed that while a strong potentiation of purine nucleobases by purine de novo nucleotide biosynthesis inhibitors was observed, the pyrimidine nucleobases were not potentiated by pyrimidine de novo nucleotide biosynthesis inhibitors, possibly highlighting a significant difference between the modulation of purine versus pyrimidine de novo pathways and their impact on nucleobase potentiation. Most significant antiviral effects and potentiation were observed for Favipiravir, T-1105, and ribavirin nucleobases combined with purine nucleotide de novo synthesis inhibitors. These results are significant because drug combinations may solve the limited efficacy observed for some antiviral nucleobase drugs such as Favipiravir.

Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5′-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5′-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.