Human cytomegalovirus (HCMV) is a prevalent herpesvirus pathogen and remains a significant cause for mortality and morbidity in immunocompromised individuals and newborns. Like all viruses, HCMV uses host cellular proteins to facilitate virus replication and spread. Sirtuin 2 (SIRT2) is an NAD+-dependent deacetylase with a variety of substrates, including α-tubulin, where acetylation can alter function and stability. We have previously developed small molecule inhibitors targeting SIRT2 and in the current study, we show that two new compounds inhibit SIRT2 activity and also inhibit HCMV replication. The combination of these SIRT2 inhibitors with standard antivirals such as ganciclovir or letermovir resulted in a generally additive profile. Using indirect immunofluorescence, we determined that SIRT2 and SIRT1 expression was reduced over time in compound treated and untreated cells. We also observed SIRT2 and SIRT1 expression was increased in HCMV infected cells, compared to surrounding uninfected cells, regardless of compound treatment. GAPDH expression was stable over time and did not increase in HCMV infected cells, indicating a possible sirtuin-specific mechanism for upregulation of SIRT2 and 1 expression in infected cells. Further investigation is required to better understand the underlying anti-HCMV mechanism of action for our SIRT2 inhibitors and how HCMV infection impacts SIRT2 expression.

Yellow fever virus (YFV) recurrently causes severe outbreaks in tropical regions of South America and Africa and an average of 30 to 40 thousand deaths worldwide each year. An effective vaccine is available but the coverage of the population in countries at risk is not optimal. No antivirals are currently approved for YFV treatment. Herein, we describe the evaluation of 6-MMPr, a de-novo-purine-nucleotide biosynthesis inhibitor, as a potentiator for enhanced activity of the broad-spectrum antiviral drug favipiravir in a hamster model of yellow fever. Administration of 6-MMPr was well-tolerated and a combination of favipiravir and 6-MMPr did not cause overt toxicity as indicated by normal weight gain of treated hamsters. Treatment with a combination of a suboptimal dose of favipiravir with 6-MMPr was significantly more effective in improving survival, weight change and virus replication when compared with monotherapy. The initiation of treatment two days after virus challenge was also effective in improving survival when compared with monotherapy. Our results demonstrate the safety and efficacy of such a combination either as a preventive or delayed treatment.

Background: p97 (also known as valosin-containing protein, VCP) is a member of the AAA+ ATPase family and is intimately associated with protein quality control and homeostasis regulation. Therefore, pharmaceutical inhibition of p97 has been actively pursued as an anticancer strategy. Recently, p97 has emerged as an important pro-viral host factor and p97 inhibitors are being evaluated as potential antiviral agents. Methods: We designed and synthesized novel p97 inhibitors based on the rearrangement of the central fused ring of our previously reported p97 inhibitors. These compounds were tested for inhibition of p97, cytotoxicity, and antiviral activity against SARS-CoV-2. Molecular docking was also performed on selected inhibitors to shed light on their binding modes. Results: Among these new p97 inhibitors, two compounds possess enhanced anti-p97 activity over their parent compounds. More significantly, these two inhibitors exhibit strong antiviral activity against SARS-CoV-2 at doses with no significant cytotoxicity. Molecular docking reveals no major change of the binding mode relative to that of their parent compounds, further supporting our design strategy. Conclusions: These compounds are structurally novel p97 inhibitors that display low toxicity and possess promising antiviral activity against SARS-CoV-2 and potentially other viruses. Further structural exploration is therefore justified and improved analogs will serve as useful tools for studying p97 as a promising host antiviral target.

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.

Nucleases are ubiquitous hydrolytic enzymes that cleave phosphodiester bond of DNA (DNases), RNA (RNases), or protein-RNA/DNA (phosphodiesterases), within the strand (endonucleases) or from the end (exonucleases) [...]

Human cytomegalovirus (HCMV) infects individuals of all ages and establishes a lifelong latency. Current antiviral drugs are suboptimal in efficacy and safety and ineffective against resistant/refractory HCMV. Therefore, there is an unmet clinical need for efficacious, safe, and mechanistically novel HCMV drugs. The recent Food and Drug Administration (FDA) approval of letermovir (LTV) validated the HCMV terminase complex as a new target for antiviral development. LTV targets terminase subunit pUL56 but not the main endonuclease enzymatic function housed in the C terminus of subunit pUL89 (pUL89-C). Structurally and mechanistically, pUL89-C is an RNase H-like viral endonuclease entailing two divalent metal ions at the active site. In recent years, numerous studies have extensively explored pUL89-C inhibition using metal-chelating chemotypes, an approach previously used for inhibiting HIV ribonuclease H (RNase H) and integrase strand transfer (INST). Collectively, the work summarized herein validates the use of metal-binding scaffolds for designing potent and specific pUL89-C inhibitors.

We report the short synthesis of novel C-nucleoside Remdesivir analogues, their cytotoxicity and an in vitro evaluation against SARS-CoV-2 (CoV2). The described compounds are nucleoside analogues bearing a nitrogen heterocycle as purine analogues. The hybrid structures described herein are designed to enhance the anti-CoV2 activity of Remdesivir. The compounds were evaluated for their cytotoxicity and their anti-CoV2 effect. We discuss the impact of combining both sugar and base modifications on the biological activities of these compounds, their lack of cytotoxicity and their antiviral efficacy.

The terminase complex of human cytomegalovirus (HCMV) is required for viral genome packaging and cleavage. Critical to the terminase functions is a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We have previously reported metal-chelating N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human immunodeficiency virus 1 (HIV-1) RNase H. In the current work, we have synthesized new analogs and resynthesized known analogs of two isomeric HtPD subtypes, anti-HtPD (13), and syn-HtPD (14), and characterized them as inhibitors of pUL89-C. Remarkably, the vast majority of analogs strongly inhibited pUL89-C in the biochemical endonuclease assay, with IC50 values in the nM range. In the cell-based antiviral assay, a few analogs inhibited HCMV in low μM concentrations. Selected analogs were further characterized in a biophysical thermal shift assay (TSA) and in silico molecular docking, and the results support pUL89-C as the protein target of these inhibitors. Collectively, the biochemical, antiviral, biophysical, and in silico data reported herein indicate that the isomeric HtPD chemotypes 13-14 can serve as valuable chemical platforms for designing improved inhibitors of HCMV pUL89-C.

Taking advantage of the uniquely constricted active site of SARS-CoV-2 Nsp14 methyltransferase, we have designed bisubstrate inhibitors interacting with the SAM and RNA substrate binding pockets. Our efforts have led to nanomolar inhibitors including compounds 3 and 10. As a prototypic inhibitor, compound 3 also has an excellent selectivity profile over a panel of human methyltransferases. Remarkably, C-nucleoside 10 exhibits high antiviral activity and low cytotoxicity, leading to a therapeutic index (CC50/EC50) greater than 139. Furthermore, a brief metabolic profiling of these two compounds suggests that they are less likely to suffer from major metabolic liabilities. Moreover, computational docking studies point to protein-ligand interactions that can be exploited to enhance inhibitory activity. In short, discovery of inhibitor 10 clearly demonstrates that potent and selective anti-SARS-CoV-2 activity can be achieved by targeting the Nsp14 methyltransferase. Therefore, the current work strongly supports the continued pursuit of Nsp14 methyltransferase inhibitors as COVID-19 therapeutics.

To search for Zika virus (ZIKV) antivirals, we have further explored previously reported 7H-pyrrolo[2,3-d]pyrimidines by examining an alternative substitution pattern of their central scaffold, leading to compound 5 with low micromolar antiviral activity. To circumvent the synthetic difficulties associated with compound 5, we have exploited a 1H-pyrazolo[3,4-d]pyrimidine scaffold and performed structure-activity relationship studies on its peripheral rings A and B. While ring B is less sensitive to structural modifications, an electron-withdrawing group at the para position of ring A is preferred for enhanced antiviral activity. Overall, we have not only discovered an alternative substitution pattern centered on a 1H-pyrazolo[3,4-d]pyrimidine scaffold but also generated anti-ZIKV compounds including 6 and 13, which possess low micromolar antiviral activity and relatively low cytotoxicity. These compounds represent new chemotypes that will be further optimized in our continued efforts to discover anti-ZIKV agents.

Human cytomegalovirus (HCMV) terminase complex entails a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We report herein the design, synthesis, and characterization of dihydroxypyrimidine (DHP) acid (14), methyl ester (13), and amide (15) subtypes as inhibitors of HCMV pUL89-C. All analogs synthesized were tested in an endonuclease assay and a thermal shift assay (TSA) and subjected to molecular docking to predict binding affinity. Although analogs inhibiting pUL89-C in the sub-μM range were identified from all three subtypes, acids (14) showed better overall potency, substantially larger thermal shift, and considerably better docking scores than esters (13) and amides (15). In the cell-based antiviral assay, six analogs inhibited HCMV with moderate activities (EC50 = 14.4-22.8 μM). The acid subtype (14) showed good in vitro ADME properties, except for poor permeability. Overall, our data support the DHP acid subtype (14) as a valuable scaffold for developing antivirals targeting HCMV pUL89-C.

Human cytomegalovirus (HCMV) replication requires a metal‐dependent endonuclease at the C‐terminus of pUL89 (pUL89‐C) for viral genome packaging and cleavage. We have previously shown that pUL89‐C can be pharmacologically inhibited with designed metal‐chelating compounds. We report herein the synthesis of a few 8‐hydroxy‐1,6‐naphthyridine subtypes, including 5‐chloro (subtype 15), 5‐aryl (subtype 16), and 5‐amino (subtype 17) variants. Analogs were studied for the inhibition of pUL89‐C in a biochemical endonuclease assay, a biophysical thermal shift assay (TSA), in silico molecular docking, and for the antiviral potential against HCMV in cell‐based assays. These studies identified eight analogs of 8‐hydroxy‐1,6‐naphthyridine‐7‐carboxamide subtypes for further characterization, most of which inhibited pUL89‐C with single‐digit μM IC50 values, and conferred antiviral activity in μM range. TSA and molecular modeling of selected analogs corroborate their binding to pUL89‐C. Collectively, our biochemical, antiviral, biophysical and in silico data suggest that 8‐hydroxy‐1,6‐naphthyridine‐7‐carboxamide subtypes can be used for designing inhibitors of HCMV pUL89‐C.

Current drugs for treating human cytomegalovirus (HCMV) infections are limited by resistance and treatment-associated toxicities. In developing mechanistically novel HCMV antivirals, we discovered an N-benzyl hydroxypyridone carboxamide antiviral hit (8a) inhibiting HCMV in submicromolar range. We describe herein the structure-activity relationship (SAR) for 8a, and the characterization of potent analogs for cytotoxicity/cytostatic property, the preliminary mechanism of action, and the absorption, distribution, metabolism and excretion (ADME) properties. The SAR revealed a few pharmacophore features conferring optimal antiviral profile, including the 5-OH, the N-1 benzyl, at least one -CH2- in the linker, and a di-halogen substituted phenyl ring in the amide moiety. In the end, we identified numerous analogs with sub-micromolar antiviral potency and good selectivity index. The preliminary mechanism of action characterization used a pUL89-C biochemical endonuclease assay, a virus entry assay, a time-of-addition assay, and a compound withdrawal assay. ADME profiling measuring aqueous solubility, plasma and liver microsomal stability, and parallel artificial membrane permeability assay (PAMPA) permeability demonstrated largely favorable drug-like properties. Together, these studies validate the N-benzyl hydroxypyridone carboxamide as a viable chemotype for potent and mechanistically distinct antivirals against HCMV.

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.

The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low μM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.

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.

The AAA+ (ATPase associated with various cellular activities) protein p97, also called valosin-containing protein, is a hexameric ring ATPase and uses ATP hydrolysis to unfold or extract proteins from biological complexes. Many cellular processes are affected by p97 including ER-associated degradation, DNA damage response, cell signaling (NF-κB), cell cycle progression, autophagy, and others. Not surprisingly, with its role in many fundamental cellular processes, p97 function is important for the replication of many viruses. We tested irreversible p97-targeting compounds for their ability to inhibit the replication of multiple viruses compared to the known p97 inhibitors NMS-873 and CB-5083. Our results indicate that overall cellular toxicity for p97 compounds provides a challenge for antivirals targeting p97. However, we identified one compound with sub-micromolar activity against human cytomegalovirus and improved cell viability to provide evidence for the potential of irreversible p97 inhibitors as antivirals.

Inhibition of viral reverse transcriptases and mammalian DNA polymerases by unnatural nucleoside analogs is a proven approach in antiviral and anticancer therapy, respectively. The majority of current nucleoside drugs retain the canonical nucleobase structure, which is fused to an unnatural sugar. In the present work, a series of novel pyrrolidine-functionalized purine and pyrimidine nucleosides was prepared via PyBOP-catalyzed S N Ar addition-elimination reactions of commercial halogenated precursors and tested for their antiviral and anticancer activity. The newly synthesized nucleoside analogs showed limited biological activity, probably as a result of their poor cellular uptake and their inefficient bioactivation to the corresponding nucleoside monophosphates. A phosphoramidate prodrug had an improved cell permeability and was metabolized to the nucleoside monophosphate form in human cells, as revealed by HPLC-MS/MS analyses.

Discovery of compound 1 as a Zika virus (ZIKV) inhibitor has prompted us to investigate its 7H-pyrrolo[2,3-d]pyrimidine scaffold, revealing structural features that elicit antiviral activity. Furthermore, we have demonstrated that 9H-purine or 1H-pyrazolo[3,4-d]pyrimidine can serve as an alternative core structure. Overall, we have identified 4,7-disubstituted 7H-pyrrolo[2,3-d]pyrimidines and their analogs including compounds 1, 8 and 11 as promising antiviral agents against flaviviruses ZIKV and dengue virus (DENV). While the molecular target of these compounds is yet to be elucidated, 4,7-disubstituted 7H-pyrrolo[2,3-d]pyrimidines and their analogs are new chemotypes in the design of small molecules against flaviviruses, an important group of human pathogens.

A variety of substituted acridones were synthesized via a one-pot, metal-free cascade reaction. In this event, the DBU-mediated addition between quinols and ortho-methoxycarbonylaryl isocyanates formed a bicyclic oxazolidinone, followed by a sequence of intramolecular condensation, tautomerization, and decarboxylation, which led to the formation of acridones. The acridones showed mild activity against the human cytomegalovirus.