Enhanced Remdesivir Analogues to Target SARS-CoV-2.

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.

Utility of an In-Vitro Micro-Neutralizing Test in Comparison to a Plaque Reduction Neutralization Test for Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Serology and Drug Screening.

Flavivirus infections, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), present significant global public health challenges. For successful vaccine design, the assessment of neutralizing antibody activity requires reliable and robust methodologies for determining antibody titers. Although the plaque reduction neutralization test (PRNT) is commonly acknowledged as the gold standard, it has limitations in terms of time and cost, and its usage may be limited in resource-limited settings. To address these challenges, we introduced the micro-neutralization test (MNT) as a simplified alternative to the PRNT. The MNT employs a 96-well plate format, conducts microscale neutralization assays, and assesses cell viability by dissolving cells to create a uniform color solution, which is measured with a spectrometer. In this study, we evaluated the utility of the MNT by contrasting the end-point titers of the MNT and PRNT using 4 monoclonal antibodies, 15 non-human primate serum samples, and 2 therapeutic drug candidates across flaviviruses. The results demonstrated a strong correlation between the MNT and PRNT titers, affirming the robustness and reproducibility of the MNT for evaluating control measures against flaviviruses. This research contributes valuable insights toward the development of a cost-effective antibody titer testing approach that is particularly suitable for resource-limited settings.

Characterization of the Second Apoptosis Inhibitor Encoded by Guinea Pig Cytomegalovirus.

Despite the usefulness of guinea pig cytomegalovirus (GPCMV) for studies on congenital CMV infection, its viral mechanisms for the evasion of host defense strategies have not been fully elucidated. We reported previously that GPCMV gp38.1 functions as a viral mitochondria-localized inhibitor of apoptosis-like function, and its weak activity suggested the presence of an additional inhibitory molecule(s). Here, we identified gp38.3-2, a 42-amino-acid (aa) reading frame embedded within the gp38.3 gene that encodes a positional homolog of murine CMV (MCMV) m41. Characterization of gp38.3-2 resulted in the following findings: (i) the aa sequence of gp38.3-2 shows some similarity to that of MCMV m41.1, a viral inhibitor of oligomerization of a member of Bcl-2 family protein BAK, but there is no correspondence in their predicted secondary structures; (ii) gp38.3-2, but not gp38.3, showed inhibitory activities against staurosporine-induced apoptosis; (iii) three-dimensional protein complex prediction suggests that the N-terminal α-helix of gp38.3-2 interacts with residues in the BH3 and BH1 motifs of BAK, and analysis of gp38.3-2 and BAK mutants supported this model; (iv) guinea pig fibroblast cells infected with gp38.3-2-deficient GPCMV strain Δ38.3-2 died earlier than cells infected with rescued strain r38.3-2, resulting in lower yields of Δ38.3-2; (v) Δ38.3-2 exhibited a partial but significant decrease in monocyte and macrophage infection in comparison with r38.3-2; and, however, (vi) little difference in the viral infection of guinea pigs was observed between these two strains. Therefore, we hypothesize that gp38.3-2 contributes little to the evasion of host defense mechanisms under the experimental conditions used. IMPORTANCE Although GPCMV provides a useful animal model for studies on the pathogenesis of congenital CMV infection and the development of CMV vaccine strategies, our understanding of the viral mechanisms by which it evades apoptosis of infected cells has been limited in comparison with those of murine and human CMVs. Here, we report a second GPCMV apoptosis inhibitor (42 amino acids in length) that interacts with BAK, a Bcl-2 family proapoptotic protein. Three-dimensional structural prediction indicated a unique BAK recognition by gp38.3-2 via the BH3 and BH1 motif sequences. Our findings suggest the potential development of BH3 mimetics that can regulate inhibition or induction of apoptosis based on short ~40-amino-acid peptide molecules as with GPCMV.

Repurposing N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human cytomegalovirus pUL89 endonuclease: Synthesis and biological characterization.

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.

Bisubstrate Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 Nsp14 Methyltransferase.

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.

8-Hydroxy-1,6-naphthyridine-7-carboxamides as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease.

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.

Characterization of a thiourea derivative that targets viral transactivators of cytomegalovirus and herpes simplex virus type 1.

Although currently available antivirals against certain herpesviruses are effective, the development of resistance during long-term use has necessitated the search for seed compounds that work against novel target molecules. In this report, we identified a thiourea derivative compound, 147B3, that inhibits the infection of human cytomegalovirus (HCMV) in fibroblasts and herpes simplex virus type 1 (HSV-1) in Vero cells at a 50% effective concentration of 0.5 µM and 1.9 µM, respectively. Characterization of the compound provided the following clues regarding its mode of action. 1) Time-of-addition and block-release assays showed that 147B3 behaved similarly to ganciclovir. 2) 147B3 reduced the expression of early and late but not immediate-early gene products and the accumulation of viral genomic DNA in both HCMV-infected and HSV-1-infected cells. 3) 147B3 inhibited the HCMV IE2-dependent activation of viral early gene promoters. 4) Four HSV-1 clones resistant to 147B3 were isolated and next-generation sequencing analysis of their genome DNA revealed that all of them had a mutation(s) in the infected cell protein 4 (ICP4) gene, which encodes a viral transcriptional factor. 5) Although 147B3 did not reduce the amount of ICP4 in an immunoblotting analysis, it changed the localization of the ICP4 from the speckles in the nuclei to diffused dots in the cytoplasm. 6) 147B3 did not affect the localization of promyelocytic leukemia (PML) bodies. Our findings suggest that 147B3 targets viral transactivators, potentially through their interaction with factors required for the viral gene expression system.

The Guinea pig cytomegalovirus GP119.1 gene encodes an IgG-binding glycoprotein that is incorporated into the virion.

Cytomegaloviruses (CMVs) encode various immunoevasins, including viral receptors for the Fc domain of host IgG (vFcγR), to evade host immune responses. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, the GPCMV genes encoding such receptors have not yet been characterized. In this study, we analyzed a locus that may encode gene products for the GPCMV immune evasion mechanisms and identified the following. (a) RACE analyses identified four transcripts in the GP117 to GP122 locus. One of the transcripts contained the GP119.1 ORF, which has weak homologies with human CMV UL119/UL118 encoding a viral FcγR and with guinea pig FcγR. (b) A transient transfection assay with plasmids expressing EGFP-tagged GP119.1 or its mutated forms identified its true translational initiation site, localization mainly in the endoplasmic reticulum, and N-glycosylation. (c) Importantly, GP119.1 bound to guinea pig IgG or the IgG-Fc fragment. (d) GP119.1 is present in the virion with a molecular mass of 15 and 23~30 kDa, and a portion of the GP119.1 products are N-glycosylated. (e) GP119.1 was dispensable for viral growth on guinea pig fibroblasts and epithelial cells in vitro. Taken together, our findings indicate that GP119.1 is an IgG-Fc binding glycoprotein incorporated into the virion, and this finding warrants further studies on the functions of GP119.1 in animal models.

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals.

Cytomegaloviruses (CMVs) employ an array of strategies designed to interfere with host defence responses against pathogens. Studies on such evasion mechanisms are important for understanding the pathogenesis of CMV diseases. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, its evasion strategies are not fully elucidated. Here, we analysed a genome locus that may encode gene products for the GPCMV evasion mechanisms and found the following. (1) RACE analyses identified five transcripts in the GP38-gp38.4 locus, one of which was a spliced product encoding gp38.1. Similarities in the splicing pattern and gene position of gp38.1 to human CMV UL37 and its exon 1 encoding vMIA (viral mitochondria-localized inhibitor of apoptosis) suggest that the gp38.1 gene encodes an apoptosis inhibitor. (2) In a transient transfection assay, gp38.1 localized in the mitochondria and relocated BAX from the cytoplasm to the mitochondria, although its co-localization with BAK was not evident. Further, the expression of gp38.1 partially reduced staurosporine-induced apoptosis. (3) GPCMV defective in the gp38.1 ORF (Δ38.1) and the virus that rescues the defect (r38.1) were generated. Guinea pig fibroblast cells infected with Δ38.1 died earlier than r38.1-infected cells, which resulted in the lower yields of Δ38.1. (4) In animals, viral loads in the spleens of r38.1-infected guinea pigs were higher than those in the spleens of Δ38.1-infected animals. In conclusion, although GPCMV gp38.1 exerts a vMIA-like function, its inhibitory effect was not robust, suggesting the presence of additional inhibitory molecule(s), such as a BAK-specific inhibitor.

Enhancement of guinea pig cytomegalovirus infection by two endogenously expressed components of the pentameric glycoprotein complex in epithelial cells.

A better understanding of the mechanisms underlying cell tropisms and the efficiency of viral infection is critical for the development of vaccines and antiviral drugs for viral diseases. In this study, we worked on the entry mechanisms of guinea pig cytomegalovirus and found that endogenous expression of a combination of two components (GP131 and GP133) of the pentameric glycoprotein complex, which is required for non-fibroblast cell tropisms, enhanced viral infection more than 10-fold. In addition, D138A alteration in GP131 increased this enhancement by an additional 10-fold. Although differences in the efficiency of viral infection among various cell types are usually explained by differences in viral entry or traffic processes, our experimental evidences dismissed such possibilities. Instead, our findings that i) endogenous expression of GP131 and GP133 after nuclear delivery of viral DNA still enhanced infection and ii) an HDAC inhibitor overcame the need of the endogenous expression led us to hypothesize a novel mechanism that controls the efficiency of viral infection through the activation of gene expression from viral DNA delivered to the nuclei. Further studies of this unexpected phenomena warrant to understand novel but also general mechanisms for cell tropisms of viral infection and determinants that control infection efficiency.

Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo.

Cytomegaloviruses (CMVs) encode cellular homologs to evade host immune functions. In this study, we analyzed the roles of GP33, a guinea pig CMV (GPCMV)-encoded G protein-coupled receptor (GPCR) homolog, in cellular signaling, viral growth and pathogenesis. The cDNA structure of GP33 was determined by RACE. The effects of GP33 on some signaling pathways were analyzed in transient transfection assays. The redET two-step recombination system for a BAC containing the GPCMV genome was used to construct a mutant GPCMV containing an early stop codon in the GP33 gene (Δ33) and a rescued GPCMV (r33). We found the following: 1) GP33 activated the CRE- and NFAT-, but not the NFκB-mediated signaling pathway. 2) GP33 was dispensable for infection in tissue cultures and in normal animals. 3) In pregnant animals, viral loads of r33 in the livers, lungs, spleens, and placentas at 6 days post-infection were higher than those of Δ33, although the viruses were cleared by 3 weeks post-infection. 4) The presence of GP33 was associated with frequent lesions, including alveolar hemorrhage in the lungs, and inflammation in the lungs, livers, and spleens of the dams. Our findings suggest that GP33 has critical roles in the pathogenesis of GPCMV during pregnancy. We hypothesize that GP33-mediated signaling activates cytokine secretion from the infected cells, which results in inflammation in some of the maternal organs and the placentas. Alternatively, GP33 may facilitate transient inflammation that is induced by the chemokine network specific to the pregnancy.

Characterization of phenyl pyrimidine derivatives that inhibit cytomegalovirus immediate-early gene expression.

Background Previously, we established a reporter cell line for human cytomegalovirus and screened anti-human cytomegalovirus compounds using the cell line. In this study, we characterized one of the identified compounds, 2,4-diamino-6-(4-methoxyphenyl)pyrimidine (coded as 35C10). Methods 50% Effective concentrations (EC50s) and 50% cytotoxic concentrations (CC50s) of 35C10 and its derivatives in human fibroblasts were determined by X-gal staining of the cells infected with human cytomegalovirus Towne strain expressing ß-galactosidase. Results EC50 and CC50 of 35C10 were 4.3 µM and >200 µM, respectively. Among several 35C10 derivatives, only one lacking 4-amino group of pyrimidine showed a similar EC50. 35C10 weakly inhibited murine cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. A "time of addition" experiment suggested that 35C10 inhibited an early phase of the infection. Although 35C10 did not inhibit viral attachment to the cells nor the delivery of viral DNA to the nuclei, it decreased the number of infected cells expressing immediate-early 1 and 2 (IE1/IE2) proteins. 35C10 also inhibited the activation of a promoter for TRL4 in the reporter cells upon human cytomegalovirus infection, but not in the same reporter cells transfected with a plasmid expressing IE2. Conclusion Our findings suggest that 35C10 is a novel compound that inhibits IE gene expression in human cytomegalovirus-infected cells.

Characterization of a thienylcarboxamide derivative that inhibits the transactivation functions of cytomegalovirus IE2 and varicella zoster virus IE62.

Previously we established reporter cell lines for human cytomegalovirus (HCMV) and varicella zoster virus (VZV) and identified several antiviral compounds against these viruses using the reporter cells. In this study, we found that one of the identified anti-HCMV compounds, a thienylcarboxamide derivative (coded as 133G4), was effective against not only HCMV but also VZV. The following findings indicate that 133G4 inhibits the activation of early gene promoters by HCMV IE2 and VZV IE62: i) 133G4 decreased the expression of HCMV early and late genes but not that of HCMV IE1/IE2 in HCMV-infected cells, ii) 133G4 inhibited the activation of several HCMV early gene promoters of transiently-transfected plasmids in HCMV-infected cells, and iii) in transient transfection assays, 133G4 decreased the activation of HCMV (or VZV) early gene promoters by HCMV IE2 (or VZV IE62) in the absence of other viral protein expression. The inhibition of early gene activation was observed in the human and African green monkey cell lines but not in the rodent cell lines, and the compound was not effective against murine CMV. In addition, VZV IE62 activated HCMV early promoters, and 133G4 still inhibited such promoter activation. Therefore, we hypothesized that 133G4 targets a cellular factor used commonly in activation of human herpesvirus promoters and examined whether 133G4 affects the functions of cellular proteins USF1, TBP, Med25 and EAP, the involvement of which in VZV IE62-dependent viral gene activation has been well characterized. Our experimental results using one-hybrid and bimolecular fluorescence complementation assays demonstrated that 133G4 did not inhibit the recruitment of USF1 or TBP to their binding sites, nor inhibited the direct interactions of VZV IE62 with Med25 and EAP. Thus, 133G4 is a unique anti-VZV and -HCMV compound, which warrants further studies to find out its inhibitory mechanism.