Enhanced efficacy of endonuclease inhibitor baloxavir acid against orthobunyaviruses when used in combination with ribavirin.

OBJECTIVES: Baloxavir acid is an endonuclease inhibitor approved for use against influenza. We evaluated whether this compound also targets the endonuclease domain of orthobunyaviruses and therefore could potentially be used against orthobunyavirus infections. METHODS: We performed a thermal shift assay and a fluorescence resonance energy transfer (FRET)-based nuclease monitoring assay using the La Crosse virus (LACV) endonuclease and baloxavir acid to prove their interaction and identify an inhibitory effect. Their interaction was further studied in a docking simulation using Glide SP. We show that baloxavir acid inhibits the viral replication of Bunyamwera virus (BUNV)-mCherry in vitro using high-content imaging and virus yield assay. Lastly, we investigated the use of baloxavir acid in combination with ribavirin in vitro by implementing the Zero Interaction Potency response surface model. RESULTS: We show that baloxavir acid augments LACV enzyme's melting temperature with ΔTm 9.5 ± 0.4°C and inhibited substrate cleavage with IC50 0.39 ± 0.03 µM. Moreover, our docking simulation suggests that baloxavir acid is able to establish an efficient binding with the LACV endonuclease. In the cell-based assay, we observed that baloxavir acid and ribavirin inhibited BUNV-mCherry with an EC50 of 0.7 ± 0.2 µM and 26.6 ± 8.9 µM, respectively. When used in combination, we found a maximum synergistic effect of 8.64. CONCLUSIONS: The influenza endonuclease inhibitor baloxavir acid is able to bind to and interfere with the endonuclease domain of orthobunyaviruses and yields a more potent antiviral effect than ribavirin against BUNV-mCherry. The combination of both compounds results in a more potent antiviral effect, suggesting that these molecules could potentially be combined to treat orthobunyavirus-infected patients.

Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

Andes virus (ANDV) is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

Inhibitory interactions of the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold with Bunyavirales cap-snatching endonucleases expose relevant drug design features.

The World Health Organization (WHO) identifies several bunyaviruses as significant threats to global public health security. Developing effective therapies against these viruses is crucial to combat future outbreaks and mitigate their impact on patient outcomes. Here, we report the synthesis of some isoindol-1-one derivatives and explore their inhibitory properties over an indispensable metal-dependent cap-snatching endonuclease (Cap-ENDO) shared among evolutionary divergent bunyaviruses. The compounds suppressed RNA hydrolysis by Cap-ENDOs, with IC50 values predominantly in the lower µM range. Molecular docking studies revealed the interactions with metal ions to be essential for the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold activity. Calorimetric analysis uncovered Mn2+ ions to have the highest affinity for sites within the targets, irrespective of aminoacidic variations influencing metal cofactor preferences. Interestingly, spectrophotometric findings unveiled sole dinuclear species formation between the scaffold and Mn2+. Moreover, the complexation of two Mn2+ ions within the viral enzymes appears to be favourable, as indicated by the binding of compound 11 to TOSV Cap-ENDO (Kd = 28 ± 3 µM). Additionally, the tendency of compound 11 to stabilize His+ more than His- Cap-ENDOs suggests exploitable differences in their catalytic pockets relevant to improving specificity. Collectively, our results underscore the isoindolinone scaffold's potential as a strategic starting point for the design of pan-antibunyavirus drugs.

Human CCAAT/enhancer-binding protein ß interacts with chromatin remodeling complexes of the imitation switch subfamily.

Transcription factor C/EBPß is involved in several cellular processes, such as proliferation, differentiation, and energy metabolism. This factor exerts its activity through recruitment of different proteins or protein complexes, including the ATP-dependent chromatin remodeling complex SWI/SNF. The C/EBPß protein is found as three major isoforms, C/EBPß1, -2, and -3. They are generated by translation at alternative AUG initiation codons of a unique mRNA, C/EBPß1 being the full-length isoform. It has been found that C/EBPß1 participates in terminal differentiation processes. Conversely, C/EBPß2 and -3 promote cell proliferation and are involved in malignant progression in a number of tissues. The mechanisms by which C/EBPß2 and -3 promote cell proliferation and tumor progression are not fully understood. In this work, we sought to identify proteins interacting with hC/EBPß using a proteomics approach. We found that all three isoforms interact with hSNF2H and hACF, components of ACF and CHRAC chromatin remodeling complexes, which belong to the imitation switch subfamily. Additional protein-protein interaction studies confirmed this finding and also showed that hC/EBPß directly interacts with hACF1. By overexpressing hC/EBPß, hSNF2H, and hACF1 in HepG2 cells and analyzing variations in expression of cyclin D1 and other C/EBPß target genes, we observed a functional interaction between C/EBPß and SNF2H/ACF1, characterized mainly by suppression of C/EBPß transactivation activity in the presence of SNF2H and ACF1. Consistent with these findings, induction of differentiation of HepG2 cells by 1% DMSO was accompanied by a reduction in the level of cyclin D1 expression and the appearance of hC/EBPß, hSNF2H, and hACF1 on the promoter region of this gene.

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Diketo acids inhibit the cap-snatching endonuclease of several Bunyavirales.

Several fatal bunyavirus infections lack specific treatment. Here, we show that diketo acids engage a panel of bunyavirus cap-snatching endonucleases, inhibit their catalytic activity and reduce viral replication of a taxonomic representative in vitro. Specifically, the non-salt form of L-742,001 and its derivatives exhibited EC50 values between 5.6 and 6.9 µM against a recombinant BUNV-mCherry virus. Structural analysis and molecular docking simulations identified traits of both the class of chemical entities and the viral target that could help the design of novel, more potent molecules for the development of pan-bunyavirus antivirals.

Differential in vitro and in vivo glycosylation of human erythropoietin expressed in adenovirally transduced mouse mammary epithelial cells.

The expression of human erythropoietin in the mammary gland is an attractive approach to diminish its current production cost. Previous attempts to produce erythropoietin in the milk of transgenic animals resulted in very low expression levels and in a detrimental effect in the health of the founder animals. Here, we show that the direct transduction of the mouse mammary gland with an adenoviral vector carrying the cDNA of erythropoietin promotes its expression in milk at a level as high as 3.5 mg/ml. The recombinant erythropoietin derived from mouse milk showed a different migration and distribution after SDS-PAGE electrophoresis as well as a low in vivo hematopoietic activity. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation compared to with its counterpart produced in CHO and HC11 cell lines. The difference between in vivo and in vitro glycosylation of human erythropoietin expressed in adenovirally transduced mammary epithelial cells suggests that key enzymes in the glycosylation pathway may be insufficient during lactation. Thus, the direct transduction of the mammary epithelium seems to be a powerful tool to express toxic proteins in milk at levels high enough for their physical, chemical and biological characterization before undertaking the generation of a transgenic mammal.