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.

Zika Virus Inhibitors Based on a 1,3-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidine-amine Scaffold.

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.

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.

Effects of Volume Changes on the Thermal Performance of PCM Layers Subjected to Oscillations of the Ambient Temperature: Transient and Steady Periodic Regimes.

The Stefan problem regarding the formation of several liquid-solid interfaces produced by the oscillations of the ambient temperature around the melting point of a phase change material has been addressed by several authors. Numerical and semi-analytical methods have been used to find the thermal response of a phase change material under these type of boundary conditions. However, volume changes produced by the moving fronts and their effects on the thermal performance of phase change materials have not been addressed. In this work, volume changes are incorporated through an additional equation of motion for the thickness of the system. The thickness of the phase change material becomes a dynamic variable of motion by imposing total mass conservation. The modified equation of motion for each interface is obtained by coupling mass conservation with a local energy-mass balance at each front. The dynamics of liquid-solid interface configurations is analyzed in the transient and steady periodic regimes. Finite element and heat balance integral methods are used to verify the consistency of the solutions to the proposed model. The heat balance integral method is modified and adapted to find approximate solutions when two fronts collide, and the temperature profiles are not smooth. Volumetric corrections to the sensible and latent heat released (absorbed) are introduced during front formation, annihilation, and in the presence of two fronts. Finally, the thermal energy released by the interior surface is estimated through the proposed model and compared with the solutions obtained through models proposed by other authors.

Glycemic control is associated with lower odds of mortality and successful extubation in severe COVID-19.

CONTEXT: Corticosteroids, specifically dexamethasone, have become the mainstay of treatment for moderate to severe COVID-19. Although the RECOVERY trial did not report adverse effects of corticosteroids, the METCOVID (Methylprednisolone as Adjunctive Therapy for Patients Hospitalized with COVID-19) study reported a higher blood glucose level in patients receiving methylprednisolone. OBJECTIVES: This study aims to analyze the association between corticosteroids and COVID-19-related outcomes in patients admitted to the medical ICU (MICU) for COVID-19 pneumonia. METHODS: This is an observational study of 141 patients admitted to the MICU between March 18 and June 7, 2020. Data on demographics, laboratory and imaging studies, and clinical course were obtained, including data on corticosteroid use. Bivariate analyses and logistic regression were performed between patient characteristics and mortality and successful extubation. RESULTS: Of the 141 patients, 86 required mechanical ventilation, 50 received steroids, and 71 died. Regarding demographics, patients had a median age of 58 (interquartile range [IQR] 48, 65), Hispanic (57.4%, n=81), and non-Hispanic Black (37.5%, n=53). The most prevalent comorbidities were hypertension (49.6%, n=70) and diabetes (48.2%, n=68). Lower blood glucose levels on admission (125.5 vs. 148 mg/dL, p=0.025) and lower peak blood glucose levels on corticosteroids (215.5 vs. 361 mg/dL, p=0.0021) were associated with lower prevalence of mortality. Patients who were successfully extubated had a lower admission blood glucose (126.5 vs. 149 mg/dL, p=0.0074) and lower peak blood glucose on corticosteroids (217 vs. 361 mg/dL, p=0.0023). CONCLUSIONS: Lower blood glucose on admission and lower maximum blood glucose on corticosteroids were associated with lower odds of mortality and successful extubation, regardless of preexisting diabetes. Hyperglycemia may be negating any potential benefit of corticosteroid therapy. These findings suggest that glucose control could be a parameter that impacts the outcome of patients receiving corticosteroids for COVID-19 pneumonia.

Enhancing the Antiviral Potency of Nucleobases for Potential Broad-Spectrum Antiviral Therapies.

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.

Reactivation of Pulmonary Tuberculosis During Treatment of Chronic Myelomonocytic Leukemia.

A 76-year-old woman from a tuberculosis (TB) endemic region with chronic myelomonocytic leukemia (CMML) on Azacitidine presented with a non-productive cough. A CT scan of the chest revealed a lobulated opacity in the right upper lobe and antibiotic therapy was initiated for a potential bacterial pneumonia. However, a high suspicion for pulmonary TB remained given her nation of origin, immunosuppression, and imaging findings. Three sputum and bronchoalveolar lavage (BAL) acid-fast bacilli (AFB) smears with PCR testing for Mycobacterium tuberculosis were negative, as were examinations for other potential fungal or bacterial etiologies of the patient's symptoms and imaging findings. While awaiting final TB culture results from BAL, her CMML underwent a transformation to acute myeloid leukemia (AML). Given the urgent need for initiation of chemotherapy, empiric treatment for TB was commenced while awaiting the final TB culture. Within 48-hours of initiating therapy for TB, the patient's fevers subsided. One week after discharge our team was notified of a positive M. tuberculosis culture from BAL. We suspect that our patient had a latent TB infection which reactivated due to her CMML. This case highlights the importance of maintaining a high clinical suspicion for TB in high-risk patients, even in the case of initially negative laboratory examinations. Further, it demonstrates the importance of screening and treating latent TB in patients with leukemias.

4,7-Disubstituted 7H-Pyrrolo[2,3-d]pyrimidines and Their Analogs as Antiviral Agents against Zika Virus.

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.

Anti-HCMV activity by an irreversible p97 inhibitor LC-1310.

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.

An antibody panel for highly specific detection and differentiation of Zika virus.

Zika virus (ZIKV) is an emerging flavivirus transmitted to humans by Aedes mosquitos. ZIKV can be transmitted from mother to fetus during pregnancy and can cause microcephaly and other birth defects. Effective vaccines for Zika are yet to be approved. Detection of the ZIKV is based on serological testing that often shows cross-reactivity with the Dengue virus (DENV) and other flaviviruses. We aimed to assemble a highly specific anti-Zika antibody panel to be utilized in the development of a highly specific and cost-effective ZIKV rapid quantification assay for viral load monitoring at point-of-care settings. To this end, we tested the affinity and specificity of twenty one commercially available monoclonal and polyclonal antibodies against ZIKV and DENV envelope proteins utilizing nine ZIKV and twelve DENV strains. We finalized and tested a panel of five antibodies for the specific detection and differentiation of ZIKV and DENV infected samples.

One-Pot Synthesis of 1-Hydroxyacridones from para-Quinols and ortho-Methoxycarbonylaryl Isocyanates.

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.

Mosquito cells persistently infected with dengue virus produce viral particles with host-dependent replication.

Dengue viruses (DENV) are important arboviruses that can establish a persistent infection in its mosquito vector Aedes. Mosquitoes have a short lifetime in nature which makes trying to study the processes that take place during persistent viral infections in vivo. Therefore, C6/36 cells have been used to study this type of infection. C6/36 cells persistently infected with DENV 2 produce virions that cannot infect BHK -21 cells. We hypothesized that the following passages in mosquito cells have a deleterious impact on DENV fitness in vertebrate cells. Here, we demonstrated that the viral particles released from persistently infected cells were infectious to mosquito but not to vertebrate cells. This host restriction occurs at the replication level and is associated with several mutations in the DENV genome. In summary, our findings provide new information about viral replication fitness in a host-dependent manner.

The ß4 subunit of Cav1.2 channels is required for an optimal interferon response in cardiac muscle cells.

The auxiliary ß4 subunit of the cardiac Cav1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the ß4 subunit in H9c2 rat cardiac cells on the abundances of Ifnb mRNA [which encodes interferon-ß (IFN-ß)] and of the IFN-ß-related genes Ddx58, Ifitm3, Irf7, Stat2, Ifih1, and Mx1, as well as on the abundances of the antiviral proteins DDX58, IRF7, STAT2, and IFITM3. Knocking down the ß4 subunit in H9c2 cells reduced the expression of IFN-ß-stimulated genes. In response to inhibition of the kinase JAK1, the abundances of ß4 subunit mRNA and protein were decreased. ß4 subunit abundance was increased, and it translocated to the nucleus, in cells treated with IFN-ß, infected with dengue virus (DENV), or transfected with poly(I:C), a synthetic analog of double-stranded RNA. Cells that surrounded the virus-infected cells showed translocation of ß4 subunit proteins to nuclei in response to spreading infection. We showed that the ß4 subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter-driven reporter was increased in cells overexpressing the ß4 subunit. Last, overexpressing ß4 in undifferentiated and differentiated H9c2 cells reduced DENV infection and decreased the abundance of the viral proteins NS1, NS3, and E-protein. DENV infection and poly(I:C) also increased the concentration of intracellular Ca2+ in these cells. These findings suggest that the ß4 subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection.

Fragile X mental retardation protein is a Zika virus restriction factor that is antagonized by subgenomic flaviviral RNA.

Subgenomic flaviviral RNA (sfRNA) accumulates during infection due to incomplete degradation of viral genomes and interacts with cellular proteins to promote infection. Here we identify host proteins that bind the Zika virus (ZIKV) sfRNA. We identified fragile X mental retardation protein (FMRP) as a ZIKV sfRNA-binding protein and confirmed this interaction in cultured cells and mouse testes. Depletion of FMRP elevated viral translation and enhanced ZIKV infection, indicating that FMRP is a ZIKV restriction factor. We further observed that an attenuated ZIKV strain compromised for sfRNA production was disproportionately stimulated by FMRP knockdown, suggesting that ZIKV sfRNA antagonizes FMRP activity. Importantly, ZIKV infection and expression of ZIKV sfRNA upregulated endogenous FMRP target genes in cell culture and ZIKV-infected mice. Together, our observations identify FMRP as a ZIKV restriction factor whose activity is antagonized by the sfRNA. Interaction between ZIKV and FMRP has significant implications for the pathogenesis of ZIKV infections.

Biochemistry and Molecular Biology of Flaviviruses.

Flaviviruses, such as dengue, Japanese encephalitis, tick-borne encephalitis, West Nile, yellow fever, and Zika viruses, are critically important human pathogens that sicken a staggeringly high number of humans every year. Most of these pathogens are transmitted by mosquitos, and not surprisingly, as the earth warms and human populations grow and move, their geographic reach is increasing. Flaviviruses are simple RNA-protein machines that carry out protein synthesis, genome replication, and virion packaging in close association with cellular lipid membranes. In this review, we examine the molecular biology of flaviviruses touching on the structure and function of viral components and how these interact with host factors. The latter are functionally divided into pro-viral and antiviral factors, both of which, not surprisingly, include many RNA binding proteins. In the interface between the virus and the hosts we highlight the role of a noncoding RNA produced by flaviviruses to impair antiviral host immune responses. Throughout the review, we highlight areas of intense investigation, or a need for it, and potential targets and tools to consider in the important battle against pathogenic flaviviruses.

Dengue subgenomic flaviviral RNA disrupts immunity in mosquito salivary glands to increase virus transmission.

Globally re-emerging dengue viruses are transmitted from human-to-human by Aedes mosquitoes. While viral determinants of human pathogenicity have been defined, there is a lack of knowledge of how dengue viruses influence mosquito transmission. Identification of viral determinants of transmission can help identify isolates with high epidemiological potential. Additionally, mechanistic understanding of transmission will lead to better understanding of how dengue viruses harness evolution to cycle between the two hosts. Here, we identified viral determinants of transmission and characterized mechanisms that enhance production of infectious saliva by inhibiting immunity specifically in salivary glands. Combining oral infection of Aedes aegypti mosquitoes and reverse genetics, we identified two 3' UTR substitutions in epidemic isolates that increased subgenomic flaviviral RNA (sfRNA) quantity, infectious particles in salivary glands and infection rate of saliva, which represents a measure of transmission. We also demonstrated that various 3'UTR modifications similarly affect sfRNA quantity in both whole mosquitoes and human cells, suggesting a shared determinism of sfRNA quantity. Furthermore, higher relative quantity of sfRNA in salivary glands compared to midgut and carcass pointed to sfRNA function in salivary glands. We showed that the Toll innate immune response was preferentially inhibited in salivary glands by viruses with the 3'UTR substitutions associated to high epidemiological fitness and high sfRNA quantity, pointing to a mechanism for higher saliva infection rate. By determining that sfRNA is an immune suppressor in a tissue relevant to mosquito transmission, we propose that 3'UTR/sfRNA sequence evolution shapes dengue epidemiology not only by influencing human pathogenicity but also by increasing mosquito transmission, thereby revealing a viral determinant of epidemiological fitness that is shared between the two hosts.

The 5' and 3' Untranslated Regions of the Flaviviral Genome.

Flaviviruses are enveloped arthropod-borne viruses with a single-stranded, positive-sense RNA genome that can cause serious illness in humans and animals. The 11 kb 5' capped RNA genome consists of a single open reading frame (ORF), and is flanked by 5' and 3' untranslated regions (UTR). The ORF is a polyprotein that is processed into three structural and seven non-structural proteins. The UTRs have been shown to be important for viral replication and immune modulation. Both of these regions consist of elements that are essential for genome cyclization, resulting in initiation of RNA synthesis. Genome mutation studies have been employed to investigate each component of the essential elements to show the necessity of each component and its role in viral RNA replication and growth. Furthermore, the highly structured 3'UTR is responsible for the generation of subgenomic flavivirus RNA (sfRNA) that helps the virus evade host immune response, thereby affecting viral pathogenesis. In addition, changes within the 3'UTR have been shown to affect transmissibility between vector and host, which can influence the development of vaccines.

DENV up-regulates the HMG-CoA reductase activity through the impairment of AMPK phosphorylation: A potential antiviral target.

Dengue is the most common mosquito-borne viral disease in humans. Changes of lipid-related metabolites in endoplasmic reticulum of dengue virus (DENV) infected cells have been associated with replicative complexes formation. Previously, we reported that DENV infection inhibits HMGCR phosphorylation generating a cholesterol-enriched cellular environment in order to favor viral replication. In this work, using enzymatic assays, ELISA, and WB we found a significant higher activity of HMGCR in DENV infected cells, associated with the inactivation of AMPK. AMPK activation by metformin declined the HMGCR activity suggesting that AMPK inactivation mediates the enhanced activity of HMGCR. A reduction on AMPK phosphorylation activity was observed in DENV infected cells at 12 and 24 hpi. HMGCR and cholesterol co-localized with viral proteins NS3, NS4A and E, suggesting a role for HMGCR and AMPK activity in the formation of DENV replicative complexes. Furthermore, metformin and lovastatin (HMGCR inhibitor) altered this co-localization as well as replicative complexes formation supporting that active HMGCR is required for replicative complexes formation. In agreement, metformin prompted a significant dose-dependent antiviral effect in DENV infected cells, while compound C (AMPK inhibitor) augmented the viral genome copies and the percentage of infected cells. The PP2A activity, the main modulating phosphatase of HMGCR, was not affected by DENV infection. These data demonstrate that the elevated activity of HMGCR observed in DENV infected cells is mediated through AMPK inhibition and not by increase in PP2A activity. Interestingly, the inhibition of this phosphatase showed an antiviral effect in an HMGCR-independent manner. These results suggest that DENV infection increases HMGCR activity through AMPK inactivation leading to higher cholesterol levels in endoplasmic reticulum necessary for replicative complexes formation. This work provides new information about the mechanisms involved in host lipid metabolism during DENV replicative cycle and identifies new potential antiviral targets for DENV replication.