CRISPR/Cas9 knock-in strategy to evaluate phospho-regulation of SAMHD1

Sterile α motif (SAM) and HD domain-containing protein 1 (SAMHD1) is a potent restriction factor for immunodeficiency virus 1 (HIV-1), active in myeloid and resting CD4+ T cells. As a dNTP triphosphate triphosphohydrolase (dNTPase), SAMHD1 is proposed to limit cellular dNTP levels correlating with inhibition of HIV-1 reverse transcription. The anti-viral activity of SAMHD1 is regulated by dephosphorylation of the residue T592. However, the impact of T592 phosphorylation on dNTPase activity is still under debate. Whether additional cellular functions of SAMHD1 impact anti-viral restriction is also not completely understood. We use BlaER1 cells as a novel human macrophage transdifferentiation model combined with CRISPR/Cas9 knock-in (KI) to study SAMHD1 mutations in a physiological context. Transdifferentiated BlaER1 cells, resembling primary human macrophages, harbor active dephosphorylated SAMHD1 that blocks HIV-1 reporter virus infection. Co-delivery of Vpx or CRISPR/Cas9-mediated SAMHD1 knock-out relieves the block to HIV-1. Using CRISPR/Cas9-mediated homologous recombination, we introduced specific mutations into the genomic SAMHD1 locus. Homozygous T592E mutation, but not T592A, leads to loss of HIV-1 restriction, confirming the role of T592 dephosphorylation in the regulation of anti-viral activity. However, T592E KI cells retain wild type dNTP levels, suggesting the antiviral state might not only rely on dNTP depletion. In conclusion, the role of the T592 phospho-site for anti-viral restriction was confirmed in an endogenous physiological context. Importantly, loss of restriction in T592E mutant cells does not correlate with increased dNTP levels, indicating that the regulation of anti-viral and dNTPase activity of SAMHD1 might be uncoupled.

Chronic Hepatitis B Viral Activity As A Prognostic Factor For Survival In Malignant Lymphoma

Background: Hepatitis B virus (HBV) infection carries a risk of liver cancer and extrahepatic malignancy. However, the incidence trend and clinical course of malignant lymphoma (ML) in HBV patients are not well known. Methods: Data about ML newly diagnosed in chronic hepatitis B (CHB) patients from 2003 to 2016 were collected from National Health Insurance Service claims. Results: A total of 13,942 CHB patients were newly diagnosed with ML from 2003 to 2016. The number of patients increased 3.8 times, from 442 in 2003 to 1,711 in 2016. The 2-year survival rate of all patients was 76.8%, and the 5-year survival rate was 69.8%. The survival rate of patients taking antivirals due to high viral activity before their diagnosis with ML was significantly lower than that of patients with lower viral activity (1 yr-77.3%, 3 yr-64.5%, and 5 yr-58.3% vs. 1 yr-84.0%, 3 yr-73.4%, and 5 yr-68.0%, respectively). The survival rate of patients with liver cirrhosis (LC) at baseline was significantly lower than that of those without LC. Cirrhotic patients taking antivirals before ML diagnosis had a worse prognosis than who did not. Conclusions: High viral activity in CHB patients with ML is useful in predicting the prognosis for survival.

Physicochemical Characteristics and In Vitro Toxicity/Anti-SARS-CoV-2 Activity of Favipiravir Solid Lipid Nanoparticles (SLNs)

The rise of coronavirus (COVID-19) cases worldwide has driven the need to discover and develop novel therapeutics with superior efficacy to treat this disease. This study aims to develop an innovative aerosolized nano-formulation of favipiravir (FPV) as an anti-viral agent against coronavirus infection. The local delivery of FPV nanoparticles (NPs) via nebulization ensures that the drug can reach the site of infection, the lungs. Solid lipid NPs of favipiravir (FPV-SLNs) were formulated utilizing the hot-evaporation method. The physicochemical formulation properties were evaluated using dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The aerosol formulation performance was evaluated using an Andersen Cascade Impactor (ACI) at a flow rate of 15 L/min. The FPV-SLN formulation’s in vitro anti-viral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was also evaluated using the SARS-CoV-2 pathogen (hCoV-19/Egypt/NRC-3/2020 isolate). The FPV-SLNs’ morphology was defined utilizing transmission electron microscopy, showing an irregular shape. By means of FPV-SLNs’ nebulization, a fine particle fraction of 60.2 ± 1.7% was produced with 60.2 ± 1.7%, and this finding suggests that FPV-SLNs were appropriate for inhalation drug delivery with a particle size of 537.6 ± 55.72 nm. Importantly, the FPV-SLNs showed anti-viral activity against SARS-CoV-2 with CC50 and IC50 values of 449.6 and 29.9 µg/mL, respectively. This study suggests that inhaled solid lipid NPs of favipiravir could potentially be used against coronavirus.

HYPOTHESIS: TETRACYCLINES FOR SEVERE COVID-19 – THE PERFECT REPURPOSING CANDIDATE?

We would like to draw attention to the potential of immunomodulatory tetracyclines for severe COVID-19. The COVID-19 pandemic is having a devastating impact on developing countries. A successful approach to manage the scarcity of cost-effective therapies worldwide is drug repurposing. Predictions of direct anti-viral activity of tetracyclines against SARS-CoV2 have been confirmed experimentally. Furthermore, their effectiveness in experimental ARDS has been proven extensively, counteracting the overt inflammatory reaction and fibrosis sequelae due to a synergic combination of pharmacological activities. Finally, a few clinical reports have confirmed their potentiall in COVID-19 patients, encouraging the development of this novel indication. We believe that the benefits of their multi-target pharmacology and their safety profile place immunomodulatory tetracyclines as gold repurposing candidates for COVID-19.

Induction of Gut Microbial Tryptamine by SARS-CoV-2 in Nonhuman Primate Model Consistent with Tryptamine-Induced Model of Neurodegeneration

The author discussed recently the possible molecular mechanisms that cause the COVID-19 disease symptoms. Here the analysis of the recent experimental data supports the hypothesis that production of the gut microbial tryptamine can be induced by the SARS-CoV-2 fecal viral activity due to the selective pressure or positive selection of tryptamine-producing microorganisms. In this report, the author suggests that the mechanism of microbial selection bases on the abilities of tryptamine to affect the viral nucleic acid. In other words, the gut microorganisms producing tryptamine are more resistant to SARS-CoV-2 fecal viral activity than microorganisms producing no tryptamine. Earlier we demonstrated the induction of neurodegeneration by tryptamine in human cells and mouse brain. Furthermore, we were able to uncover the human gut bacteria associated with Alzheimer’s disease (AD) using PCR testing of human fecal samples with the new-designed primers targeting the tryptophan-tryptamine pathway. Likely, SARS-CoV-2 is one of the selective pressure factors in the cascade accelerating the neurodegenerative process in AD. This suggestion is consistent with a higher proportion of AD patients among COVID-19 related victims. Gut microbial tryptamine increase due to the viral infection-induced dysbiosis can synergize and potentiate the tryptamine cytotoxicity, necrotizing ability and other properties as a virulence factor.

Computational Chemistry to Repurposing Drugs for the Control of COVID-19

Thus far, in 2021, 219 countries with over 175 million people have been infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a positive sense, single-stranded RNA virus, and is the causal agent for coronavirus disease (COVID-19). Due to the urgency of the situation, virtual screening as a computational modeling method offers a fast and effective modality of identifying drugs that may be effective against SARS-CoV-2. There has been an overwhelming abundance of molecular docking against SARS-CoV-2 in the last year. Due to the massive volume of computational studies, this systematic review has been created to evaluate and summarize the findings of existing studies. Herein, we report on computational articles of drugs which target, (1) viral protease, (2) Spike protein-ACE 2 interaction, (3) RNA-dependent RNA polymerase, and (4) other proteins and nonstructural proteins of SARS-CoV-2. Based on the studies presented, there are 55 identified natural or drug compounds with potential anti-viral activity. The next step is to show anti-viral activity in vitro and translation to determine effectiveness into human clinical trials.

Simple rapid in vitro screening method for SARS-CoV-2 anti-virals that identifies potential cytomorbidity-associated false positives

Background The international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19. The initial step to identifying potential candidates usually involves in vitro screening that includes standard cytotoxicity controls. Under-appreciated is that viable, but stressed or otherwise compromised cells, can also have a reduced capacity to replicate virus. A refinement proposed herein for in vitro drug screening thus includes a simple growth assay to identify drug concentrations that cause cellular stress or “cytomorbidity”, as distinct from cytotoxicity or loss of viability. Methods A simple rapid bioassay is presented for antiviral drug screening using Vero E6 cells and inhibition of SARS-CoV-2 induced cytopathic effects (CPE) measured using crystal violet staining. We use high cell density for cytotoxicity assays, and low cell density for cytomorbidity assays. Results The assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. In contrast, nitazoxanide, oleuropein, cyclosporine A and ribavirin all showed no ability to inhibit SARS-CoV-2 CPE. Hydroxychloroquine, cyclohexamide, didemnin B, γ-mangostin and linoleic acid were all able to inhibit viral CPE at concentrations that did not induce cytotoxicity. However, these drugs inhibited CPE at concentrations that induced cytomorbidity, indicating non-specific anti-viral activity. Conclusions We describe the methodology for a simple in vitro drug screening assay that identifies potential anti-viral drugs via their ability to inhibit SARS-CoV-2-induced CPE. The additional growth assay illustrated how several drugs display anti-viral activity at concentrations that induce cytomorbidity. For instance, hydroxychloroquine showed anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities. The cytomorbidity assay can therefore rapidly exclude potential false positives.