AI-Driven Discovery of SARS-CoV-2 Main Protease Fragment-like Inhibitors with Antiviral Activity In Vitro.

SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp's, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 µM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case.

BACKGROUND: According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak. OBJECTIVE: To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies. METHODS: We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). FINDINGS: We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM. MAIN CONCLUSIONS: Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.

Anti-SARS-CoV-2 Activity of Ampelozizyphus amazonicus (Saracura-Mirá): Focus on the Modulation of the Spike-ACE2 Interaction by Chemically Characterized Bark Extracts by LC-DAD-APCI-MS/MS.

Traditional medicine shows several treatment protocols for COVID-19 based on natural products, revealing its potential as a possible source of anti-SARS-CoV-2 agents. Ampelozizyphus amazonicus is popularly used in the Brazilian Amazon as a fortifier and tonic, and recently, it has been reported to relieve COVID-19 symptoms. This work aimed to investigate the antiviral potential of A. amazonicus, focusing on the inhibition of spike and ACE2 receptor interaction, a key step in successful infection. Although saponins are the major compounds of this plant and often reported as its active principles, a polyphenol-rich extract was the best inhibitor of the spike and ACE2 interaction. Chemical characterization of A. amazonicus bark extracts by LC-DAD-APCI-MS/MS before and after clean-up steps for polyphenol removal showed that the latter play an essential role in maintaining this activity. The effects of the extracts on viral replication were also assessed, and all samples (aqueous and ethanol extracts) demonstrated in vitro activity, inhibiting viral titers in the supernatant of Calu-3 cells after 24 hpi. By acting both in the SARS-CoV-2 cell entry process and its replication, A. amazonicus bark extracts stand out as a multitarget agent, highlighting the species as a promising candidate in the development of anti-SARS-CoV-2 drugs.

Antiviral Effect of 5'-Arylchalcogeno-3-aminothymidine Derivatives in SARS-CoV-2 Infection.

The understanding that zidovudine (ZDV or azidothymidine, AZT) inhibits the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 and that chalcogen atoms can increase the bioactivity and reduce the toxicity of AZT has directed our search for the discovery of novel potential anti-coronavirus compounds. Here, the antiviral activity of selenium and tellurium containing AZT derivatives in human type II pneumocytes cell model (Calu-3) and monkey kidney cells (Vero E6) infected with SARS-CoV-2, and their toxic effects on these cells, was evaluated. Cell viability analysis revealed that organoselenium (R3a-R3e) showed lower cytotoxicity than organotellurium (R3f, R3n-R3q), with CC50 ≥ 100 µM. The R3b and R3e were particularly noteworthy for inhibiting viral replication in both cell models and showed better selectivity index. In Vero E6, the EC50 values for R3b and R3e were 2.97 ± 0.62 µM and 1.99 ± 0.42 µM, respectively, while in Calu-3, concentrations of 3.82 ± 1.42 µM and 1.92 ± 0.43 µM (24 h treatment) and 1.33 ± 0.35 µM and 2.31 ± 0.54 µM (48 h) were observed, respectively. The molecular docking calculations were carried out to main protease (Mpro), papain-like protease (PLpro), and RdRp following non-competitive, competitive, and allosteric inhibitory approaches. The in silico results suggested that the organoselenium is a potential non-competitive inhibitor of RdRp, interacting in the allosteric cavity located in the palm region. Overall, the cell-based results indicated that the chalcogen-zidovudine derivatives were more potent than AZT in inhibiting SARS-CoV-2 replication and that the compounds R3b and R3e play an important inhibitory role, expanding the knowledge about the promising therapeutic capacity of organoselenium against COVID-19.

Myrtucommulones and Related Acylphloroglucinols from Myrtaceae as a Promising Source of Multitarget SARS-CoV-2 Cycle Inhibitors.

The LABEXTRACT plant extract bank, featuring diverse members of the Myrtaceae family from Brazilian hot spot regions, provides a promising avenue for bioprospection. Given the pivotal roles of the Spike protein and 3CLpro and PLpro proteases in SARS-CoV-2 infection, this study delves into the correlations between the Myrtaceae species from the Atlantic Forest and these targets, as well as an antiviral activity through both in vitro and in silico analyses. The results uncovered notable inhibitory effects, with Eugenia prasina and E. mosenii standing out, while E. mosenii proved to be multitarget, presenting inhibition values above 72% in the three targets analyzed. All extracts inhibited viral replication in Calu-3 cells (EC50 was lower than 8.3 µg·mL-1). Chemometric analyses, through LC-MS/MS, encompassing prediction models and molecular networking, identified potential active compounds, such as myrtucommulones, described in the literature for their antiviral activity. Docking analyses showed that one undescribed myrtucommulone (m/z 841 [M - H]-) had a higher fitness score when interacting with the targets of this study, including ACE2, Spike, PLpro and 3CLpro of SARS-CoV-2. Also, the study concludes that Myrtaceae extracts, particularly from E. mosenii and E. prasina, exhibit promising inhibitory effects against crucial stages in SARS-CoV-2 infection. Compounds like myrtucommulones emerge as potential anti-SARS-CoV-2 agents, warranting further exploration.

Ebselen and Diphenyl Diselenide Inhibit SARS-CoV-2 Replication at Non-Toxic Concentrations to Human Cell Lines.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the causative agent of the COVID-19 pandemic, a global public health problem. Despite the numerous studies for drug repurposing, there are only two FDA-approved antiviral agents (Remdesivir and Nirmatrelvir) for non-hospitalized patients with mild-to-moderate COVID-19 symptoms. Consequently, it is pivotal to search for new molecules with anti-SARS-CoV-2 activity and to study their effects in the human immune system. Ebselen (Eb) is an organoselenium compound that is safe for humans and has antioxidant, anti-inflammatory, and antimicrobial properties. Diphenyl diselenide ((PhSe)2) shares several pharmacological properties with Eb and is of low toxicity to mammals. Herein, we investigated Eb and (PhSe)2 anti-SARS-CoV-2 activity in a human pneumocytes cell model (Calu-3) and analyzed their toxic effects on human peripheral blood mononuclear cells (PBMCs). Both compounds significantly inhibited the SARS-CoV-2 replication in Calu-3 cells. The EC50 values for Eb and (PhSe)2 after 24 h post-infection (hpi) were 3.8 µM and 3.9 µM, respectively, and after 48 hpi were 2.6 µM and 3.4 µM. These concentrations are safe for non-infected cells, since the CC50 values found for Eb and (PhSe)2 on Calu-3 were greater than 200 µM. Importantly, the concentration rates tested on viral replication were not toxic to human PBMCs. Therefore, our findings reinforce the efficacy of Eb and demonstrate (PhSe)2 as a new candidate to be tested in future trials against SARS-CoV-2 infection/inflammation conditions.

Monkeypox (Mpox) virus isolation and ultrastructural characterisation from a Brazilian human sample case

BACKGROUND According to the last 2023 Monkeypox (Mpox) Outbreak Global Map from the Centres for Disease Control and Prevention (CDC), more than 100 countries with no Mpox infection report cases. Brazil stands out in this group and is the second country with the highest number of cases in the last outbreak. OBJECTIVE To contribute to knowledge of the virus infection effects in a cellular model, which is important for diagnosis infections not yet included in a provider´s differential diagnosis and for developing viral inhibition strategies. METHODS We describe a virus isolation protocol for a human clinical sample from a patient from Brazil, the viral growth in a cell model through plaque forming units (PFU) assay, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy (TEM). FINDINGS We follow the viral isolation in Vero cell culture from a Mpox positive clinically diagnosed sample and show the infection effects on cellular structures using a TEM. MAIN CONCLUSIONS Understanding the impact of viral growth on cellular structures and its replication kinetics may offer better strategies for the development of new drugs with antiviral properties.