Efficient in vitro assay for evaluating drug efficacy and synergy against emerging SARS-CoV-2 strains

Antimicrobial Agents and Chemotherapy, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 17, 2024

ABSTRACT Novel and repurposed antiviral drugs are available for the treatment of coronavirus disease 2019 (COVID-19). However, combinations may be more potent lead to faster viral clearance, but methods screening against respiratory viruses not well established labor-intensive. Here, we describe a time-efficient (72–96 h) simple in vitro drug-sensitivity assay severe acute syndrome 2 (SARS-CoV-2) using standard 96-well plates. We employ different synergy models (zero interaction potency, highest single agent, Loewe, Bliss) determine efficacy therapies synergistic ancestral emerging clinical SARS-CoV-2 strains. found that monotherapy remdesivir, nirmatrelvir, active metabolite molnupiravir (EIDD-1931) demonstrated baseline EC50s within clinically achievable levels 4.34 mg/L (CI: 3.74–4.94 mg/L), 1.25 1.10–1.45 0.25 0.20–0.30 respectively, strain. their varied newer Omicron variants BA.1.1.15 BA.2, particularly with protease inhibitor nirmatrelvir. also remdesivir nirmatrelvir have consistent, strong effect (Bliss score >10) at relevant drug concentrations (nirmatrelvir 0.25–1 1–4 mg/L) across all strains tested. This method offers practical tool streamlines identification effective combination detection resistance. Our findings support use targeting multiple components enhance COVID-19 efficacy, context

Language: Английский

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Viral Entry Inhibitors Protect against SARS-CoV-2-Induced Neurite Shortening in Differentiated SH-SY5Y Cells

Viruses, Journal Year: 2023, Volume and Issue: 15(10), P. 2020 - 2020

Published: Sept. 28, 2023

The utility of human neuroblastoma cell lines as in vitro model to study neuro-invasiveness and neuro-virulence SARS-CoV-2 has been demonstrated by our laboratory others. aim this report is further characterize the associated cellular responses caused a pre-alpha strain on differentiated SH-SY5Y prevent its cytopathic effect using set entry inhibitors. susceptibility was confirmed at high multiplicity-of-infection, without viral replication or release. Infection reduction length neuritic processes, occurrence plasma membrane blebs, clustering, changes lipid droplets electron density. No expression cytoskeletal proteins, such tubulins tau, could explain neurite shortening. To counteract toxic neurites, inhibitors targeting TMPRSS2, ACE2, NRP1 receptors, Spike RBD were co-incubated with inoculum. shortening be prevented highest concentration camostat mesylate, anti-RBD antibody, inhibitor, but not soluble ACE2. According degree inhibition, average amount intracellular RNA negatively correlated length. This that specific host receptors reverse neurocytopathic SH-SY5Y.

Language: Английский

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A Cell-Based Papain-like Protease (PLpro) Activity Assay for Rapid Detection of Active SARS-CoV-2 Infections and Antivirals

medRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 21, 2024

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life prosperity. An urgent need remains for simple fast tests that reliably detect active infections with SARS-CoV-2 in asymptomatic presymptomatic carriers. Here we introduce rapid activity-based diagnostic (ABDx) test identifies by measuring the activity of viral enzyme, Papain-Like protease (PLpro). The system consists peptide fluoresces when cleaved SARS PLpro is crude, unprocessed lysates from tongue scrapes saliva. Test results obtained 30 minutes or less using widely available fluorescence plate readers, battery-operated portable instrument testing at point care (PoC) sites low-resource hard reach populations. Proof-of-concept was clinical study on specimens collected patients COVID-19 like symptoms who tested positive (n=10) negative LIAT RT-PCR nasal mid turbinate swabs. When saliva these in-house endpoint only 3 were 17 positive. correlated cases (3 out negatives 14 16 positives, one invalid specimen. Despite small number samples, agreement significant (p value = 0.01). In presumptive false patient RNA detected 5 days later RT-PCR. Two detected, sample late Ct 35 RT-PCR, indicating an infection no longer present. assay easily scalable expected all viable recombinants, making it attractive as screening surveillance tool. Additionally, show feasibility platform new homogeneous phenotypic antiviral drugs neutralizing antibodies.

Language: Английский

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Phosphoglycerate mutase regulates Treg differentiation through control of serine synthesis and one-carbon metabolism

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: June 28, 2024

The differentiation and suppressive functions of regulatory CD4 T cells (Tregs) are supported by a broad array metabolic changes, providing potential therapeutic targets for immune modulation. In this study, we focused on the role glycolytic enzymes in Tregs identified phosphoglycerate mutase (PGAM) as being differentially overexpressed associated with highly phenotype. Pharmacologic or genetic inhibition PGAM reduced Treg function while reciprocally inducing markers pro-inflammatory, helper 17 (Th17)-like state. was dependent contribution 3-phosphoglycerate (3PG), substrate, to de novo serine synthesis. Blocking synthesis from 3PG reversed effect polarization, exogenous directly inhibited polarization. Additionally, altering levels

Language: Английский

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A cell-based Papain-like Protease (PLpro) activity assay for rapid detection of active SARS-CoV-2 infections and antivirals

PLoS ONE, Journal Year: 2024, Volume and Issue: 19(12), P. e0309305 - e0309305

Published: Dec. 26, 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are a continuous threat to human life. An urgent need remains for simple fast tests that reliably detect active infections with SARS-CoV-2 in the early stage of infection. Here we introduce rapid activity-based diagnostic (ABDx) test identifies by measuring activity viral enzyme, Papain-Like protease (PLpro). The system consists peptide fluoresces when cleaved SARS PLpro is crude, unprocessed lysates from tongue scrapes saliva. Test results obtained 30 minutes or less using widely available fluorescence plate readers, battery-operated portable instrument on-site testing. Proof-of-concept was study on clinical specimens collected patients COVID-19 like symptoms who tested positive (n = 10) negative LIAT RT-PCR nasal mid turbinate swabs. When saliva these in-house endpoint RT-PCR, 17 were only 5 negative, which became days later. correlated cases (3 out 3 negatives 14 16 positives, one invalid specimen). Despite small number samples, agreement significant (p value 0.01). Two false detected, sample late Ct 35 indicating an infection no longer present. assay easily scalable expected all viable variants, making it attractive as screening surveillance tool. Additionally, show feasibility platform new homogeneous phenotypic antiviral drugs neutralizing antibodies.

Language: Английский

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The Trimeric Artesunate Analog TF27, a Broadly Acting Anti-Infective Model Drug, Exerts Pronounced Anti-SARS-CoV-2 Activity Spanning Variants and Host Cell Types

Pharmaceutics, Journal Year: 2022, Volume and Issue: 15(1), P. 115 - 115

Published: Dec. 29, 2022

Starting in 2019, the spread of respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated pandemic corona virus disease (COVID-19) has led to enormous efforts development medical countermeasures. Although innovative vaccines have scaled back number severe COVID cases, emergence omicron variant (B.1.1.529) illustrates how vaccine struggles keep pace with viral evolution. On other hand, while recently approved antiviral drugs remdesivir, molnupiravir, Paxlovid are considered as broadly acting anti-coronavirus therapeutics, only molnupiravir orally available none these recommended for prophylactic use. Thus, so far unexploited small molecules, targeting strategies, mechanisms urgently needed address issues current putative future outbreaks newly emerging variants concern. Recently, we others described anti-infective potential particularly pronounced activity artesunate related compounds trioxane/sesquiterpene class. In particular, trimeric derivative TF27 demonstrated strong anti-cytomegalovirus at nanomolar concentrations vitro well vivo efficacy after oral administration therapeutic even treatment settings. Here, extended this analysis by evaluating its anti-SARS-CoV-2 potential. Our main findings follows: (i) compound exerted (EC50 = 0.46 ± 0.20 µM), (ii) was clearly distinct from induction cytotoxicity, (iii) pretreatment prevented replication cultured cells, (iv) likewise been Calu-3 human lung Caco-2 colon cells infected wild-type, delta, or SARS-CoV-2, respectively, (v) combination treatments revealed synergistic interaction GC376, but antagonistic EIDD-1931. Combined, data thus highlight trioxane further pharmacologic towards improved options COVID-specific medication.

Language: Английский

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