Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 61
Filter
1.
Signal Transduction and Targeted Therapy ; 8(1):42, 2023.
Article in English | ProQuest Central | ID: covidwho-2211939

ABSTRACT

The Omicron variants of SARS-CoV-2, primarily authenticated in November 2021 in South Africa, has initiated the 5th wave of global pandemics. Here, we systemically examined immunological and metabolic characteristics of Omicron variants infection. We found Omicron resisted to neutralizing antibody targeting receptor binding domain (RBD) of wildtype SARS-CoV-2. Omicron could hardly be neutralized by sera of Corona Virus Disease 2019 (COVID-19) convalescents infected with the Delta variant. Through mass spectrometry on MHC-bound peptidomes, we found that the spike protein of the Omicron variants could generate additional CD8 + T cell epitopes, compared with Delta. These epitopes could induce robust CD8 + T cell responses. Moreover, we found booster vaccination increased the cross-memory CD8 + T cell responses against Omicron. Metabolic regulome analysis of Omicron-specific T cell showed a metabolic profile that promoted the response of memory T cells. Consistently, a greater fraction of memory CD8 + T cells existed in Omicron stimulated peripheral blood mononuclear cells (PBMCs). In addition, CD147 was also a receptor for the Omicron variants, and CD147 antibody inhibited infection of Omicron. CD147-mediated Omicron infection in a human CD147 transgenic mouse model induced exudative alveolar pneumonia. Taken together, our data suggested that vaccination booster and receptor blocking antibody are two effective strategies against Omicron.

2.
Frontiers in endocrinology ; 13, 2022.
Article in English | EuropePMC | ID: covidwho-2147820

ABSTRACT

Aims The global COVID-19 pandemic has required a drastic transformation of prenatal care services. Whether the reformulation of the antenatal care systems affects maternal and infant outcomes remains unknown. Particularly, women with gestational diabetes mellitus (GDM) are among those who bear the greatest brunt. Thus, this study aimed to evaluate the impact of COVID-19 lockdown during late pregnancy on maternal and infant outcomes in women stratified by the GDM status in China. Study design The participants were women who experienced the COVID-19 lockdown during late pregnancy (3185 in the 2020 cohort) or not (2540 in the 2019 cohort) that were derived from the Beijing Birth Cohort Study. Maternal metabolic indicators, neonatal outcomes, and infant anthropometrics at 12 months of age were compared between the two cohorts, stratified by the GDM status. Results Participants who experienced COVID-19 lockdown in late pregnancy showed lower gestational weight gain than those in the control cohort. Nevertheless, they displayed a worse metabolic profile. COVID-19 lockdown during pregnancy was associated with higher glycosylated hemoglobin (HbA1c) (β= 0.11, 95% CI = 0.05–0.16, q-value = 0.002) and lower high density lipoprotein cholesterol level (HDL-C) level (β=–0.09, 95% CI = –0.14 to –0.04, q-value = 0.004) in women with GDM, adjusted for potential confounders. In normoglycemic women, COVID-19 lockdown in late pregnancy was associated with higher fasting glucose level (β= 0.10, 95% CI = 0.08–0.12, q-value <0.0001), lower HDL-C level (β=–0.07, 95% CI = –0.08 to –0.04, q-value <0.0001), and increased risk of pregnancy-induced hypertension (adjusted OR=1.80, 95%CI=1.30–2.50, q-value=0.001). The fasting glucose level decreased less from early to late pregnancy in women who experienced COVID-19 lockdown than in the controls, regardless of the GDM status. The HDL-C has risen less with COVID-19 lockdown in the normoglycemic subgroup. In contrast, no significant differences regarding neonatal outcomes or infant weight were found between the two cohorts. Conclusion Experiencing the COVID-19 lockdown in pregnancy was associated with worse maternal metabolic status but similar neonatal outcomes and infant weight.

3.
Front Pharmacol ; 13: 918083, 2022.
Article in English | MEDLINE | ID: covidwho-2009894

ABSTRACT

Preclinical pharmacokinetics (PK) and In Vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.4 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

4.
Water Res ; 223: 119021, 2022 Sep 01.
Article in English | MEDLINE | ID: covidwho-2004603

ABSTRACT

Due to the Covid-19 pandemic, the worldwide biocides application has been increased, which will eventually result in enhanced residuals in treated wastewater. At the same time, chlorine disinfection of secondary effluents and hospital wastewaters has been intensified. With respect to predicted elevated exposure in wastewater, the chlorination kinetics, transformation pathways and toxicity evolution were investigated in this study for two typical isothiazolinone biocides, methyl-isothiazolinone (MIT) and chloro-methyl-isothiazolinone (CMIT). Second-order rate constants of 0.13 M-1·s-1, 1.95 × 105 M-1·s-1 and 5.14 × 105 M-1·s-1 were determined for the reaction of MIT with HOCl, Cl2O and Cl2, respectively, while reactivity of CMIT was around 1-2 orders of magnitude lower. While chlorination of isothiazolinone biocides at pH 7.1 was dominated by Cl2O-oxidation, acidic pH and elevated Cl- concentration favored free active chlorine (FAC) speciation into Cl2 and increased overall isothiazolinone removal. Regardless of the dominant FAC species, the elimination of MIT and CMIT resulted in an immediate loss of acute toxicity under all experimental conditions, which was attributed to a preferential attack at the S-atom resulting in subsequent formation of sulfoxides and sulfones and eventually an S-elimination. However, chlorination of isothiazolinone biocides in secondary effluent only achieved <10% elimination at typical disinfection chlorine exposure 200 mg·L-1·min, but was predicted to be remarkably increased by acidizing solution to pH 5.5. Alternative measures might be needed to minimize the discharge of these toxic chemicals into the aquatic environment.


Subject(s)
COVID-19 , Disinfectants , Water Pollutants, Chemical , Water Purification , Chlorine , Disinfectants/toxicity , Halogenation , Halogens , Humans , Hydrogen-Ion Concentration , Kinetics , Pandemics , Sulfones , Sulfoxides , Thiazoles , Waste Water , Water Pollutants, Chemical/analysis , Water Purification/methods
5.
PLoS One ; 17(8): e0272364, 2022.
Article in English | MEDLINE | ID: covidwho-1987156

ABSTRACT

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.


Subject(s)
Bacteriophages , COVID-19 , Single-Domain Antibodies , Antibodies, Neutralizing , Antibodies, Viral , Bacteriophages/metabolism , Humans , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
6.
Tetrahedron Lett ; 104: 154012, 2022 Aug 17.
Article in English | MEDLINE | ID: covidwho-1984119

ABSTRACT

The COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to spread around the world. GS-441524 is the parent nucleoside of remdesivir which is the first drug approved for the treatment of COVID-19, and demonstrates strong activity against SARS-Cov-2 in vitro and in vivo. Herein, we reported the synthesis of a series of deuterated GS-441524 analogs, which had deuterium atoms up to five at the ribose and the nucleobase moieties. Compared to GS-441524, all the deuterated compounds showed similar inhibitory activities against SARS-CoV-2 in vitro.

7.
Nature computational science ; 2(4):265-275, 2022.
Article in English | EuropePMC | ID: covidwho-1940349

ABSTRACT

Progress in cryo-electron microscopy has provided the potential for large-size protein structure determination. However, the success rate for solving multi-domain proteins remains low because of the difficulty in modelling inter-domain orientations. Here we developed domain enhanced modeling using cryo-electron microscopy (DEMO-EM), an automatic method to assemble multi-domain structures from cryo-electron microscopy maps through a progressive structural refinement procedure combining rigid-body domain fitting and flexible assembly simulations with deep-neural-network inter-domain distance profiles. The method was tested on a large-scale benchmark set of proteins containing up to 12 continuous and discontinuous domains with medium- to low-resolution density maps, where DEMO-EM produced models with correct inter-domain orientations (template modeling score (TM-score) >0.5) for 97% of cases and outperformed state-of-the-art methods. DEMO-EM was applied to the severe acute respiratory syndrome coronavirus 2 genome and generated models with average TM-score and root-mean-square deviation of 0.97 and 1.3 Å, respectively, with respect to the deposited structures. These results demonstrate an efficient pipeline that enables automated and reliable large-scale multi-domain protein structure modelling from cryo-electron microscopy maps.

8.
Int J Infect Dis ; 122: 622-627, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1926531

ABSTRACT

OBJECTIVES: Here, we retrospectively described the diagnosis and treatment of 32 cases diagnosed with Chlamydia psittaci pneumonia during the COVID-19 pandemic. METHODS: Clinical information was collected from all the patients. Reverse transcription-PCR and ELISAs were conducted for the detection of COVID-19 using nasal swabs and bronchoalveolar lavage fluid (BALF) samples. Metagenomic next-generation sequencing (mNGS) was performed for the identification of causative pathogens using BALF, peripheral blood and sputum samples. End-point PCR was performed to confirm the mNGS results. RESULTS: All 32 patients showed atypical pneumonia and had infection-like symptoms that were similar to COVID-19. Results of reverse transcription-PCR and ELISAs ruled out COVID-19 infection. mNGS identified C. psittaci as the suspected pathogen in these patients within 48 hours, which was validated by PCR, except for three blood samples. The sequence reads that covered fragments of C. psittaci genome were detected more often in BALF than in sputum or blood samples. All patients received doxycycline-based treatment regimens and showed favorable outcomes. CONCLUSION: This retrospective study, with the highest number of C. psittaci pneumonia enrolled cases in China so far, suggests that human psittacosis may be underdiagnosed and misdiagnosed clinically, especially in the midst of the COVID-19 pandemic.


Subject(s)
COVID-19 , Chlamydophila psittaci , Influenza, Human , Mycoses , Pneumonia, Mycoplasma , Pneumonia , Psittacosis , COVID-19/diagnosis , Chlamydophila psittaci/genetics , Humans , Pandemics , Psittacosis/diagnosis , Psittacosis/drug therapy , Psittacosis/epidemiology , Retrospective Studies
9.
J Affect Disord ; 307: 108-114, 2022 06 15.
Article in English | MEDLINE | ID: covidwho-1920983

ABSTRACT

OBJECTIVE: To systematically examine the efficacy and safety of antidepressants for the treatment of coronavirus disease 2019 (COVID-19). METHODS: A systematic search was performed independently by two researchers based on Chinese Journal Net, WanFang, PsycINFO, Cochrane Library, PubMed, and EMBASE. RESULTS: Seven studies (n = 92,947) including three retrospective studies (n = 91,083), two randomized clinical trials (RCTs, n = 1649), two prospective cohort study (n = 215) involving (n = 92,947) patients with COVID-19 were examined. For RCTs, fluvoxamine outperformed placebo in reducing clinical deterioration and hospitalisation for COVID-19 patients. For retrospective studies, antidepressants (2 studies) and fluoxetine (1 study) possibly reduced the risk of mortality in patients with COVID-19. Results from two remaining studies supported the superiority of fluvoxamine in reducing risk of mortality in COVID-19 patients. The two RCTs that examined the safety of fluvoxamine for COVID-19 patients found inconsistent results but no significant group differences in the dropout rate. CONCLUSION: This systematic review found emerging evidence for fluvoxamine in reducing the risk of mortality and hospitalisation in COVID-19 patients, but inconsistent evidence for the safety of fluvoxamine in COVID-19 patients. More studies are needed to determine the efficacy and safety of antidepressants for the treatment of COVID-19.


Subject(s)
COVID-19 , Antidepressive Agents/adverse effects , Fluvoxamine/adverse effects , Humans , Prospective Studies , Retrospective Studies
10.
ACS Infect Dis ; 8(6): 1191-1203, 2022 06 10.
Article in English | MEDLINE | ID: covidwho-1873405

ABSTRACT

SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allows for rapid movement of the existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites by TMPRSS2. Therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.


Subject(s)
COVID-19 , Percutaneous Coronary Intervention , Angiotensin-Converting Enzyme 2 , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning/methods , Humans , Pandemics , SARS-CoV-2 , Serine Endopeptidases
11.
JASA Express Lett ; 2(5): 055202, 2022 05.
Article in English | MEDLINE | ID: covidwho-1854216

ABSTRACT

Medical masks have become necessary of late because of the COVID-19 outbreak; however, they tend to attenuate the energy of speech signals and affect speech quality. Therefore, this study proposes an optical-based microphone approach to obtain speech signals from speakers' medical masks. Experimental results showed that the optical-based microphone approach achieved better performance (85.61%) than the two baseline approaches, namely, omnidirectional (24.17%) and directional microphones (31.65%), in the case of long-distance speech and background noise. The results suggest that the optical-based microphone method is a promising approach for acquiring speech from a medical mask.


Subject(s)
COVID-19 , Hearing Aids , Speech Perception , COVID-19/prevention & control , Equipment Design , Humans , Masks , Speech , Vibration
12.
Sci Rep ; 12(1): 6294, 2022 04 15.
Article in English | MEDLINE | ID: covidwho-1805651

ABSTRACT

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a heparan sulfate-spike complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar heparan sulfate-binding activities but with reduced affinity for DNA topoisomerases may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , COVID-19/drug therapy , Heparin/metabolism , Heparitin Sulfate/metabolism , Humans , Mitoxantrone/pharmacology , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism
13.
J Chem Inf Model ; 62(8): 1988-1997, 2022 04 25.
Article in English | MEDLINE | ID: covidwho-1783923

ABSTRACT

The cell entry of SARS-CoV-2 has emerged as an attractive drug development target. We previously reported that the entry of SARS-CoV-2 depends on the cell surface heparan sulfate proteoglycan (HSPG) and the cortex actin, which can be targeted by therapeutic agents identified by conventional drug repurposing screens. However, this drug identification strategy requires laborious library screening, which is time consuming, and often limited number of compounds can be screened. As an alternative approach, we developed and trained a graph convolutional network (GCN)-based classification model using information extracted from experimentally identified HSPG and actin inhibitors. This method allowed us to virtually screen 170,000 compounds, resulting in ∼2000 potential hits. A hit confirmation assay with the uptake of a fluorescently labeled HSPG cargo further shortlisted 256 active compounds. Among them, 16 compounds had modest to strong inhibitory activities against the entry of SARS-CoV-2 pseudotyped particles into Vero E6 cells. These results establish a GCN-based virtual screen workflow for rapid identification of new small molecule inhibitors against validated drug targets.


Subject(s)
Antiviral Agents , SARS-CoV-2 , Virus Internalization , Actins , Antiviral Agents/chemistry , COVID-19/drug therapy , Heparan Sulfate Proteoglycans , Humans , SARS-CoV-2/drug effects , Virus Internalization/drug effects
14.
Drug Discov Today ; 27(7): 1983-1993, 2022 07.
Article in English | MEDLINE | ID: covidwho-1773250

ABSTRACT

Drug repurposing is an appealing method to address the Coronavirus 2019 (COVID-19) pandemic because of the low cost and efficiency. We analyzed our in-house database of approved drug screens and compared their activity profiles with results from a severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) cytopathic effect (CPE) assay. The activity profiles of the human ether-à-go-go-related gene (hERG), phospholipidosis (PLD), and many cytotoxicity screens were found significantly correlated with anti-SARS-CoV-2 activity. hERG inhibition is a nonspecific off-target effect that has contributed to promiscuous drug interactions, whereas drug-induced PLD is an undesirable effect linked to hERG blockers. Thus, this study identifies preferred drug candidates as well as chemical structures that should be avoided because of their potential to induce toxicity. Lastly, we highlight the hERG liability of anti-SARS-CoV-2 drugs currently enrolled in clinical trials.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/adverse effects , COVID-19/drug therapy , Drug Repositioning/methods , Humans , Pandemics
16.
J Med Chem ; 65(6): 4590-4599, 2022 03 24.
Article in English | MEDLINE | ID: covidwho-1740391

ABSTRACT

Identification of anti-SARS-CoV-2 compounds through traditional high-throughput screening (HTS) assays is limited by high costs and low hit rates. To address these challenges, we developed machine learning models to identify compounds acting via inhibition of the entry of SARS-CoV-2 into human host cells or the SARS-CoV-2 3-chymotrypsin-like (3CL) protease. The optimal classification models achieved good performance with area under the receiver operating characteristic curve (AUC-ROC) values of >0.78. Experimental validation showed that the best performing models increased the assay hit rate by 2.1-fold for viral entry inhibitors and 10.4-fold for 3CL protease inhibitors compared to those of the original drug repurposing screens. Twenty-two compounds showed potent (<5 µM) antiviral activities in a SARS-CoV-2 live virus assay. In conclusion, machine learning models can be developed and used as a complementary approach to HTS to expand compound screening capacities and improve the speed and efficiency of anti-SARS-CoV-2 drug discovery.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning , Humans , Protease Inhibitors/pharmacology
17.
Microbiol Spectr ; 10(1): e0278021, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1700612

ABSTRACT

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S), and open reading frame (ORF)3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad preexisting immunity in the community. IMPORTANCE T cells play a critical role in protection against SARS-CoV-2. Despite being highly topical, the protective role of preexisting memory CD8+ T cells, induced by prior exposure to circulating common coronavirus strains, remains less clear. In this study, we established a robust approach to specifically assess T cell responses to highly conserved regions within SARS-CoV-2. Consistent with recent observations we demonstrate that recognition of these highly conserved regions is associated with an increased likelihood of milder disease. However, extending these observations we observed that recognition of these conserved regions is rare in both exposed and unexposed volunteers, which we believe is associated with the low abundance of these proteins in SARS-CoV-2 infected cells. These observations have important implications for the likely role preexisting immunity plays in controlling severe disease, further emphasizing the importance of vaccination to generate the immunodominant T cells required for immune protection.


Subject(s)
COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , SARS-CoV-2/immunology , Amino Acid Sequence , CD8-Positive T-Lymphocytes/immunology , COVID-19/genetics , COVID-19/virology , Conserved Sequence , Coronavirus/chemistry , Coronavirus/classification , Coronavirus/genetics , Coronavirus/immunology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Cross Reactions , Epitope Mapping , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/genetics , Humans , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
19.
ACS Pharmacol Transl Sci ; 5(1): 8-19, 2022 Jan 14.
Article in English | MEDLINE | ID: covidwho-1621207

ABSTRACT

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have been put forth, only a few compounds are in late-stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here, we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies conjugated to donor and acceptor fluorophores that produce a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for remdesivir of 9.3 µM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, ß), Brazilian/Japanese P.1 (Gamma, γ), and Californian (Epsilon, ε) variants of concern (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

20.
SLAS Discov ; 27(2): 86-94, 2022 03.
Article in English | MEDLINE | ID: covidwho-1586501

ABSTRACT

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.


Subject(s)
Antiviral Agents/pharmacology , High-Throughput Screening Assays/methods , SARS-CoV-2/drug effects , Virus Internalization/drug effects , HEK293 Cells , Humans
SELECTION OF CITATIONS
SEARCH DETAIL