scholarly journals Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bjoern Meyer ◽  
Jeanne Chiaravalli ◽  
Stacy Gellenoncourt ◽  
Philip Brownridge ◽  
Dominic P. Bryne ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Viral Proteins ◽  
Cellular Proteins ◽  
Cleavage Sites ◽  
Antibody Testing ◽  
High Confidence ◽  
Cellular Targets ◽  
Main Protease ◽  
Basic Biology ◽  
Dose Dependent

AbstractSARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.

scholarly journals Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets for drug repurposing

2020 ◽  
Author(s):  
Bjoern Meyer ◽  
Jeanne Chiaravalli ◽  
Philip Brownridge ◽  
Dominic P. Bryne ◽  
Leonard A. Daly ◽  
...  
Keyword(s):  
Protease Activity ◽  
Drug Repurposing ◽  
Cellular Proteins ◽  
Cleavage Sites ◽  
Antibody Testing ◽  
Cellular Targets ◽  
Main Protease ◽  
Antigenic Proteins ◽  
Culture Models ◽  
Basic Biology

AbstractSARS-CoV-2 is the causative agent behind the COVID-19 pandemic, and responsible for tens of millions of infections, and hundreds of thousands of deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify multiple previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts. We discovered significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We showed that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19 disease.

scholarly journals In Silico study of Wheatgrass constituents against Coronavirus COVID-19 Proteins.

2021 ◽  
Author(s):  
Deshmukh Srus ◽  
Badhe Pravin ◽  
Otari Kishor ◽  
Badhe Ashwini
Keyword(s):  
Virtual Screening ◽  
Screening Tool ◽  
Therapeutic Potential ◽  
Chemical Constituents ◽  
Viral Proteins ◽  
Chemical Components ◽  
Vaccine Production ◽  
Main Protease ◽  
In Silico Study

Abstract As a result of COVID-19, the coronavirus associated with SARS-CoV-2 has emerged as the most lethal and infectious pandemic in history. Vaccines alone cannot assure us of a healthy future. As a result, drug production must go hand in hand with vaccine production. The purpose of this study is to evaluate the therapeutic potential of certain chemical constituents of Wheatgrass (Triticum aestivum Linn.,) that may be useful for treatment of COVID19. Seven chemical constituents of Wheatgrass, including Ascobic acid (SWA00A), Rutin (SWA00B), Ferulic acid (SWA00C), quercetin (SWA00D), Luteolin (SWA00E), Apigenin (SWA00F), and Kaempferol (SWA00G), were used for virtual screening. Covid19 viral proteins such as 6lu7-SARS-CoV2 main protease, 6zsl-SARS-CoV-2 helicase, 6w9c-papain-like protease of SARS-CoV-2, and 6m71-RNA-dependent RNA polymerase were selected for study. Drugs used in the treatment of COVID-19 namely Remdesivir, Darunavir, Ralimetinib, Berzosertib, Alpha-interferon, Arabinol, Chloroquine phosphate, Indinavir, Lopinavir, Ritonavir, Plegylated alfa interferon, 2-chloro-2-deoxy-D-glucose are taken as standards. Molecular docking was performed using the PyRx Virtual Screening tool. Among all 7 chemical components, Rutin (SWA00B) had the strongest binding affinity. According to the present study, Rutin present in Wheatgrass shows the highest potential to inhibit SARS-CoV-2 proteins. Wheatgrass has promising anti-SARS-CoV-2 properties, but further research is needed to prove their efficacy in vivo.

scholarly journals Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

2020 ◽  
Vol 21 (24) ◽  
pp. 9523
Author(s):  
Márió Miczi ◽  
Mária Golda ◽  
Balázs Kunkli ◽  
Tibor Nagy ◽  
József Tőzsér ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Severe Pneumonia ◽  
Cellular Protein ◽  
Target Sequence ◽  
Cleavage Sites ◽  
Cellular Targets ◽  
Main Protease ◽  
Human Proteins ◽  
Successful Replication

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.

Anticoagulant Activity of Hirulog™, a Direct Thrombin Inhibitor, in Humans

1993 ◽  
Vol 69 (02) ◽  
pp. 157-163 ◽  
Author(s):  
Irving Fox ◽  
Adrian Dawson ◽  
Peter Loynds ◽  
Jane Eisner ◽  
Kathleen Findlen ◽  
...  
Keyword(s):  
Rational Design ◽  
Therapeutic Potential ◽  
Anticoagulant Activity ◽  
Subcutaneous Administration ◽  
Direct Thrombin Inhibitor ◽  
Controlled Study ◽  
Thrombin Inhibitor ◽  
Thrombin Time ◽  
Thrombotic Disease ◽  
Dose Dependent

SummaryHirulog™ (BG8967) is a direct thrombin inhibitor built by rational design using the protein hirudin as a model (Maraganore et al. [1990]; Biochemistry 29: 7095–101). In order to evaluate the therapeutic potential for hirulog in the management of thrombotic disease, the tolerability and anticoagulant activity of the agent were examined in a study of human volunteers.In a randomized, placebo-controlled study (n = 54), the intravenous infusion of hirulog over 15 min showed a rapid, dose-dependent prolongation of activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). There was a corresponding dose-dependent increase in plasma hirulog levels. The peptide was rapidly cleared with a half-life of 36 min and a total body clearance rate for the peptide of 0.43 1 kg−1 h−1. Similar activity was observed following subcutaneous injection but with sustained pharmacodynamic and pharmacokinetic behavior. There was a significant correlation between pharmacokinetic and pharmacodynamic variables for both intravenous (r = 0.8, p <0.001) and subcutaneous administration (r = 0.7, p = 0.002).To evaluate the possible interactions of aspirin on the tolerability and anticoagulant activity of intravenous hirulog, a cross-over design was employed in eight subjects. Aspirin administration did not modify the peptide’s activity. At the administered dose of 0.6 mg kg−1 h−1 for 2 h, hirulog infusion prolonged APTT from 230 to 260% baseline. The infusion of hirulog in subjects who had received aspirin was not associated with any significant changes in the template bleeding time.The final phase of the study examined the activity and tolerability of hirulog in ten subjects during prolonged intravenous infusions for up to 24 h. The peptide (0.3 mg kg−1 h−1) exhibited sustained anticoagulant activity with no evidence for a cumulative effect. During hirulog infusion, APTT was prolonged from 210 to 250% baseline.In all phases of the study, hirulog administration was generally well-tolerated.Our observations show that hirulog is an active antithrombin agent with excellent tolerability in humans. As a direct thrombin inhibitor, hirulog provides a novel approach for the management of thrombotic disease.

β-1, 3-Glucan attenuated chronic unpredictable mild stress induced cognitive impairment in rodents via normalizing corticosterone levels

2020 ◽  
Vol 20 ◽  
Author(s):  
Saniya Hashim Khan ◽  
Sheraz Khan ◽  
Inamullah Khan ◽  
Narmeen Hashim
Keyword(s):  
Water Maze ◽  
Memory Impairment ◽  
Glucocorticoid Receptors ◽  
Therapeutic Potential ◽  
Mild Stress ◽  
Chronic Unpredictable Mild Stress ◽  
Neuroprotective Effects ◽  
Injurious Effect ◽  
Naturally Occurring ◽  
Dose Dependent

Background: Chronic stress elevates the cortisol beyond normal levels, which affects cognition including learning & memory. This injurious effect is primarily mediated via over excitation of metabotropic glucocorticoid receptors (mGR). Methods: The present study was aimed appraise the neuroprotective effects of naturally occurring molecule β-1,3-glucan by interfering with stress-cortisol-mGR axis. Our data of virtual screening (in silico) exhibited the promising interactions of βglucan with the mGR. Therefore, the study was extended to evaluate its efficacy (2.5, 5 and 10 mg/kg/ i.p) in an animal model of chronic unpredictable mild stress (CUMS, 28 days) induced memory impairment. Results: Results of the current study revealed the β-glucan provided dose dependent protection against deleterious effects of stress on learning and memory associated parameters observed in Morris water maze (MWM) task. At higher tested doses, it has also significantly antagonized the stress induced weight loss and corticosterone elevation. Conclusion: From these findings, it can be deduced that the β-glucan possesses therapeutic potential against stress induced memory impairment, and this effect can be attributed to its normalizing effect on corticosterone levels.

Abstract 619: Alpha Keto Acids Decompose Peroxides and Prevents Oxidation of Lipoproteins

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Chandrakala Aluganti Narasimhulu ◽  
Kathryn Young Burge ◽  
Yu Yuan ◽  
Sampath Parthasarathy
Keyword(s):  
Therapeutic Potential ◽  
Lipid Peroxides ◽  
Enzymatic Oxidation ◽  
Raw 264.7 Cells ◽  
Dependent Manner ◽  
Gene Expressions ◽  
Dual Effect ◽  
Keto Acids ◽  
Hydroperoxyoctadecadienoic Acid ◽  
Dose Dependent

Background: Alpha keto acids are unstable and decompose rapidly. In this study, we tested the ability of alpha keto acids to reduce peroxides and inhibit oxidation of lipoproteins. Methods: Keto salicylic acid (KSA) and Keto Octanoicacid (KoA) were synthesized and their ability to reduce hydrogen peroxides as well as lipid peroxides (LOOH) was measured using 13-hydroperoxyoctadecadienoic acid (13-HPODE). Lipoproteins (LDL and HDL) were isolated from human plasma and oxidation of liporproteins was performed using copper and MPO in the presence or absence of the keto compounds. RAW 264.7 cells and HUVECS were incubated with LPS and mm-LDL respectively either in the presence or absence of the keto compounds. RNA was isolated from treated cells and real time PCR was performed to analyze IL-1α, IL-6, MCP-1 and VCAM1 gene expressions. Reactive oxygen species were evaluated using DCF fluorescence in presence and absence of the keto compounds. Results: KSA reduced both H2O2 and 13-HPODE whereas KoA is able to reduce the former but not the latter. Both compounds inhibited the lipoprotein oxidation in a dose dependent manner and were able to reduce ROS production by H2O2. KSA is able to inhibit both LPS as well as mm-LDL induced inflammation. However, KoA showed a dual effect as it induced inflammatory markers in the presence of LPS, but inhibited the mm-LDL-induced inflammatory gene expressions. Conclusion: The results of our studies suggest that these keto compounds a) inhibit both enzymatic and non enzymatic oxidation of lipoproteins; b) reduce peroxides and ROS and c) have inhibitory and inducing effect on inflammatory cytokine/gene production in presence of mm-LDL and LPS respectively. Based on these results, we predict that these keto compounds could have therapeutic potential in reducing CVD/atherosclerosis-associated inflammation.

Natural products targeting cancer stem cells: a revisit

2021 ◽  
Vol 28 ◽  
Author(s):  
Jiahua Cui ◽  
Jiajun Qian ◽  
Larry Ming-Cheung Chow ◽  
Jinping Jia
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Natural Products ◽  
Cancer Stem Cells ◽  
Therapeutic Potential ◽  
Tumor Development ◽  
Biologically Active ◽  
Cellular Targets ◽  
Drug Candidates ◽  
Enhanced Expression

Background: The proposed central role of cancer stem cells (CSCs) in tumor development has been extended to explain the diverse oncologic phenomena such as multidrug resistance, metastasis and tumor recurrence in clinics. Due to the enhanced expression of ATP-binding cassette transporters and anti-apoptotic factors, stagnation on G0 phase and the strong ability of self-renewal, the CSCs were highly resistant to clinical anticancer drugs. Therefore, the discovery of new drug candidates that could effectively eradicate cancer stem cells afforded promising outcomes in cancer therapy. Introduction: Natural products and their synthetic analogues are a rich source of biologically active compounds and several of them have already been recognized as potent CSCs killers. We aim to provide a collection of recently identified natural products that suppressed the survival of the small invasive CSC populations and combated the drug resistance of these cells in chemotherapy. Results and Conclusion: These anti-CSCs natural products included flavonoids, stilbenes, quinones, terpenoids, polyketide antibiotics, steroids and alkaloids. In the present review, we highlighted the therapeutic potential of natural products and their derivatives against the proliferation and drug resistance of CSCs, their working mechanisms and related structure-activity relationships. Meanwhile, in this survey, several natural products with diverse cellular targets such as the naphthoquinone shikonin and the stilbene resveratrol were characterized as promising lead compounds for future development.

scholarly journals Screening of Chloroquine, Hydroxychloroquine and Its Derivatives for Their Binding Affinity to Multiple SARS-CoV-2 Protein Drug Targets

2020 ◽  
Author(s):  
Mallikarjuna Nimgampalle ◽  
Vasudharani Devanthan ◽  
Ambrish Saxena
Keyword(s):  
Binding Affinity ◽  
In Silico ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Viral Proteins ◽  
Protein Drug ◽  
Potential Treatment ◽  
Health Authorities ◽  
Derivatives Of

Recently Chloroquine and its derivative Hydroxychloroquine have garnered enormous interest amongst the clinicians and health authorities’ world over as a potential treatment to contain COVID-19 pandemic. The present research aims at investigating the therapeutic potential of Chloroquine and its potent derivative Hydroxychloroquine against SARS-CoV-2 viral proteins. At the same time we have screened some chemically synthesized derivatives of Chloroquine and compared their binding efficacy with chemically synthesized Chloroquine derivatives through <i>in silico</i>approaches. For the purpose of the study, we have selected some essential viral proteins and enzymes implicated in SARS-CoV-2 replication and multiplication as putative drug targets.<br>

scholarly journals Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen

2020 ◽  
Author(s):  
Maria Kuzikov ◽  
Elisa Costanzi ◽  
Jeanette Reinshagen ◽  
Francesca Esposito ◽  
Laura Vangeel ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Small Molecules ◽  
Drug Target ◽  
Treatment Options ◽  
Cleavage Sites ◽  
X Ray ◽  
Binding Characteristics ◽  
Main Protease ◽  
Ic50 Values

Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 uM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Angs., showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.

scholarly journals The Fc-mediated effector functions of a potent SARS-CoV-2 neutralizing antibody, SC31, isolated from an early convalescent COVID-19 patient, are essential for the optimal therapeutic efficacy of the antibody

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253487
Author(s):  
Conrad E. Z. Chan ◽  
Shirley G. K. Seah ◽  
De Hoe Chye ◽  
Shane Massey ◽  
Maricela Torres ◽  
...  
Keyword(s):  
Therapeutic Efficacy ◽  
Neutralizing Antibodies ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Rhesus Macaques ◽  
Neutralizing Antibody ◽  
Effector Functions ◽  
Viral Loads ◽  
Therapeutic Doses ◽  
Dose Dependent

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.

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