scholarly journals Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir

2021 ◽  
Vol 102 (7) ◽  
Author(s):  
Marie O. Pohl ◽  
Idoia Busnadiego ◽  
Francesco Marrafino ◽  
Lars Wiedmer ◽  
Annika Hunziker ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Therapeutic Option ◽  
Cell Entry ◽  
Protease Inhibition ◽  
Antiviral Compounds ◽  
Main Protease ◽  
Anti Cancer ◽  
Synergistic Mechanism ◽  
Host Cell Entry

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

scholarly journals Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir

2021 ◽  
Author(s):  
Marie O. Pohl ◽  
Idoia Busnadiego ◽  
Francesco Marrafino ◽  
Lars Wiedmer ◽  
Annika Hunziker ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Therapeutic Option ◽  
Protease Inhibition ◽  
Antiviral Compounds ◽  
Main Protease ◽  
Ic50 Value ◽  
Anti Cancer ◽  
Synergistic Mechanism ◽  
Host Cell Entry

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we screened by high-throughput docking 6,000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3CL main protease inhibitor in vitro (IC50 value of 29 μM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication in Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our study shows that combining computational and cellular screening is an efficient means to identify existing drugs with repurposing potential as antiviral compounds. Future studies should aim at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

scholarly journals A structure-based rationale for sialic acid independent host-cell entry of Sosuga virus

2019 ◽  
Vol 116 (43) ◽  
pp. 21514-21520 ◽  
Author(s):  
Alice J. Stelfox ◽  
Thomas A. Bowden
Keyword(s):  
Sialic Acid ◽  
Host Cell ◽  
Receptor Binding ◽  
Cell Attachment ◽  
Cell Entry ◽  
Head Region ◽  
Close Proximity ◽  
Host Cell Attachment ◽  
Host Cell Entry

The bat-borne paramyxovirus, Sosuga virus (SosV), is one of many paramyxoviruses recently identified and classified within the newly established genus Pararubulavirus, family Paramyxoviridae. The envelope surface of SosV presents a receptor-binding protein (RBP), SosV-RBP, which facilitates host-cell attachment and entry. Unlike closely related hemagglutinin neuraminidase RBPs from other genera of the Paramyxoviridae, SosV-RBP and other pararubulavirus RBPs lack many of the stringently conserved residues required for sialic acid recognition and hydrolysis. We determined the crystal structure of the globular head region of SosV-RBP, revealing that while the glycoprotein presents a classical paramyxoviral six-bladed β-propeller fold and structurally classifies in close proximity to paramyxoviral RBPs with hemagglutinin-neuraminidase (HN) functionality, it presents a receptor-binding face incongruent with sialic acid recognition. Hemadsorption and neuraminidase activity analysis confirms the limited capacity of SosV-RBP to interact with sialic acid in vitro and indicates that SosV-RBP undergoes a nonclassical route of host-cell entry. The close overall structural conservation of SosV-RBP with other classical HN RBPs supports a model by which pararubulaviruses only recently diverged from sialic acid binding functionality.

Novel Drugs Targeting the SARS-CoV-2/COVID-19 Machinery

2020 ◽  
Vol 20 (16) ◽  
pp. 1423-1433 ◽  
Author(s):  
Ariane Sternberg ◽  
Dwight L. McKee ◽  
Cord Naujokat
Keyword(s):  
Host Cell ◽  
Protein Processing ◽  
Cell Entry ◽  
Genome Replication ◽  
Rna Dependent Rna Polymerase ◽  
Main Protease ◽  
Novel Drugs ◽  
Pathogenic Viruses ◽  
Repurposed Drugs ◽  
Host Cell Entry

Like other human pathogenic viruses, coronavirus SARS-CoV-2 employs sophisticated macromolecular machines for viral host cell entry, genome replication and protein processing. Such machinery encompasses SARS-CoV-2 envelope spike (S) glycoprotein required for host cell entry by binding to the ACE2 receptor, viral RNA-dependent RNA polymerase (RdRp) and 3-chymotrypsin-like main protease (3Clpro/Mpro). Under the pressure of the accelerating COVID-19 pandemic caused by the outbreak of SARS-CoV-2 in Wuhan, China in December 2019, novel and repurposed drugs were recently designed and identified for targeting the SARS-CoV-2 reproduction machinery, with the aim to limit the spread of SARS-CoV-2 and morbidity and mortality due to the COVID-19 pandemic.

scholarly journals Kunitz type protease inhibitor EgKI-1 from the canine tapeworm Echinococcus granulosus as a promising anti-cancer therapeutic

2018 ◽  
Author(s):  
Shiwanthi L Ranasinghe ◽  
Glen M Boyle ◽  
Katja Fischer ◽  
Jeremy Potriquet ◽  
Jason P Mulvenna ◽  
...  
Keyword(s):  
Cell Growth ◽  
Protease Inhibitor ◽  
Hydatid Cyst ◽  
Echinococcus Granulosus ◽  
Cancer Cell ◽  
Dependent Manner ◽  
Anti Cancer ◽  
Kunitz Type ◽  
Kunitz Type Protease Inhibitor

AbstractEgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.

scholarly journals Cysteine biosynthesis is a determinant of Brucella ovis stress survival and fitness in the intracellular niche

2020 ◽  
Author(s):  
Lydia M. Varesio ◽  
Aretha Fiebig ◽  
Sean Crosson
Keyword(s):  
Stationary Phase ◽  
Cell Entry ◽  
Host Cells ◽  
Cysteine Biosynthesis ◽  
Brucella Ovis ◽  
Cysteine Synthesis ◽  
Host Cell Entry ◽  
Phase Entry ◽  
Male Genital

AbstractBrucella ovis is an ovine intracellular pathogen with tropism for the male genital tract. To establish and maintain infection, B. ovis must survive stressful conditions inside host cells, including low pH, nutrient limitation, and reactive oxygen species. These same conditions are often encountered in stationary phase cultures. Studies of stationary phase may thus inform understanding of Brucella infection biology, yet the genes that are important in Brucella stationary phase physiology remain poorly defined. We measured fitness of a barcoded pool of B. ovis Tn-himar mutants as a function of growth phase and identified cysE as a determinant of fitness in stationary phase. CysE catalyzes the first step in cysteine biosynthesis from serine. We provide genetic evidence that two related enzymes, CysK1 and CysK2, function redundantly to catalyze cysteine synthesis downstream of CysE. Deleting either cysE or both cysK1 and cysK2 leads to premature entry into stationary phase and reduced culture yield. These phenotypes are rescued by addition of cysteine or glutathione to the medium. We further show that deletion of cysE results in sensitivity to exogenous hydrogen peroxide. Finally, we demonstrate that B. ovis ΔcysE has no defect in host cell entry but is attenuated in macrophage-like cells and in ovine testis epithelial cells at one- and two-days post infection. Our study uncovered unexpected redundancy at the CysK step of cysteine biosynthesis in B. ovis, and demonstrated that cysteine anabolism is an important determinant of stationary phase entry in vitro and fitness in the intracellular niche.

scholarly journals Favipiravir May Acts as COVID-19 Main Protease PDB ID 6LU7 Inhibitor: Docking Analysis

2020 ◽  
Vol 10 (6) ◽  
pp. 6821-6828 ◽  
Keyword(s):  
Amino Acids ◽  
Protease Inhibitor ◽  
Essential Amino Acids ◽  
Antiviral Drugs ◽  
Binding Pattern ◽  
Protease Inhibition ◽  
Docking Analysis ◽  
Protease Enzyme ◽  
Main Protease ◽  
Polymerase Inhibitor

Coronavirus is a well-known threat to the human being in the form of COVID-19. Virus replication may be controlled by inhibition of protease enzyme. Hence, well known 13 antiviral drugs have been observed by docking analysis for understanding the binding pattern of drugs with COVID-19 main protease PDB ID: 6LU7 for any possibilities of protease inhibition. For docking analysis PyRx- Python Prescription 0.8 was used. This analysis reveals that the essential amino acids involved in binding of antiviral drugs to COVID-19 main protease PDB ID: 6LU7 are Glycine (Gly), Serine (Ser), Cysteine (Cys), Leucine (Leu), Asparagine (Asn), Glutamine (Gln), Glutamic acid (Glu) and Threonine (Thr). After docking analysis, it was observed that Favipiravir maybe act as COVID-19 main protease inhibitor despite being vRNA polymerase inhibitor and may further be used in the treatment of COVID-19 infection.

scholarly journals Suppressors of a Host Range Mutation in the Rabbitpox Virus Serpin SPI-1 Map to Proteins Essential for Viral DNA Replication

2005 ◽  
Vol 79 (14) ◽  
pp. 9168-9179 ◽  
Author(s):  
Benjamin G. Luttge ◽  
Richard W. Moyer
Keyword(s):  
Dna Replication ◽  
Protease Inhibitor ◽  
Host Range ◽  
Suppressor Mutation ◽  
Protease Inhibition ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Range Function ◽  
Rabbitpox Virus

ABSTRACT The orthopoxvirus serpin SPI-1 is an intracellular serine protease inhibitor that is active against cathepsin G in vitro. Rabbitpox virus (RPV) mutants with deletions of the SPI-1 gene grow on monkey kidney cells (CV-1) but do not plaque on normally permissive human lung carcinoma cells (A549). This reduced-host-range (hr) phenotype suggests that SPI-1 may interact with cellular and/or other viral proteins. We devised a genetic screen for suppressors of SPI-1 hr mutations by first introducing a mutation into SPI-1 (T309R) at residue P14 of the serpin reactive center loop. The SPI-1 T309R serpin is inactive as a protease inhibitor in vitro. Introduction of the mutation into RPV leads to the same restricted hr phenotype as deletion of the SPI-1 gene. Second-site suppressors were selected by restoration of growth of the RPV SPI-1 T309R hr mutant on A549 cells. Both intragenic and extragenic suppressors of the T309R mutation were identified. One novel intragenic suppressor mutation, T309C, restored protease inhibition by SPI-1 in vitro. Extragenic suppressor mutations were mapped by a new procedure utilizing overlapping PCR products encompassing the entire genome in conjunction with marker rescue. One suppressor mutation, which also rendered the virus temperature sensitive for growth, mapped to the DNA polymerase gene (E9L). Several other suppressors mapped to gene D5R, an NTPase required for DNA replication. These results unexpectedly suggest that the host range function of SPI-1 may be associated with viral DNA replication by an as yet unknown mechanism.

scholarly journals Structure-Based Virtual Screening: Identification of a Novel NS2B-NS3 Protease Inhibitor with Potent Antiviral Activity against Zika and Dengue Viruses

2021 ◽  
Vol 9 (3) ◽  
pp. 545
Author(s):  
Hye Jin Shin ◽  
Mi-Hwa Kim ◽  
Joo-Youn Lee ◽  
Insu Hwang ◽  
Gun Young Yoon ◽  
...  
Keyword(s):  
Virtual Screening ◽  
Protease Activity ◽  
Therapeutic Option ◽  
Denv Infection ◽  
Antiviral Drugs ◽  
Protease Inhibition ◽  
Docking Analysis ◽  
Ns3 Protease ◽  
Protease Inhibition Assay

Zika virus (ZIKV), which is associated with severe diseases in humans, has spread rapidly and globally since its emergence. ZIKV and dengue virus (DENV) are closely related, and antibody-dependent enhancement (ADE) of infection between cocirculating ZIKV and DENV may exacerbate disease. Despite these serious threats, there are currently no approved antiviral drugs against ZIKV and DENV. The NS2B-NS3 viral protease is an attractive antiviral target because it plays a pivotal role in polyprotein cleavage, which is required for viral replication. Thus, we sought to identify novel inhibitors of the NS2B-NS3 protease. To that aim, we performed structure-based virtual screening using 467,000 structurally diverse chemical compounds. Then, a fluorescence-based protease inhibition assay was used to test whether the selected candidates inhibited ZIKV protease activity. Among the 123 candidate inhibitors selected from virtual screening, compound 1 significantly inhibited ZIKV NS2B-NS3 protease activity in vitro. In addition, compound 1 effectively inhibited ZIKV and DENV infection of human cells. Molecular docking analysis suggested that compound 1 binds to the NS2B-NS3 protease of ZIKV and DENV. Thus, compound 1 could be used as a new therapeutic option for the development of more potent antiviral drugs against both ZIKV and DENV, reducing the risks of ADE.

scholarly journals Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease

2021 ◽  
Vol 478 (13) ◽  
pp. 2499-2515 ◽  
Author(s):  
Jennifer C. Milligan ◽  
Theresa U. Zeisner ◽  
George Papageorgiou ◽  
Dhira Joshi ◽  
Christelle Soudy ◽  
...  
Keyword(s):  
Economic Effects ◽  
Calpain Inhibitor ◽  
Chemical Library ◽  
Antiviral Compounds ◽  
Main Protease ◽  
The World ◽  
Ic50 Values ◽  
Global Population ◽  
Micromolar Range

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

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