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  1. Article ; Online: SARS-CoV-2 3CLpro mutations confer resistance to Paxlovid (nirmatrelvir/ritonavir) in a VSV-based, non-gain-of-function system

    Heilmann, Emmanuel / Costacurta, Francesco / Volland, Andre / von Laer, Dorothee

    bioRxiv

    Abstract: Protease inhibitors are among the most powerful antiviral drugs. A first protease inhibitor against the SARS-CoV-2 protease 3CLpro, Paxlovid (nirmatrelvir / ritonavir), has recently been authorized by the U.S. FDA for emergency use (EUA 105 Pfizer ... ...

    Abstract Protease inhibitors are among the most powerful antiviral drugs. A first protease inhibitor against the SARS-CoV-2 protease 3CLpro, Paxlovid (nirmatrelvir / ritonavir), has recently been authorized by the U.S. FDA for emergency use (EUA 105 Pfizer Paxlovid). To find resistant mutants against the protease-inhibitor-component of Paxlovid, nirmatrelvir, we engineered a chimeric Vesicular Stomatitis Virus (VSV). By replacing an intergenic region, which is essential for separate gene transcription, with 3CLpro, this chimeric VSV became dependent on the protease to process two of its genes. We then applied selective pressure with nirmatrelvir to induce mutations. The effect of those mutants was confirmed by re-introduction in the 3CLpro and testing with a recently developed cellular assay. Furthermore, we found that mutations predicted by our method already exist in SARS-CoV-2 sequence depositions in NCBI and GISAID data bases. These may represent emerging resistant virus variants or a natural heterogeneity in the susceptibility to nirmatrelvir.
    Keywords covid19
    Language English
    Publishing date 2022-07-04
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2022.07.02.495455
    Database COVID19

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  2. Article ; Online: Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants.

    Volland, André / Lohmüller, Michael / Heilmann, Emmanuel / Kimpel, Janine / Herzog, Sebastian / von Laer, Dorothee

    PLoS pathogens

    2021  Volume 17, Issue 10, Page(s) e1009996

    Abstract: Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 ... ...

    Abstract Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).
    MeSH term(s) HEK293 Cells ; Heparan Sulfate Proteoglycans/metabolism ; Humans ; Lymphocytic Choriomeningitis/metabolism ; Lymphocytic Choriomeningitis/transmission ; Lymphocytic choriomeningitis virus/metabolism ; Lymphocytic choriomeningitis virus/pathogenicity ; Receptors, Virus/metabolism
    Chemical Substances Heparan Sulfate Proteoglycans ; Receptors, Virus
    Language English
    Publishing date 2021-10-14
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2205412-1
    ISSN 1553-7374 ; 1553-7366
    ISSN (online) 1553-7374
    ISSN 1553-7366
    DOI 10.1371/journal.ppat.1009996
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: A VSV-based assay quantifies coronavirus Mpro/3CLpro/Nsp5 main protease activity and chemical inhibition.

    Heilmann, Emmanuel / Costacurta, Francesco / Geley, Stephan / Mogadashi, Seyad Arad / Volland, Andre / Rupp, Bernhard / Harris, Reuben Stewart / von Laer, Dorothee

    Communications biology

    2022  Volume 5, Issue 1, Page(s) 391

    Abstract: Protease inhibitors are among the most powerful antiviral drugs. However, for SARS-CoV-2 only a small number of protease inhibitors have been identified thus far and there is still a great need for assays that efficiently report protease activity and ... ...

    Abstract Protease inhibitors are among the most powerful antiviral drugs. However, for SARS-CoV-2 only a small number of protease inhibitors have been identified thus far and there is still a great need for assays that efficiently report protease activity and inhibition in living cells. Here, we engineer a safe VSV-based system to report both gain- and loss-of-function of coronavirus main protease (M
    MeSH term(s) COVID-19 ; Cysteine Endopeptidases ; Humans ; Indoles ; Lactams ; Leucine ; Nitriles ; Peptide Hydrolases ; Proline ; Protease Inhibitors/pharmacology ; Pyrrolidinones ; SARS-CoV-2 ; Viral Proteins/chemistry
    Chemical Substances Indoles ; Lactams ; Nitriles ; Protease Inhibitors ; Pyrrolidinones ; Viral Proteins ; nirmatrelvir (7R9A5P7H32) ; Proline (9DLQ4CIU6V) ; Peptide Hydrolases (EC 3.4.-) ; Cysteine Endopeptidases (EC 3.4.22.-) ; Leucine (GMW67QNF9C) ; lufotrelvir (XJ51YOB1SC)
    Language English
    Publishing date 2022-04-27
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 2399-3642
    ISSN (online) 2399-3642
    DOI 10.1038/s42003-022-03277-0
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: A VSV-based assay quantifies coronavirus Mpro/3CLpro/Nsp5 main protease activity and chemical inhibition

    Emmanuel Heilmann / Francesco Costacurta / Stephan Geley / Seyad Arad Mogadashi / Andre Volland / Bernhard Rupp / Reuben Stewart Harris / Dorothee von Laer

    Communications Biology, Vol 5, Iss 1, Pp 1-

    2022  Volume 12

    Abstract: A vesicular stomatitis virus (VSV)-based assay enables high-throughput screening for small molecular protease inhibitors that can block viral proteases, like the Mpro/3CLpro/Nsp5 in SARS-CoV-2. ...

    Abstract A vesicular stomatitis virus (VSV)-based assay enables high-throughput screening for small molecular protease inhibitors that can block viral proteases, like the Mpro/3CLpro/Nsp5 in SARS-CoV-2.
    Keywords Biology (General) ; QH301-705.5
    Language English
    Publishing date 2022-04-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants.

    André Volland / Michael Lohmüller / Emmanuel Heilmann / Janine Kimpel / Sebastian Herzog / Dorothee von Laer

    PLoS Pathogens, Vol 17, Iss 10, p e

    2021  Volume 1009996

    Abstract: Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 ... ...

    Abstract Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).
    Keywords Immunologic diseases. Allergy ; RC581-607 ; Biology (General) ; QH301-705.5
    Subject code 570
    Language English
    Publishing date 2021-10-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: SARS-CoV-2 3CL

    Heilmann, Emmanuel / Costacurta, Francesco / Moghadasi, Seyed Arad / Ye, Chengjin / Pavan, Matteo / Bassani, Davide / Volland, Andre / Ascher, Claudia / Weiss, Alexander Kurt Hermann / Bante, David / Harris, Reuben S / Moro, Stefano / Rupp, Bernhard / Martinez-Sobrido, Luis / von Laer, Dorothee

    Science translational medicine

    2023  Volume 15, Issue 678, Page(s) eabq7360

    Abstract: Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease ... ...

    Abstract Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CL
    MeSH term(s) Humans ; SARS-CoV-2/genetics ; COVID-19 ; Mutation/genetics ; Protease Inhibitors/chemistry ; Antiviral Agents/pharmacology ; Antiviral Agents/chemistry
    Chemical Substances GC376 (H1NMJ5XDG5) ; ensitrelvir (PX665RAA3H) ; Protease Inhibitors ; Antiviral Agents
    Language English
    Publishing date 2023-01-11
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2518854-9
    ISSN 1946-6242 ; 1946-6234
    ISSN (online) 1946-6242
    ISSN 1946-6234
    DOI 10.1126/scitranslmed.abq7360
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

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    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)

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  7. Article ; Online: Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors.

    Moghadasi, Seyed Arad / Heilmann, Emmanuel / Khalil, Ahmed Magdy / Nnabuife, Christina / Kearns, Fiona L / Ye, Chengjin / Moraes, Sofia N / Costacurta, Francesco / Esler, Morgan A / Aihara, Hideki / von Laer, Dorothee / Martinez-Sobrido, Luis / Palzkill, Timothy / Amaro, Rommie E / Harris, Reuben S

    Science advances

    2023  Volume 9, Issue 13, Page(s) eade8778

    Abstract: Vaccines and drugs have helped reduce disease severity and blunt the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential ... ...

    Abstract Vaccines and drugs have helped reduce disease severity and blunt the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease [M
    MeSH term(s) Humans ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; COVID-19 ; Protease Inhibitors/chemistry ; Phylogeny ; Peptide Hydrolases
    Chemical Substances nirmatrelvir and ritonavir drug combination ; Protease Inhibitors ; ensitrelvir (PX665RAA3H) ; Peptide Hydrolases (EC 3.4.-)
    Language English
    Publishing date 2023-03-29
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2810933-8
    ISSN 2375-2548 ; 2375-2548
    ISSN (online) 2375-2548
    ISSN 2375-2548
    DOI 10.1126/sciadv.ade8778
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article: A comprehensive study of SARS-CoV-2 main protease (M

    Costacurta, Francesco / Dodaro, Andrea / Bante, David / Schöppe, Helge / Sprenger, Bernhard / Moghadasi, Seyed Arad / Fleischmann, Jakob / Pavan, Matteo / Bassani, Davide / Menin, Silvia / Rauch, Stefanie / Krismer, Laura / Sauerwein, Anna / Heberle, Anne / Rabensteiner, Toni / Ho, Joses / Harris, Reuben S / Stefan, Eduard / Schneider, Rainer /
    Kaserer, Teresa / Moro, Stefano / von Laer, Dorothee / Heilmann, Emmanuel

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease ( ... ...

    Abstract Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CL
    Language English
    Publishing date 2023-10-04
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.09.22.558628
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors

    Moghadasi, Seyed Arad / Heilmann, Emmanuel / Moraes, Sofia N / Kearns, Fiona L. / von Laer, Dorothee / Amaro, Rommie E / Harris, Reuben

    bioRxiv

    Abstract: First-generation vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission and evolution and increasing selective pressures have the potential to yield viral variants capable of ... ...

    Abstract First-generation vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission and evolution and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease (Mpro/3CLpro) of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional inhibitor in clinical development, ensitrelvir, shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that nirmatrelvir-resisting variants have pre-existed the introduction of this drug into the human population and are capable of spreading. A similarly strong argument can be made for ensitrelvir. These results caution against broad administration of protease inhibitors as stand-alone therapies and encourage the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles.
    Keywords covid19
    Language English
    Publishing date 2022-08-08
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2022.08.07.503099
    Database COVID19

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    Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

  10. Article: Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors.

    Moghadasi, Seyed Arad / Heilmann, Emmanuel / Khalil, Ahmed Magdy / Nnabuife, Christina / Kearns, Fiona L / Ye, Chengjin / Moraes, Sofia N / Costacurta, Francesco / Esler, Morgan A / Aihara, Hideki / von Laer, Dorothee / Martinez-Sobrido, Luis / Palzkill, Timothy / Amaro, Rommie E / Harris, Reuben S

    bioRxiv : the preprint server for biology

    2022  

    Abstract: Vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these ...

    Abstract Vaccines and drugs have helped reduce disease severity and blunt the spread of SARS-CoV-2. However, ongoing virus transmission, continuous evolution, and increasing selective pressures have the potential to yield viral variants capable of resisting these interventions. Here, we investigate the susceptibility of natural variants of the main protease (Mpro/3CLpro) of SARS-CoV-2 to protease inhibitors. Multiple single amino acid changes in Mpro confer resistance to nirmatrelvir (the active component of Paxlovid). An additional clinical-stage inhibitor, ensitrelvir (Xocova), shows a different resistance mutation profile. Importantly, phylogenetic analyses indicate that several of these resistant variants have pre-existed the introduction of these drugs into the human population and are capable of spreading. These results encourage the monitoring of resistance variants and the development of additional protease inhibitors and other antiviral drugs with different mechanisms of action and resistance profiles for combinatorial therapy.
    Language English
    Publishing date 2022-12-26
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2022.08.07.503099
    Database MEDical Literature Analysis and Retrieval System OnLINE

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