Betulonic acid derivatives inhibiting coronavirus replication in cell culture via the nsp15 endoribonuclease

ABSTRACTThe lack of medication to suppress coronavirus infections is a main reason for the dramatic course of the COVID-19 pandemic. There is an urgent need to identify suitable coronavirus drug targets and corresponding lead molecules. Here we describe the discovery of a class of coronavirus inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. SAR exploration of these 1,2,3-triazolo fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 μM) of human coronavirus 229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

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Human Coronavirus 229E Papain-Like Proteases Have Overlapping Specificities but Distinct Functions in Viral Replication

Journal of Virology ◽

10.1128/jvi.02091-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 3922-3932 ◽

Cited By ~ 36

Author(s):

John Ziebuhr ◽

Barbara Schelle ◽

Nadja Karl ◽

Ekaterina Minskaia ◽

Sonja Bayer ◽

...

Keyword(s):

Cell Culture ◽

Viral Replication ◽

Active Site ◽

Reverse Genetics ◽

Cysteine Proteases ◽

Proteolytic Processing ◽

Human Coronavirus ◽

Mutant Genotype ◽

Amino Terminal ◽

Human Coronavirus 229E

ABSTRACT Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PLpros). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PLpro activities. Reverse-genetics data revealed that replacement of the PL2pro active-site cysteine was lethal. By contrast, the PL1pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1pro activity. Further experiments showed that both PL1pro and PL2pro were able to cleave the nsp1-nsp2 cleavage site, with PL2pro cleaving the site less efficiently. The PL1pro-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PLpro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2pro plays a universal and essential proteolytic role that appears to be assisted by the PL1pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.

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Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease

Journal of Medicinal Chemistry ◽

10.1021/acs.jmedchem.0c02124 ◽

2021 ◽

Author(s):

Annelies Stevaert ◽

Besir Krasniqi ◽

Benjamin Van Loy ◽

Tien Nguyen ◽

Joice Thomas ◽

...

Keyword(s):

Betulonic Acid ◽

Human Coronavirus ◽

Human Coronavirus 229E

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Lessons Learned From Serial Isolations Of Human Coronavirus 229E From Environmental Surfaces Of A Classroom During Influenza Season

10.31988/scitrends.16688 ◽

2018 ◽

Author(s):

John Lednicky

Keyword(s):

Influenza Season ◽

Lessons Learned ◽

Human Coronavirus ◽

Environmental Surfaces ◽

Human Coronavirus 229E

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Faculty Opinions recommendation of Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1024182.290792 ◽

2005 ◽

Author(s):

Arieh Warshel

Keyword(s):

Active Site ◽

Substrate Binding ◽

Computational Prediction ◽

Binding Mode

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Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽

10.3390/cancers13071679 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1679

Author(s):

Vishnu Mohan ◽

Jean P. Gaffney ◽

Inna Solomonov ◽

Maxim Levin ◽

Mordehay Klepfish ◽

...

Keyword(s):

Monoclonal Antibody ◽

Active Site ◽

Drug Targets ◽

Fas Ligand ◽

Specific Activity ◽

Matrix Metalloproteases ◽

Ductal Adenocarcinoma ◽

Enzyme Active Site ◽

Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

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A protocol for adding knowledge to Wikidata: aligning resources on human coronaviruses

BMC Biology ◽

10.1186/s12915-020-00940-y ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Andra Waagmeester ◽

Egon L. Willighagen ◽

Andrew I. Su ◽

Martina Kutmon ◽

Jose Emilio Labra Gayo ◽

...

Keyword(s):

Common Ground ◽

Knowledge Bases ◽

Public Knowledge ◽

Medical Problems ◽

Human Coronavirus ◽

Data Schema ◽

Helping Others ◽

Human Coronaviruses ◽

The Right ◽

Human Coronavirus 229E

Abstract Background Pandemics, even more than other medical problems, require swift integration of knowledge. When caused by a new virus, understanding the underlying biology may help finding solutions. In a setting where there are a large number of loosely related projects and initiatives, we need common ground, also known as a “commons.” Wikidata, a public knowledge graph aligned with Wikipedia, is such a commons and uses unique identifiers to link knowledge in other knowledge bases. However, Wikidata may not always have the right schema for the urgent questions. In this paper, we address this problem by showing how a data schema required for the integration can be modeled with entity schemas represented by Shape Expressions. Results As a telling example, we describe the process of aligning resources on the genomes and proteomes of the SARS-CoV-2 virus and related viruses as well as how Shape Expressions can be defined for Wikidata to model the knowledge, helping others studying the SARS-CoV-2 pandemic. How this model can be used to make data between various resources interoperable is demonstrated by integrating data from NCBI (National Center for Biotechnology Information) Taxonomy, NCBI Genes, UniProt, and WikiPathways. Based on that model, a set of automated applications or bots were written for regular updates of these sources in Wikidata and added to a platform for automatically running these updates. Conclusions Although this workflow is developed and applied in the context of the COVID-19 pandemic, to demonstrate its broader applicability it was also applied to other human coronaviruses (MERS, SARS, human coronavirus NL63, human coronavirus 229E, human coronavirus HKU1, human coronavirus OC4).

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Examining the persistence of human Coronavirus 229E on fresh produce

Food Microbiology ◽

10.1016/j.fm.2021.103780 ◽

2021 ◽

pp. 103780

Author(s):

Madeleine Blondin-Brosseau ◽

Jennifer Harlow ◽

Tanushka Doctor ◽

Neda Nasheri

Keyword(s):

Fresh Produce ◽

Human Coronavirus ◽

Human Coronavirus 229E

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Effect of pH and temperature on the infectivity of human coronavirus 229E

Canadian Journal of Microbiology ◽

10.1139/m89-160 ◽

1989 ◽

Vol 35 (10) ◽

pp. 972-974 ◽

Cited By ~ 40

Author(s):

Alain Lamarre ◽

Pierre J. Talbot

Keyword(s):

Incubation Period ◽

Storage Conditions ◽

Viral Infectivity ◽

Human Coronavirus ◽

Virus Growth ◽

The Stability ◽

And Storage ◽

Influence Of Ph ◽

Human Coronavirus 229E

The stability of human coronavirus 229E infectivity was maximum at pH 6.0 when incubated at either 4 or 33 °C. However, the influence of pH was more pronounced at 33 °C. Viral infectivity was completely lost after a 14-day incubation period at 22, 33, or 37 °C but remained relatively constant at 4 °C for the same length of time. Finally, the infectious titer did not show any significant reduction when subjected to 25 cycles of thawing and freezing. These studies will contribute to optimize virus growth and storage conditions, which will facilitate the molecular characterization of this important pathogen.Key words: coronavirus, pH, temperature, infectivity, human coronavirus.

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Recent insights into lytic polysaccharide monooxygenases (LPMOs)

Biochemical Society Transactions ◽

10.1042/bst20170549 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1431-1447 ◽

Cited By ~ 39

Author(s):

Tobias Tandrup ◽

Kristian E. H. Frandsen ◽

Katja S. Johansen ◽

Jean-Guy Berrin ◽

Leila Lo Leggio

Keyword(s):

Active Site ◽

Room Temperature ◽

Experimental Studies ◽

Binding Mode ◽

Structural Features ◽

Oxygen Species ◽

Animal Kingdom ◽

X Ray ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

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