Aprotinin Inhibits SARS-CoV-2 Replication

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air–liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease

Proteome dynamics under proteotoxic stress and disease

Die Funktion aller Zellen wird durch ein komplexes Netzwerk von Proteinen aufrechterhalten, welches nach Bedarf neue Proteine synthetisiert und beschädigte, beziehungsweise nicht benötigte Enzyme/Proteine abbaut. Dieses Netzwerk der sogenannte Proteinhomeostase benötigt Regulation auf unterschiedlichen Ebenen um korrekt zu arbeiten. Störungen dieser Maschinerie sind an der Pathogenese von malignen Neoplasien und neurodegenerativen Erkrankungen beteiligt. Außerdem beeinträchtigen verschiedene humanpathogene Bakterien und Viren dieses Netzwerk der Proteinhomeostase. Um diese Krankheitsbilder besser zu verstehen ist es daher notwendig die Regulation dieses Netzwerks und die Auswirkungen von Pertubationen auf zellulärer Ebene zu beobachten um Rückschlüsse auf die Pathogenese ziehen zu können. Es konnten bereits für einige Krankheiten Therapieansätze entwickelt werden, die das Proteinhomeostase-Netzwerk gezielt modulieren. Trotz dieser Fortschritte ist weiterhin für viele Störfaktoren unklar, wie sie das Proteinnetzwerk der Zelle beeinflussen. Dies ist teilweise dem Umstand geschuldet, dass die resultierenden Effekte innerhalb der Zelle hochdynamisch und daher oft schwer messbar sind. Teil dieser Arbeit war es eine Messmethode zu entwickeln, die es ermöglicht die Auswirkungen von proteotoxischem Stress, d.h. Störungen der Proteinhomeostase, auf die Dynamik von Proteinsynthese und –abbau in der Zelle mit Hilfe von Massenspektrometrie (MS) zu untersuchen. Um diese Messungen zu ermöglichen wurde eine neue Methode entwickelt, multiplexed enhanced protein dynamic (mePROD) MS, welche durch besondere Probenzusammenstellung und Datenanalyse ermöglicht, schnelle und transiente Veränderungen (im Zeitraum von Minuten) in der Proteinbiosynthese von tausenden Proteine zu messen. Zwei der prominentesten Regulatoren der Proteinbiosynthese die Integrated Stress Response (ISR) und mammalian target of rapamycin (mTOR) werden mit vielen verschiedenen Krankheiten in Verbindung gebracht und sind unter anderem Ziele für neue Therapien in der Onkologie. Obwohl beide die Proteinbiosynthese beeinflussen, wurden sie bisher als Signalwege mit unterschiedlichen Effekten auf die Proteinsynthese gesehen. Mittels der neu entwickelten mePROD-Methode konnte im Rahmen dieser Arbeit erstmals der globale Effekt beider Regulationswege auf das Proteinnetzwerk gezeigt werden. Es fanden sich überlappende Muster zwischen mTOR- und ISR-vermittelter Regulation der Proteinsynthese. Dies verändert die bisherige Modellvorstellung für diese zellulären Vorgänge und stellt eine wichtige Ressource für das Forschungsfeld dar. Des Weiteren wurde die entwickelte mePROD Methodik verbessert um den Probendurchsatz zu erhöhen. Durch die Kombination mit logik-basierter Messmethoden, konnte die Anzahl der durch mePROD quantifizierten Proteinen bei gleicher Messzeit verdreifacht werden. Die Dynamik von Proteinsynthese und -abbau wird häufig während Infektionen verändert um die Vermehrung des Krankheitserreger zu ermöglichen oder selbiges zu bekämpfen. Für eine rationale Entwicklung von Therapien gegen diese Infektionen ist es daher unumgänglich die Veränderungen ausgehend von dem Erreger zu erfassen, zu verstehen und die Antwort der Wirtszelle auf die Infektion zu charakterisieren. Ende des Jahres 2019 wurde ein neues Virus entdeckt, SARS-CoV-2, welches sich, hochinfektiös, rasant verbreitet hat und zu einer weltweiten Pandemie geführt hat. Um das neuartige Virus zu verstehen und auf Basis molekularbiologischer Erkenntnisse Therapieansätze zu entwickeln, haben wir die zuvor entwickelte mePROD Methode angewandt um die dynamischen Veränderungen im Wirtsproteom zu erforschen. Es konnte erstmals beobachtet werden, dass nach Infektion mit SARS-CoV-2 verschiedenste Prozesse dereguliert wurden (mRNA Splicing, Glykolyse, DNS Synthese oder Proteinhomeostase. Durch gezielte Inhibierung dieser Prozesse konnte gezeigt werden, dass diese Funktionen der Wirtszelle wichtig für die Replikation von SARS-CoV-2 und durch Medikation modulierbar sind. In einer weiteren Studie konnte zudem durch Analyse von zellulären Signaltransduktionswegen nachgewiesen werden, dass die Aktivierung von Wachstumsfaktor-Signalwegen für die effiziente Replikation der Viren notwendig ist. Die getestete Anwendung von Medikamenten, die bereits zugelassen (Ribavirin, Sorafenib) oder sich in klinischen Studien befinden, blockierte die Virusreplikation vollständig in vitro und zeigt somit mögliche, in die Klinik übertragbare, Therapieansätze.Correct cellular function is ensured by a complex network of proteins and enzymes, regulating protein synthesis and degradation. This protein network, maintaining the so-called protein homeostasis, regulates those processes on multiple levels, producing new or degrading old proteins to cope with changing intra- and extracellular environments. Disturbance of this tightly regulated machinery can have severe effects on the cell and can lead to a variety of pathologies on organism level. Diseases including cancer, neurodegeneration and infections are associated with causative or consequent alterations in protein homeostasis. To understand the pathologies of these diseases, it is therefore critical to examine how perturbations of protein homeostasis affect cellular pathways and physiology. In the recent years, analysis of protein homeostasis networks has resulted in the development of novel therapeutic approaches. However, for many factors it remains unclear how the cell is affected, if they are disturbed. Protein synthesis and degradation represent immediate responses of the cell to changes and need to be studied in the right timeframe, making them difficult to access by common methodology. In this work we developed a new mass spectrometry (MS) based method to study protein synthesis and degradation on a system-wide scale. Multiplexed enhanced protein dynamic (mePROD) MS was developed, overcoming these limitations by special sample mixing and novel data analysis protocols. MePROD thereby enables the measurement of rapid and transient (e.g. minutes) changes in protein synthesis of thousands of proteins. During responses of the cell to stressors (e.g. protein misfolding, oxidation or infection), two major pathways regulate the protein synthesis: the Integrated Stress Response (ISR) and mammalian target of rapamycin (mTOR). Both pathways have been connected with various diseases in the past and are common therapy targets. Although both pathways target protein synthesis in stress responses, the set of targets regulated by these pathways was believed to differ. Through the new mePROD MS method we could measure a comprehensive comparison of both pathways for the first time, revealing comparable system-wide patterns of regulation between the two pathways. This changed the current view on the regulation elicited by these pathways and furthermore represents a useful resource for the whole field of research. We could further develop the mePROD method and decrease MS measurement time needed to obtain an in-depth dataset. Through implementation of logic based instrument methods, it was possible to enhance the number of measured proteins by approximately three-fold within the same measurement time. The dynamics of protein synthesis and degradation are frequently modulated by pathogens infecting the cell to promote pathogen replication. At the same time, the cell counteracts the infection by modulating protein dynamics as well. To develop useful therapy approaches to fight infections, it therefore is necessary to understand the complex changes within the host cell during infections on a system-wide scale. In 2019, a novel coronavirus spread around the world, causing a world-wide health-crisis. To better understand this novel virus and its infection of the host cell we conducted a study applying the mePROD methodology and classical proteomics to characterize the dynamic changes during the infection course in vitro. We discovered that the infection remodeled a diverse set of host cell pathways (e.g. mRNA splicing, glycolysis, DNA synthesis and protein homeostasis) and thereby showed possible targets for antiviral therapy. By targeted inhibition of these pathways, we could observe that these pathways indeed are necessary for SARS-CoV-2 replication and their inhibition could reduce viral load in the cells. Another experimental approach focused on the dynamic changes of protein modification, namely phosphorylation, after infection with SARS-CoV-2. Here, we could show the very important participation of growth factor signaling pathways in viral proliferation. Both studies together revealed critical pathways that are needed for the viral proliferation and hence are promising candidates for further therapies. Subsequent targeting of these pathways by either already approved drugs (Ribavirin and Sorafenib) or drugs in clinical trials (2-deoxyglucose, Pladienolide-B, NMS-873, Pictilisib, Omipalisib, RO5126766 and Lonafarnib) could block viral replication in vitro and suggests important clinical approaches targeting SARS-COV-2 infection

Den molekularen Wirtszellveränderungen durch SARS-CoV-2 auf der Spur

Upon infection with SARS-CoV-2, a variety of changes happen inside the host cell. The virus hijacks host cell pathways for driving its own replication, while the host counteracts with response mechanisms. To gain a comprehensive understanding of COVID-19, caused by SARS-CoV-2 infection, and develop therapeutic strategies, it is crucial to observe these systematic changes in their entirety. In our recent studies, we followed the effects of SARS-CoV-2 infection on the human proteome, which led to the identification of several drugs that abolished viral proliferation in cells

PBLMM: Peptide-based linear mixed models for differential expression analysis of shotgun proteomics data

Here, we present a peptide-based linear mixed models tool—PBLMM, a standalone desktop application for differential expression analysis of proteomics data. We also provide a Python package that allows streamlined data analysis workflows implementing the PBLMM algorithm. PBLMM is easy to use without scripting experience and calculates differential expression by peptide-based linear mixed regression models. We show that peptide-based models outperform classical methods of statistical inference of differentially expressed proteins. In addition, PBLMM exhibits superior statistical power in situations of low effect size and/or low sample size. Taken together our tool provides an easy-to-use, high-statistical-power method to infer differentially expressed proteins from proteomics data

Unbiased translation proteomics upon cell stress

The mammalian target of rapamycin and the integrated stress response are central cellular hubs regulating translation upon stress. The precise proteins and pathway specificity of translation targets of these pathways remained largely unclear. We recently described a new method for quantitative translation proteomics and found that both pathways control translation of the same sets of proteins

DynaTMT: a user-friendly tool to process combined SILAC/TMT data

The measurement of protein dynamics by proteomics to study cell remodeling has seen increased attention over the last years. This development is largely driven by a number of technological advances in proteomics methods. Pulsed stable isotope labeling in cell culture (SILAC) combined with tandem mass tag (TMT) labeling has evolved as a gold standard for profiling protein synthesis and degradation. While the experimental setup is similar to typical proteomics experiments, the data analysis proves more difficult: After peptide identification through search engines, data extraction requires either custom scripted pipelines or tedious manual table manipulations to extract the TMT-labeled heavy and light peaks of interest. To overcome this limitation, which deters researchers from using protein dynamic proteomics, we developed a user-friendly, browser-based application that allows easy and reproducible data analysis without the need for scripting experience. In addition, we provide a python package that can be implemented in established data analysis pipelines. We anticipate that this tool will ease data analysis and spark further research aimed at monitoring protein translation and degradation by proteomics

Functional translatome proteomics reveal converging and dose-dependent regulation by mtorc1 and eif2α

Regulation of translation is essential during stress. However, the precise sets of proteins regulated by the key translational stress responses—the integrated stress response (ISR) and mTORC1—remain elusive. We developed multiplexed enhanced protein dynamics (mePROD) proteomics, adding signal amplification to dynamic-SILAC and multiplexing, to enable measuring acute changes in protein synthesis. Treating cells with ISR/mTORC1-modulating stressors, we showed extensive translatome modulation with ∼20% of proteins synthesized at highly reduced rates. Comparing translation-deficient sub-proteomes revealed an extensive overlap demonstrating that target specificity is achieved on protein level and not by pathway activation. Titrating cap-dependent translation inhibition confirmed that synthesis of individual proteins is controlled by intrinsic properties responding to global translation attenuation. This study reports a highly sensitive method to measure relative translation at the nascent chain level and provides insight into how the ISR and mTORC1, two key cellular pathways, regulate the translatome to guide cellular survival upon stress

Growth factor receptor signaling inhibition prevents SARS-CoV-2 replication

SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinder therapy development. We employed a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phospho-proteomics. We identified viral protein phosphorylation and defined phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways were activated. Drug-protein network analyses revealed GFR signaling as key pathway targetable by approved drugs. Inhibition of GFR downstream signaling by five compounds prevented SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as central pathway essential for SARS-CoV-2 replication. It provides with novel strategies for COVID-19 treatment

Data model harmonization for the All Of Us Research Program: Transforming i2b2 data into the OMOP common data model.

BackgroundThe All Of Us Research Program (AOU) is building a nationwide cohort of one million patients' EHR and genomic data. Data interoperability is paramount to the program's success. AOU is standardizing its EHR data around the Observational Medical Outcomes Partnership (OMOP) data model. OMOP is one of several standard data models presently used in national-scale initiatives. Each model is unique enough to make interoperability difficult. The i2b2 data warehousing and analytics platform is used at over 200 sites worldwide, which uses a flexible ontology-driven approach for data storage. We previously demonstrated this ontology system can drive data reconfiguration, to transform data into new formats without site-specific programming. We previously implemented this on our 12-site Accessible Research Commons for Health (ARCH) network to transform i2b2 into the Patient Centered Outcomes Research Network model.Methods and resultsHere, we leverage our investment in i2b2 high-performance transformations to support the AOU OMOP data pipeline. Because the ARCH ontology has gained widespread national interest (through the Accrual to Clinical Trials network, other PCORnet networks, and the Nebraska Lexicon), we leveraged sites' existing investments into this standard ontology. We developed an i2b2-to-OMOP transformation, driven by the ARCH-OMOP ontology and the OMOP concept mapping dictionary. We demonstrated and validated our approach in the AOU New England HPO (NEHPO). First, we transformed into OMOP a fake patient dataset in i2b2 and verified through AOU tools that the data was structurally compliant with OMOP. We then transformed a subset of data in the Partners Healthcare data warehouse into OMOP. We developed a checklist of assessments to ensure the transformed data had self-integrity (e.g., the distributions have an expected shape and required fields are populated), using OMOP's visual Achilles data quality tool. This i2b2-to-OMOP transformation is being used to send NEHPO production data to AOU. It is open-source and ready for use by other research projects

Aprotinin Inhibits SARS-CoV-2 Replication

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air–liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease