Plitidepsin Is a Broad-Spectrum Antiviral Shaping the Cell Proteostatic Balance

Background: Different viruses employ similar pathways for replication, revealing key intracellular hotspots to target with host-directed therapies and achieve a broad-spectrum antiviral activity. Plitidepsin is a clinically approved antitumoral agent that blocks the elongation factor eEF1A required for protein translation. This drug counteracts SARS-CoV-2 replication and shows a favorable safety profile in COVID-19 patients. Yet, the precise antiviral mechanism of action of plitidepsin remains unknown. Method(s): Here we used a deep quantitative proteomic analysis to measure the impact of plitidepsin on the proteome of SARS-CoV-2-infected Vero E6 cells. This was complemented with transmission electron microscopy assays, which unraveled the subcellular and morphological changes associated to plitidepsin treatment. In addition, we performed functional in vitro assays to dissect the antiviral activity of plitidepsin against SARS-CoV-2 and other viruses. Result(s): We found that this drug inhibited the synthesis of all SARS-CoV-2 proteins in a dose-dependent manner. These included the R1AB polyproteins, which facilitate the synthesis of non-structural proteins involved in the formation of double membrane vesicles (DMV) required for viral replication. Plitidepsin reduced DMV formation and the morphogenesis of new viruses, having a greater impact on viral than on host proteins. Less than 14% of the cellular proteome was significantly affected by plitidepsin, inducing the up-regulation of key molecules associated with protein biosynthesis, such as the translation initiation factors eIF4A2 and eIF2S3. Therefore, plitidepsin induced a compensatory state that rescued protein translation. This proteostatic response explains how cells preserve the cellular proteome after treatment with a translation inhibitor such as plitidepsin. In addition, it suggests that plitidepsin could inhibit other RNA-dependent and non-integrated DNA viruses, as we confirmed in vitro using Zika virus, Hepatitis C virus replicon and Herpes simplex virus. However, the compensatory proteostasis induced by plitidespin also explains why this drug failed to inhibit the replication of integrated DNA proviruses such as HIV-1. Conclusion(s): Unraveling the mechanism of action of host-directed therapies like plitidepsin is imperative to define the indications and antiviral profile of these compounds. This knowledge will be key to develop broad-spectrum treatments and have them ready to deploy when future pandemic viruses break through.

Jahrestagung der Gesellschaft fur Padiatrische Pneumologie.

The proceedings contain 54 papers. The topics discussed include: cytokines in severe childhood asthma;transcriptional gene regulation of interleukin-6 in epithelial cells in viral-induced asthma exacerbation;assessment of the long-term safety and efficacy of dupilumab in children with asthma: LIBERTY ASTHMA EXCURSION;impulse oscillometry bronchodilator response in preschool children;pulmonary function in non-hospitalized adults and children after mild Covid-19;exhaled aerosols in PCR-confirmed SARS-CoV-2-infected children;early respiratory infectious diseases have an influence on the gut microbiome;comparison of three eradication treatment protocols for pseudomonas aeruginosa in children and adolescents with cystic fibrosis;neutrophilic airway inflammation in children with repaired esophageal atresia-tracheoesophageal fistula (EA/TEF);and multiplex immunofluorescence and multispectral imaging as a tool to evaluate host directed therapy.

Applying Lessons Learned From COVID-19 Therapeutic Trials to Improve Future ALI/ARDS Trials.

Host-directed therapeutics targeting immune dysregulation are considered the most promising approach to address the unmet clinical need for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) related to coronavirus disease 2019 (COVID-19). To better understand the current clinical study landscape and gaps in treating hospitalized patients with severe or critical COVID-19, we identified COVID-19 trials developing host-directed therapies registered at ClinicalTrials.gov and discussed the factors contributing to the success vs failure of these studies. We have learned, instead of the one-size-fits-all approach, future clinical trials evaluating a targeted immunomodulatory agent in heterogeneous patients with ALI/ARDS due to COVID-19 or other infectious diseases can use immune-based biomarkers in addition to clinical and demographic characteristics to improve patient stratification and inform clinical decision-making. Identifying distinct patient subgroups based on immune profiles across the disease trajectory, regardless of the causative pathogen, may accelerate evaluating host-directed therapeutics in trials of ALI/ARDS and related conditions (eg, sepsis).

Inhibition of glycogen synthase kinase-3-beta (GSK3ß) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication.

GSK3ß has been proposed to have an essential role in Coronaviridae infections. Screening of a targeted library of GSK3ß inhibitors against both SARS-CoV-2 and HCoV-229E to identify broad-spectrum anti-Coronaviridae inhibitors resulted in the identification of a high proportion of active compounds with low toxicity to host cells. A selected lead compound, T-1686568, showed low micromolar, dose-dependent activity against SARS-CoV-2 and HCoV-229E. T-1686568 showed efficacy in viral-infected cultured cells and primary 2D organoids. T-1686568 also inhibited SARS-CoV-2 variants of concern Delta and Omicron. Importantly, while inhibition by T-1686568 resulted in the overall reduction of viral load and protein translation, GSK3ß inhibition resulted in cellular accumulation of the nucleocapsid protein relative to the spike protein. Following identification of potential phosphorylation sites of Coronaviridae nucleocapsid, protein kinase substrate profiling assays combined with Western blotting analysis of nine host kinases showed that the SARS-CoV-2 nucleocapsid could be phosphorylated by GSK3ß and PKCa. GSK3ß phosphorylated SARS-CoV-2 nucleocapsid on the S180/S184, S190/S194 and T198 phospho-sites, following previous priming in the adjacent S188, T198 and S206, respectively. Such inhibition presents a compelling target for broad-spectrum anti-Coronaviridae compound development, and underlies the mechanism of action of GSK3ß host-directed therapy against this class of obligate intracellular pathogens.

Deep variational graph autoencoders for novel host-directed therapy options against COVID-19.

The COVID-19 pandemic has been keeping asking urgent questions with respect to therapeutic options. Existing drugs that can be repurposed promise rapid implementation in practice because of their prior approval. Conceivably, there is still room for substantial improvement, because most advanced artificial intelligence techniques for screening drug repositories have not been exploited so far. We construct a comprehensive network by combining year-long curated drug-protein/protein-protein interaction data on the one hand, and most recent SARS-CoV-2 protein interaction data on the other hand. We learn the structure of the resulting encompassing molecular interaction network and predict missing links using variational graph autoencoders (VGAEs), as a most advanced deep learning technique that has not been explored so far. We focus on hitherto unknown links between drugs and human proteins that play key roles in the replication cycle of SARS-CoV-2. Thereby, we establish novel host-directed therapy (HDT) options whose utmost plausibility is confirmed by realistic simulations. As a consequence, many of the predicted links are likely to be crucial for the virus to thrive on the one hand, and can be targeted with existing drugs on the other hand.

Development of Inhalable ATRA-Loaded PLGA Nanoparticles as Host-Directed Immunotherapy against Tuberculosis.

Developing new effective treatment strategies to overcome the rise in multi-drug resistant tuberculosis cases (MDR-TB) represents a global challenge. A host-directed therapy (HDT), acting on the host immune response rather than Mtb directly, could address these resistance issues. We developed an HDT for targeted TB treatment, using All Trans Retinoic Acid (ATRA)-loaded nanoparticles (NPs) that are suitable for nebulization. Efficacy studies conducted on THP-1 differentiated cells infected with the H37Ra avirulent Mycobacterium tuberculosis (Mtb) strain, have shown a dose-dependent reduction in H37Ra growth as determined by the BACT/ALERT® system. Confocal microscopy images showed efficient and extensive cellular delivery of ATRA-PLGA NPs into THP-1-derived macrophages. A commercially available vibrating mesh nebulizer was used to generate nanoparticle-loaded droplets with a mass median aerodynamic diameter of 2.13 µm as measured by cascade impaction, and a volumetric median diameter of 4.09 µm as measured by laser diffraction. In an adult breathing simulation experiment, 65.1% of the ATRA PLGA-NP dose was inhaled. This targeted inhaled HDT could offer a new adjunctive TB treatment option that could enhance current dosage regimens leading to better patient prognosis and a decreasing incidence of MDR-TB.

Andrographolide induces anti-SARS-CoV-2 response through host-directed mechanism: an in silico study.

Aim: Considering the present alarming situation of COVID-19 pandemic, we concentrated on evaluating the efficacy of a novel natural antiviral drug-candidate andrographolide against SARS-CoV-2 through an in silico model of study. Materials & methods: Interaction of andrographolide against the major host molecules that are responsible for SARS-CoV-2 pathogenesis were determined using bio-computational tools, in other words, molecular docking, molecular dynamics simulation and pharmacodynamics-pharmacokinetics analysis. Result: Computational findings represent that andrographolide efficiently interacts with the major human-host-associated putative drug-targets of viral-entry points like furin (-10.54 kcal/mol), TMPRSS-2 (-9.50 kcal/mol), ACE2 (-8.99 kcal/mol) and Cathepsin L (-8.98 kcal/mol). Moreover, it also blocks the inflammatory regulators including TLR4-MD2 and IL-6, which promote virus-induced inflammation leading to cytokine storm in the host body. Conclusion: This work elucidates that, the candidature of andrographolide can be utilized as a potent natural agent for the therapeutic intervention of SARS-CoV-2 through host-directed treatment.

Red Blood Cell BCL-xL Is Required for Plasmodium falciparum Survival: Insights into Host-Directed Malaria Therapies.

The development of antimalarial drug resistance is an ongoing problem threatening progress towards the elimination of malaria, and antimalarial treatments are urgently needed for drug-resistant malaria infections. Host-directed therapies (HDT) represent an attractive strategy for the development of new antimalarials with untapped targets and low propensity for resistance. In addition, drug repurposing in the context of HDT can lead to a substantial decrease in the time and resources required to develop novel antimalarials. Host BCL-xL is a target in anti-cancer therapy and is essential for the development of numerous intracellular pathogens. We hypothesised that red blood cell (RBC) BCL-xL is essential for Plasmodium development and tested this hypothesis using six BCL-xL inhibitors, including one FDA-approved compound. All BCL-xL inhibitors tested impaired proliferation of Plasmodium falciparum 3D7 parasites in vitro at low micromolar or sub-micromolar concentrations. Western blot analysis of infected cell fractions and immunofluorescence microscopy assays revealed that host BCL-xL is relocated from the RBC cytoplasm to the vicinity of the parasite upon infection. Further, immunoprecipitation of BCL-xL coupled with mass spectrometry analysis identified that BCL-xL forms unique molecular complexes with human µ-calpain in uninfected RBCs, and with human SHOC2 in infected RBCs. These results provide interesting perspectives for the development of host-directed antimalarial therapies and drug repurposing efforts.

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

Host-directed therapy, an untapped opportunity for antimalarial intervention.

Host-directed therapy (HDT) is gaining traction as a strategy to combat infectious diseases caused by viruses and intracellular bacteria, but its implementation in the context of parasitic diseases has received less attention. Here, we provide a brief overview of this field and advocate HDT as a promising strategy for antimalarial intervention based on untapped targets. HDT provides a basis from which repurposed drugs could be rapidly deployed and is likely to strongly limit the emergence of resistance. This strategy can be applied to any intracellular pathogen and is particularly well placed in situations in which rapid identification of treatments is needed, such as emerging infections and pandemics, as starkly illustrated by the current COVID-19 crisis.

Host directed therapies: COVID-19 and beyond.

The global spread of SARS-CoV-2 has necessitated the development of novel, safe and effective therapeutic agents against this virus to stop the pandemic, however the development of novel antivirals may take years, hence, the best alternative available, is to repurpose the existing antiviral drugs with known safety profile in humans. After more than one year into this pandemic, global efforts have yielded the fruits and with the launch of many vaccines in the market, the world is inching towards the end of this pandemic, nonetheless, future pandemics of this magnitude or even greater cannot be denied. The preparedness against viruses of unknown origin should be maintained and the broad-spectrum antivirals with activity against range of viruses should be developed to curb future viral pandemics. The majority of antivirals developed till date are pathogen specific agents, which target critical viral pathways and lack broad spectrum activity required to target wide range of viruses. The surge in drug resistance among pathogens has rendered a compelling need to shift our focus towards host directed factors in the treatment of infectious diseases. This gains special relevance in the case of viral infections, where the pathogen encodes a handful of genes and predominantly depends on host factors for their propagation and persistence. Therefore, future antiviral drug development should focus more on targeting molecules of host pathways that are often hijacked by many viruses. Such cellular proteins of host pathways offer attractive targets for the development of broad-spectrum anticipatory antivirals. In the present article, we have reviewed the host directed therapies (HDTs) effective against viral infections with a special focus on COVID-19. This article also discusses the strategies involved in identifying novel host targets and subsequent development of broad spectrum HDTs.

TFEB - at the crossroads of host-pathogen interactions.

The existence of constantly evolving dynamic interactions between the host and the pathogen determines their fate in this continuous arms race. Hence, identifying the molecular basis of processes that reinforce host defensive strategies to eliminate intracellular pathogens is of utmost significance. Pathogenic intrusion activates autophagy and phagocytic pathways that culminate in the lysosome, a vital organelle responsible for pathogen clearance. The transcription factor TFEB plays a pivotal role in autophagy-lysosomal function. Although TFEB is an emerging transcription factor in the field of immune signaling pathways, its role in infectious diseases remains contentious. Recent evidence suggests that infection with certain bacterial and viral pathogens causes TFEB, which is normally located in the cytoplasm, to translocate to the nucleus. There, it activates the transcription of genes that trigger the autophagy-lysosomal and inflammatory pathways to target intracellular pathogens. It is known that some pathogens modulate TFEB to establish themselves inside the host; in some cases, pathogens restrict TFEB to the cytoplasm, whereas in others, functional TFEB fuels pathogen survival and replication. However, the key regulators and molecular mechanisms that decide the outcome of TFEB function during intracellular infection are not clear. In this Review, we attempt to dissect the complex functions of TFEB in host-pathogen interactions and explore the suitability of TFEB as a therapeutic target of clinical relevance.

Broad Anti-Viral Capacities of Lian-Hua-Qing-Wen Capsule and Jin-Hua-Qing-Gan Granule and Rational use Against COVID-19 Based on Literature Mining.

The novel coronavirus disease 2019 (COVID-19) has become a matter of international concern as the disease is spreading exponentially. Statistics showed that infected patients in China who received combined treatment of Traditional Chinese Medicine and modern medicine exhibited lower fatality rate and relatively better clinical outcomes. Both Lian-Hua-Qing-Wen Capsule (LHQWC) and Jin-Hua-Qing-Gan Granule (JHQGG) have been recommended by China Food and Drug Administration for the treatment of COVID-19 and have played a vital role in the prevention of a variety of viral infections. Here, we desired to analyze the broad-spectrum anti-viral capacities of LHQWC and JHQGG, and to compare their pharmacological functions for rational clinical applications. Based on literature mining, we found that both LHQWC and JHQGG were endowed with multiple antiviral activities by both targeting viral life cycle and regulating host immune responses and inflammation. In addition, from literature analyzed, JHQGG is more potent in modulating viral life cycle, whereas LHQWC exhibits better efficacies in regulating host anti-viral responses. When translating into clinical applications, oral administration of LHQWC could be more beneficial for patients with insufficient immune functions or for patients with alleviated symptoms after treatment with JHQGG.

Enhancement of the IFN-ß-induced host signature informs repurposed drugs for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a causative agent for the outbreak of coronavirus disease 2019 (COVID-19). This global pandemic is now calling for efforts to develop more effective COVID-19 therapies. Here we use a host-directed approach, which focuses on cellular responses to diverse small-molecule treatments, to identify potentially effective drugs for COVID-19. This framework looks at the ability of compounds to elicit a similar transcriptional response to IFN-ß, a type I interferon that fails to be induced at notable levels in response to SARS-CoV-2 infection. By correlating the perturbation profiles of ~3,000 small molecules with a high-quality signature of IFN-ß-responsive genes in primary normal human bronchial epithelial cells, our analysis revealed four candidate COVID-19 compounds, namely homoharringtonine, narciclasine, anisomycin, and emetine. We experimentally confirmed that the predicted compounds significantly inhibited SARS-CoV-2 replication in Vero E6 cells at nanomolar, relatively non-toxic concentrations, with half-maximal inhibitory concentrations of 165.7 nM, 16.5 nM, and 31.4 nM for homoharringtonine, narciclasine, and anisomycin, respectively. Together, our results corroborate a host-centric strategy to inform protective antiviral therapies for COVID-19.

[Treatment of COVID-19 guided by holistic view of traditional Chinese medicine--therapy aimed at both viral and host].

The global outbreak of coronavirus disease 2019(COVID-19) has further spread, and there is an increasing number of confirmed cases in many countries. On February 28, 2020 of Geneva time, the World Health Organization has raised global risk level to the very high level in view of outbreak of COVID-19. Since some patients' condition appeared to deteriorate rapidly after infection of this 2019 novel coronavirus(2019-nCoV), a variety of treatments should be considered. Holistic view and syndrome differentiation are the two characteristics of traditional Chinese medicine(TCM). Therefore, under the guidance of the holistic view, syndrome diffe-rentiation of TCM has achieved good effects in the treatment of COVID-19. This treatment mainly aimed at eliminating pathogens and strengthening overall health, regulating the balance of body and coordinating various of functions of Zangfu organs. In addition, modern medical proposes host-directed therapy(HDT), a strategy aims to interfere with host cell mechanism, enhance immune responses, and reduce exacerbated inflammation. To some extent, the combined application of HDT and antiviral therapy is highly consistent with the therapeutic concept of the holistic view of TCM. Therefore, under the guidance of the holistic view, syndrome differentiation of TCM uses treatments, such as clearing heat, detoxification, relieving asthma, clearing damp and phlegm, together with Lianhua Qingwen Capsules, Maxing Shigan Decoction, and Haoqin Qingdan Decoction under the guidance of these therapeutic methods. These therapeutic methods and prescriptions intervened with both virus and host at the same time in the treatment of COVID-19, which has important implications for the effective clinical treatment of COVID-19.