Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2.

Currently, human infections with the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are accelerating the ongoing spread of the pandemic. Several innovative types of vaccines have already been developed, whereas effective options of antiviral treatments still await a scientific implementation. The development of novel anti-SARS-CoV-2 drug candidates demands skillful strategies and analysis systems. Promising results have been achieved with first generation direct-acting antivirals targeting the viral polymerase RdRp or the protease 3CLpro. Such recently approved or investigational drugs like remdesivir and GC376 represent a basis for further development and optimization. Here, we establish a multi-readout assay (MRA) system that enables the antiviral assessment and mechanistic characterization of novel test compounds, drug repurposing and combination treatments. Our SARS-CoV-2-specific MRA combines the quantitative measurement of several parameters of virus infection, such as the intracellular production of proteins and genomes, enzymatic activities and virion release, as well as the use of reporter systems. In this regard, the antiviral efficacy of remdesivir and GC376 has been investigated in human Caco-2 cells. The readouts included the use of spike- and double-strand RNA-specific monoclonal antibodies for in-cell fluorescence imaging, a newly generated recombinant SARS-CoV-2 reporter virus d6YFP, the novel 3CLpro-based FRET CFP::YFP and the previously reported FlipGFP reporter assays, as well as viral genome-specific RT-qPCR. The data produced by our MRA confirm the high antiviral potency of these two drugs in vitro. Combined, this MRA approach may be applied for broader analyses of SARS-CoV-2-specific antivirals, including compound screenings and the characterization of selected drug candidates.

Collective forward-looking responsibility of patient advocacy organizations: conceptual and ethical analysis.

BACKGROUND: Patient advocacy organizations (PAOs) have an increasing influence on health policy and biomedical research, therefore, questions about the specific character of their responsibility arise: Can PAOs bear moral responsibility and, if so, to whom are they responsible, for what and on which normative basis? Although the concept of responsibility in healthcare is strongly discussed, PAOs particularly have rarely been systematically analyzed as morally responsible agents. The aim of the current paper is to analyze the character of PAOs' responsibility to provide guidance to themselves and to other stakeholders in healthcare. METHODS: Responsibility is presented as a concept with four reference points: (1) The subject, (2) the object, (3) the addressee and (4) the underlying normative standard. This four-point relationship is applied to PAOs and the dimensions of collectivity and prospectivity are analyzed in each reference point. RESULTS: Understood as collectives, PAOs are, in principle, capable of intentionality and able to act and, thus, fulfill one prerequisite for the attribution of moral responsibility. Given their common mission to represent those affected, PAOs can be seen as responsible for patients' representation and advocacy, primarily towards a certain group but secondarily in a broader social context. Various legal and political statements and the bioethical principles of justice, beneficence and empowerment can be used as a normative basis for attributing responsibility to PAOs. CONCLUSIONS: The understanding of responsibility as a four-point relation incorporating collective and forward-looking dimensions helps one to understand the PAOs' roles and responsibilities better. The analysis, thus, provides a basis for the debate about PAOs' contribution and cooperation in the healthcare sector.

Chemically sulfated polysaccharides from natural sources: Assessment of extraction-sulfation efficiencies, structural features and antiviral activities.

The provisioning of compound libraries with a high degree of diversity and attractive pharmacological properties is a limiting step in drug development. This study reports the production of highly bioactive sulfated polysaccharides, originally present in a nonsulfated, dormant state in natural sources, and demonstrates their antiviral activity (human cytomegalovirus EC50 values of 2.34-7.77 µg/mL) at a low degree of cytotoxicity. Furthermore, data strongly suggested the inhibition of virus entry as the main mode of antiviral action. Remarkably, the utilized oleum-DMF reagent was able to generate a range of sulfated polysaccharides from various natural sources, possessing varying saccharide compositions, degrees of sulfation (0.4-1.7) and molecular masses (38-94,000 g/mol). Typically, in a matter of minutes, this reagent not only solubilized polysaccharides but also chemically converted their hydroxyl functionality into sulfates. The most active sulfated polysaccharide (EC50 of 2.62 µg/mL) proved to be a 94,000 g/mol branched glucan with sulfates at C-6/C-3,6/C-2,3,6 positions. In conclusion, the important determinants of such compounds' antiviral activity are: (i) degree of sulfation, (ii) molecular mass and (iii) structural features. Thus, our approach offers a huge prospect for the improvement of natural source-derived libraries based on biologically active polysaccharides with diversified chemical profiles.