The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Biomolecular condensation of the neuronal microtubule‐associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate‐mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate‐like density for nuclear‐envelope Tau. These findings suggest that a complex interplay between interaction partners, post‐translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding‐competent Tau and lead to distinct cellular Tau accumulations.

Application of in situ dynamic light scattering to solutions of protein–detergent complexes permits characterization of these complexes in samples as small as 2 µl in volume.

Liquid-liquid phase separation (LLPS) in cells is known as a complex physicochemical process causing the formation of membrane-less organelles (MLOs). Cells have welldefined different membrane-surrounded organelles like mitochondria, endoplasmic reticulum, lysosomes, peroxisomes, etc., however, on demand they can create MLOs

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the host’s innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 Å and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 Å, show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Background The transition to a biobased economy involving the depolymerization and fermentation of renewable agro-industrial sources is a challenge that can only be met by achieving the efficient hydrolysis of biomass to monosaccharides. In nature, lignocellulosic biomass is mainly decomposed by fungi. We recently identified six efficient cellulose degraders by screening fungi from Vietnam. Results We characterized a high-performance cellulase-producing strain, with an activity of 0.06 U/mg, which was identified as a member of the Fusarium solani species complex linkage 6 (Fusarium metavorans), isolated from mangrove wood (FW16.1, deposited as DSM105788). The genome, representing nine potential chromosomes, was sequenced using PacBio and Illumina technology. In-depth secretome analysis using six different synthetic and artificial cellulose substrates and two agro-industrial waste products identified 500 proteins, including 135 enzymes assigned to five different carbohydrate-active enzyme (CAZyme) classes. The F. metavorans enzyme cocktail was tested for saccharification activity on pre-treated sugarcane bagasse, as well as untreated sugarcane bagasse and maize leaves, where it was complemented with the commercial enzyme mixture Accellerase 1500. In the untreated sugarcane bagasse and maize leaves, initial cell wall degradation was observed in the presence of at least 196 µg/mL of the in-house cocktail. Increasing the dose to 336 µg/mL facilitated the saccharification of untreated sugarcane biomass, but had no further effect on the pre-treated biomass. Conclusion Our results show that F. metavorans DSM105788 is a promising alternative pre-treatment for the degradation of agro-industrial lignocellulosic materials. The enzyme cocktail promotes the debranching of biopolymers surrounding the cellulose fibers and releases reduced sugars without process disadvantages or loss of carbohydrates.

Protein phase separation and protein liquid cluster formation have been observed and analysed in protein crystallization experiments and, in recent years, have been reported more frequently, especially in studies related to membraneless organelles and protein cluster formation in cells. A detailed understanding about the phase separation process preceding liquid dense cluster formation will elucidate what has, so far, been poorly understood—despite intracellular crowding and phase separation being very common processes—and will also provide more insights into the early events of in vitro protein crystallization. In this context, the phase separation and crystallization kinetics of concanavalin A were analysed in detail, which applies simultaneous dynamic light scattering and depolarized dynamic light scattering to obtain insights into metastable intermediate states between the soluble phase and the crystalline form. A multi-step mechanism was identified for ConA phase separation, according to the resultant ACF decay, acquired after an increase in the concentration of the crowding agent until a metastable ConA gel intermediate between the soluble and final crystalline phases was observed. The obtained results also revealed that ConA is trapped in a macromolecular network due to short-range intermolecular protein interactions and is unable to transform back into a non-ergodic solution.