Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

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 repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus 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 binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

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 (M^(pro)), 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 M^(pro). 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

Estudo do potencial enzimático hidrolítico e oxidativo do microorganismo Myceliophthora thermophila M.7.7

In terms of the here presented PhD research project the hydrolytic and oxidative enzymatic properties from the ascomycota and themotolerant fungae Myceliophthora thermophila M.7.7. were investigated in detail. Six different secretomes were analysed by mass spectroscopy and 187 proteins were identified. Within these proteins beside already known and expected enzymes also several oxidative active enzymes were confirmed. Enzymes belonging to the cellobiose dehydrogenase and glycoside hydrolase family 7 were most abundant, when in natura and steam exploded sugar cane bagasses were used as carbon source. Particular a high abundance of glycoside hydrolases was identified when a mixture of hemicelluloses was applied as sole carbon source. In this case the secretome analysis showed a substrate-specific secretion for this fungae. The isolation, purification and characterization of a β-glycosidase from M. thermophila M.7.7. showed and confirmed excellent enzymatic charateristics and parameters for future potential applications as a supplemental enzyme in enzyme cocktails, or implimanting by molecular biology in organisms deficient in β-glucosidase. Even at low protein concentration the enzymatic hydrolysis, utilizing the crude extract, resulted in a conversion of approx. 21% in natura bagasse and approx. 16% in commercial cellulose, indicating a high specificity and synergy of the enzyme complex to cellulose polymers with a higher degree of polymerization and crystallinity index in its structureNeste trabalho foram estudados os sistemas enzimáticos hidrolítico e oxidativo do ascomiceto termotolerante Myceliophthora thermophila M.7.7. Seis diferentes secretomas foram avaliados por espectroscopia de massas que identificou o total de 187 proteínas confirmando a presença do clássico sistema enzimático hidrólitico revelando também a produção de enzimas do sistema oxidativo de degradação. As enzimas mais abundantes foram celobiose desidrogenase e glicosídeo hidrolase família 7 secretadas principalmente nos bagaços de cana-de-açúcar in natura e explodido a vapor. A abundante produção de glicosídeo hidrolases foi identificada quando polissacarídeos de hemiceluloses foram a fonte de carbono empregada, mostrando que o produção de enzimas é substrato específica. O isolamento, a caracterização bioquímica e o estudo cinético enzimático de uma β-glicosidase do M. thermophila M.7.7. também foram realizados. A β-glicosidase purificada apresentou excelentes parâmetros para sua aplicabilidade como enzima suplementar a coquetéis enzimáticos ou até mesmo sua clonagem e expressão em organismos deficientes em β-glicosidase. Experimentos de hidrolíse enzimática também foram executados empregando o extrato bruto de M. thermophila, que resultou em uma conversão de 21% em bagaço in natura e 16% em celulose comercial mesmo com baixa carga de proteínas, indicando maior especificidade e sinergia do complexo enzimático à polímeros de celulose com maior índice de cristalinidade e grau de polimerização em sua estruturaCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Multi-Step Concanavalin A Phase Separation and Early-Stage Nucleation Monitored Via Dynamic and Depolarized Light Scattering

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

Exploring Nucleation Pathways in Distinct Physicochemical Environments Unveiling Novel Options to Modulate and Optimize Protein Crystallization

The scientific discussion about classical and nonclassical nucleation theories has lasted for two decades so far. Recently, multiple nucleation pathways and the occurrence and role of metastable intermediates in crystallization processes have attracted increasing attention, following the discovery of functional phase separation, which is now under investigation in different fields of cellular life sciences, providing interesting and novel aspects for conventional crystallization experiments. In this context, more systematic investigations need to be carried out to extend the current knowledge about nucleation processes. In terms of the data we present, a well-studied model protein, glucose isomerase (GI), was employed first to investigate systematically the early stages of the crystallization process, covering condensing and prenucleation ordering of protein molecules in diverse scenarios, including varying ionic and crowding agent conditions, as well as the application of a pulsed electric field (pEF). The main method used to characterize the early events of nucleation was synchronized polarized and depolarized dynamic light scattering (DLS/DDLS), which is capable of collecting the polarized and depolarized component of scattered light from a sample suspension in parallel, thus monitoring the time-resolved evolution of the condensation and geometrical ordering of proteins at the early stages of nucleation. A diffusion interaction parameter, KD, of GI under varying salt conditions was evaluated to discuss how the proportion of specific and non-specific protein–protein interactions affects the nucleation process. The effect of mesoscopic ordered clusters (MOCs) on protein crystallization was explored further by adding different ratios of MOCs induced by a pEF to fresh GI droplets in solution with different PEG concentrations. To emphasize and complement the data and results obtained with GI, a recombinant pyridoxal 5-phosphate (vitamin B6) synthase (Pdx) complex of Staphylococcus aureus assembled from twelve monomers of Pdx1 and twelve monomers of Pdx2 was employed to validate the ability of the pEF influencing the nucleation of complex macromolecules and the effect of MOCs on adjusting the crystallization pathway. In summary, our data revealed multiple nucleation pathways by tuning the proportion of specific and non-specific protein interactions, or by utilizing a pEF which turned out to be efficient to accelerate the nucleation process. Finally, a novel and reproducible experimental strategy, which can adjust and facilitate a crystallization process by pEF-induced MOCs, was summarized and reported for the first time

Exploring Nucleation Pathways in Distinct Physicochemical Environments Unveiling Novel Options to Modulate and Optimize Protein Crystallization

The scientific discussion about classical and nonclassical nucleation theories has lasted for two decades so far. Recently, multiple nucleation pathways and the occurrence and role of metastable intermediates in crystallization processes have attracted increasing attention, following the discovery of functional phase separation, which is now under investigation in different fields of cellular life sciences, providing interesting and novel aspects for conventional crystallization experiments. In this context, more systematic investigations need to be carried out to extend the current knowledge about nucleation processes. In terms of the data we present, a well-studied model protein, glucose isomerase (GI), was employed first to investigate systematically the early stages of the crystallization process, covering condensing and prenucleation ordering of protein molecules in diverse scenarios, including varying ionic and crowding agent conditions, as well as the application of a pulsed electric field (pEF). The main method used to characterize the early events of nucleation was synchronized polarized and depolarized dynamic light scattering (DLS/DDLS), which is capable of collecting the polarized and depolarized component of scattered light from a sample suspension in parallel, thus monitoring the time-resolved evolution of the condensation and geometrical ordering of proteins at the early stages of nucleation. A diffusion interaction parameter, KD, of GI under varying salt conditions was evaluated to discuss how the proportion of specific and non-specific protein–protein interactions affects the nucleation process. The effect of mesoscopic ordered clusters (MOCs) on protein crystallization was explored further by adding different ratios of MOCs induced by a pEF to fresh GI droplets in solution with different PEG concentrations. To emphasize and complement the data and results obtained with GI, a recombinant pyridoxal 5-phosphate (vitamin B6) synthase (Pdx) complex of Staphylococcus aureus assembled from twelve monomers of Pdx1 and twelve monomers of Pdx2 was employed to validate the ability of the pEF influencing the nucleation of complex macromolecules and the effect of MOCs on adjusting the crystallization pathway. In summary, our data revealed multiple nucleation pathways by tuning the proportion of specific and non-specific protein interactions, or by utilizing a pEF which turned out to be efficient to accelerate the nucleation process. Finally, a novel and reproducible experimental strategy, which can adjust and facilitate a crystallization process by pEF-induced MOCs, was summarized and reported for the first time