Computer-aided drug screening by molecular docking, molecular dynamics (MD) and structural-activity relationship (SAR) can offer an efficient approach to identify promising drug repurposing candidates for COVID-19 treatment. In this study, computational screening is performed by molecular docking of 1615 Food and Drug Administration (FDA) approved drugs against the main protease (Mpro) of SARS-CoV-2. Several promising approved drugs, including Simeprevir, Ergotamine, Bromocriptine and Tadalafil, stand out as the best candidates based on their binding energy, fitting score and noncovalent interactions at the binding sites of the receptor. All selected drugs interact with the key active site residues, including His41 and Cys145. Various noncovalent interactions including hydrogen bonding, hydrophobic interactions, pi-sulfur and pi-pi interactions appear to be dominant in drug-Mpro complexes. MD simulations are applied for the most promising drugs. Structural stability and compactness are observed for the drug-Mpro complexes. The protein shows low flexibility in both apo and holo form during MD simulations. The MM/PBSA binding free energies are also measured for the selected drugs. For pattern recognition, structural similarity and binding energy prediction, multiple linear regression (MLR) models are used for the quantitative structural-activity relationship. The binding energy predicted by MLR model shows an 82% accuracy with the binding energy determined by molecular docking. Our details results can facilitate rational drug design targeting the SARS-CoV-2 main protease.

Tardigrades, also known as water bears, make up a phylum of small but extremely robust animals renowned for their ability to survive extreme stresses including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins such as gelatin, our data support a mechanism in which gelation of CAHS D is driven by intermolecular beta-beta interactions. We find that gelation of CAHS D promotes the slowing of diffusion, and coordination of residual water. Slowed diffusion and increased water coordination correlate with the ability of CAHS D to provide robust stabilization of an enzyme, lactate dehydrogenase, which otherwise unfolds when dried. Conversely, slowed diffusion and water coordination do not promote the prevention of protein aggregation during drying. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as "molecular Swiss army knives" capable of providing protection through several different mechanisms simultaneously.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins that form gels. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of gelation for CAHS D similar to that of intermediate filaments. We show that gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fiber-like condensates during osmotic stress. Condensation of CAHS D improves survival of osmotically shocked cells through at least two mechanisms: reduction of cell volume change and reduction of metabolic activity. Importantly, condensation of CAHS D is reversible and metabolic rates return to control levels after CAHS condensates are resolved. This work provides insights into how tardigrades induce biostasis through the self-assembly of CAHS gels.

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Biologics, pharmaceuticals containing or derived from living organisms, such as vaccines, antibodies, stem cells, blood, and blood products are a cornerstone of modern medicine. However, nearly all biologics have a major deficiency: they are inherently unstable, requiring storage under constant cold conditions. The so-called ‘cold-chain’, while effective, represents a serious economic and logistical hurdle for deploying biologics in remote, underdeveloped, or austere settings where access to cold-chain infrastructure ranging from refrigerators and freezers to stable electricity is limited. To address this issue, we explore the possibility of using anhydrobiosis, the ability of organisms such as tardigrades to enter a reversible state of suspended animation brought on by extreme drying, as a jumping off point in the development of dry storage technology that would allow biologics to be kept in a desiccated state under not only ambient but elevated temperatures. Here we examine the ability of different protein and sugar-based mediators of anhydrobiosis derived from tardigrades and other anhydrobiotic organisms to stabilize Human Blood Clotting Factor VIII under repeated dehydration/rehydration cycles, thermal stress, and long-term dry storage conditions. We find that while both protein and sugar-based protectants can stabilize the biologic pharmaceutical Human Blood Clotting Factor VIII under all these conditions, protein-based mediators offer more accessible avenues for engineering and thus tuning of protective function. Using classic protein engineering approaches, we fine tune the biophysical properties of a protein-based mediator of anhydrobiosis derived from a tardigrade, CAHS D. Modulating the ability of CAHS D to form hydrogels make the protein better or worse at providing protection to Human Blood Clotting Factor VIII under different conditions. This study demonstrates the effectiveness of tardigrade CAHS proteins and other mediators of desiccation tolerance at preserving the function of a biologic without the need for the cold-chain. In addition, our study demonstrates that engineering approaches can tune natural products to serve specific protective functions, such as coping with desiccation cycling versus thermal stress. Ultimately, these findings provide a proof of principle that our reliance on the cold-chain to stabilize life-saving pharmaceuticals can be broken using natural and engineered mediators of desiccation tolerance.