ABSTRACT
The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).
Subject(s)
Coronavirus 3C Proteases/chemistry , Models, Molecular , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/genetics , Crystallization , Crystallography, X-Ray , HumansABSTRACT
Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing;therefore, confirmation of in silico drug screening studies have been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with nine drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.
ABSTRACT
An improved analysis for single-particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the Atomic, Molecular and Optical Science instrument at the Linac Coherent Light Source as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from half of the detector and a small fraction of single hits. The general SPI analysis workflow was upgraded with the expectation-maximization based classification of diffraction patterns and mode decomposition on the final virus-structure determination step. The presented processing pipeline allowed us to determine the 3D structure of bacteriophage PR772 without symmetry constraints with a spatial resolution of 6.9â nm. The obtained resolution was limited by the scattering intensity during the experiment and the relatively small number of single hits.
ABSTRACT
Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, and virus neutralization assay using xCELLigence MP real-time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of montelukast both on the main protease enzyme inhibition and virus entry into the host cell (spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with montelukast for 20 h on the infected cells. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and, if its effect is proved in clinical phase studies, it should be used against coronavirus disease 2019 (COVID-19).
Subject(s)
Acetates/pharmacology , Angiotensin-Converting Enzyme 2/metabolism , Cyclopropanes/pharmacology , Quinolines/pharmacology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Sulfides/pharmacology , A549 Cells , Acetates/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Animals , Cell Survival/drug effects , Chlorocebus aethiops , Cyclopropanes/chemistry , Drug Repositioning , HEK293 Cells , Humans , Models, Molecular , Molecular Docking Simulation , Molecular Structure , Neutralization Tests , Protein Conformation , Quinolines/chemistry , SARS-CoV-2/drug effects , Serine Endopeptidases/chemistry , Sulfides/chemistry , Vero Cells , Virus Internalization/drug effectsABSTRACT
The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.
Subject(s)
COVID-19 Drug Treatment , Coronavirus 3C Proteases/chemistry , Drug Design , Drug Repositioning , SARS-CoV-2 , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Dimerization , Molecular Conformation , Molecular Docking Simulation , Principal Component Analysis , Protein Conformation , Recombinant Proteins/chemistry , TemperatureABSTRACT
OBJECTIVE: To compare the effectiveness of different mask types in limiting the dispersal of coughed air. METHOD: The Schlieren method with a single curved mirror was used in this study. Coughed air has a slightly higher temperature than ambient air, which generates a refractive index gradient. A curved mirror with a radius of curvature of 10 m and a diameter of 60 cm was used. The spread of the cough wavefront was investigated among five subjects wearing: (1) no mask; (2) a single surgical mask; (3) a double surgical mask; (4) a cloth mask; (5) a valveless N95 mask; and (6) a valved N95 mask. RESULTS: All mask types reduced the size of the contaminated region significantly. The percentage reduction in the cross-sectional area of the contaminated region for the same mask types on different subjects revealed by normalized data suggests that the fit of a mask plays an important role. CONCLUSIONS: No significant difference in the spread of coughed air was found between the use of a single surgical mask or a double surgical mask. Cloth masks may be effective, depending on the quality of the cloth. Valved N95 masks exclusively protect the user. The fit of a mask is an important factor to minimize the contaminated region.