Synthesis and Stereochemical Determination of the Peptide Antibiotic Novo29.

This paper describes the synthesis and stereochemical determination of Novo29 (clovibactin), a new peptide antibiotic that is related to teixobactin and is active against Gram-positive bacteria. Novo29 is an eight-residue depsipeptide that contains the noncanonical amino acid hydroxyasparagine of hitherto undetermined stereochemistry in a macrolactone ring. The amino acid building blocks Fmoc-(2R,3R)-hydroxyasparagine-OH and Fmoc-(2R,3S)-hydroxyasparagine-OH were synthesized from (R,R)- and (S,S)-diethyl tartrate. Novo29 and epi-Novo29 were then prepared by solid-phase peptide synthesis using these building blocks. Correlation with an authentic sample of Novo29 through 1H NMR spectroscopy, LC-MS, and in vitro antibiotic activity established that Novo29 contains (2R,3R)-hydroxyasparagine. X-ray crystallography reveals that epi-Novo29 adopts an amphiphilic conformation, with a hydrophobic surface and a hydrophilic surface. Four sets of epi-Novo29 molecules pack in the crystal lattice to form a hydrophobic core. The macrolactone ring adopts a conformation in which the main-chain amide NH groups converge to create a cavity, which binds ordered water and acetate anion. The amphiphilic conformation of epi-Novo29 is reminiscent of the amphiphilic conformation adopted by the related antibiotic teixobactin and its derivatives, which contains a hydrophobic surface that interacts with the lipids of the bacterial cell membrane and a hydrophilic surface that interacts with the aqueous environment. Molecular modeling suggests that Novo29 can adopt an amphiphilic conformation similar to teixobactin, suggesting that Novo29 may interact with bacteria in a similar fashion to teixobactin.

A cyclic peptide inhibitor of the SARS-CoV-2 main protease.

This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (Mpro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of Mpro. The cyclic peptide contains a [4-(2-aminoethyl)phenyl]-acetic acid (AEPA) linker that is designed to enforce a conformation that mimics a peptide substrate of Mpro. In vitro evaluation of the cyclic peptide inhibitor reveals that the inhibitor exhibits modest activity against Mpro and does not appear to be cleaved by the enzyme. Conformational searching predicts that the cyclic peptide inhibitor is fairly rigid, adopting a favorable conformation for binding to the active site of Mpro. Computational docking to the SARS-CoV-2 Mpro suggests that the cyclic peptide inhibitor can bind the active site of Mpro in the predicted manner. Molecular dynamics simulations provide further insights into how the cyclic peptide inhibitor may bind the active site of Mpro. Although the activity of the cyclic peptide inhibitor is modest, its design and study lays the groundwork for the development of additional cyclic peptide inhibitors against Mpro with improved activities.

Structure-based drug design of an inhibitor of the SARS-CoV-2 (COVID-19) main protease using free software: A tutorial for students and scientists.

This paper describes the structure-based design of a preliminary drug candidate against COVID-19 using free software and publicly available X-ray crystallographic structures. The goal of this tutorial is to disseminate skills in structure-based drug design and to allow others to unleash their own creativity to design new drugs to fight the current pandemic. The tutorial begins with the X-ray crystallographic structure of the main protease (Mpro) of the SARS coronavirus (SARS-CoV) bound to a peptide substrate and then uses the UCSF Chimera software to modify the substrate to create a cyclic peptide inhibitor within the Mpro active site. Finally, the tutorial uses the molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV Mpro and the highly homologous SARS-CoV-2 Mpro. The supporting information provides an illustrated step-by-step protocol, as well as a video showing the inhibitor design process, to help readers design their own drug candidates for COVID-19 and the coronaviruses that will cause future pandemics. An accompanying preprint in bioRxiv [https://doi.org/10.1101/2020.08.03.234872] describes the synthesis of the cyclic peptide and the experimental validation as an inhibitor of SARS-CoV-2 Mpro.

Structure-Based Drug Design of an Inhibitor of the SARS-CoV-2 (COVID-19) Main Protease Using Free Software: A Tutorial for Students and Scientists.

This paper describes the structure-based design of a preliminary drug candidate against COVID-19 using free software and publicly available X-ray crystallographic structures. The goal of this tutorial is to disseminate skills in structure-based drug design and to allow others to unleash their own creativity to design new drugs to fight the current pandemic. The tutorial begins with the X-ray crystallographic structure of the main protease (Mpro) of the SARS coronavirus (SARS-CoV) bound to a peptide substrate and then uses the UCSF Chimera software to modify the substrate to create a cyclic peptide inhibitor within the Mpro active site. Finally, the tutorial uses the molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV Mpro and the highly homologous SARS-CoV-2 Mpro. The supporting information (supplementary material) provides an illustrated step-by-step guide for the inhibitor design, to help readers design their own drug candidates for COVID-19 and the coronaviruses that will cause future pandemics. An accompanying preprint in bioRxiv [https://doi.org/10.1101/2020.08.03.234872] describes the synthesis of the cyclic peptide and the experimental validation as an inhibitor of SARS-CoV-2 Mpro.

Glial scars are permeable to the neurotoxic environment of chronic stroke infarcts.

Following stroke, the damaged tissue undergoes liquefactive necrosis, a stage of infarct resolution that lasts for months although the exact length of time is currently unknown. One method of repair involves reactive astrocytes and microglia forming a glial scar to compartmentalize the area of liquefactive necrosis from the rest of the brain. The formation of the glial scar is a critical component of the healing response to stroke, as well as other central nervous system (CNS) injuries. The goal of this study was to evaluate the toxicity of the extracellular fluid present in areas of liquefactive necrosis and determine how effectively it is segregated from the remainder of the brain. To accomplish this goal, we used a mouse model of stroke in conjunction with an extracellular fluid toxicity assay, fluorescent and electron microscopy, immunostaining, tracer injections into the infarct, and multiplex immunoassays. We confirmed that the extracellular fluid present in areas of liquefactive necrosis following stroke is toxic to primary cortical and hippocampal neurons for at least 7 weeks following stroke, and discovered that although glial scars are robust physical and endocytic barriers, they are nevertheless permeable. We found that molecules present in the area of liquefactive necrosis can leak across the glial scar and are removed by a combination of paravascular clearance and microglial endocytosis in the adjacent tissue. Despite these mechanisms, there is delayed atrophy, cytotoxic edema, and neuron loss in regions adjacent to the infarct for weeks following stroke. These findings suggest that one mechanism of neurodegeneration following stroke is the failure of glial scars to impermeably segregate areas of liquefactive necrosis from surviving brain tissue.