Inhibition of glycogen synthase kinase-3-beta (GSK3β) blocks nucleocapsid phosphorylation and SARS-CoV-2 replication

GSK3β has been proposed to have an essential role in Coronaviridae infection. Screening of a targeted library of GSK3β inhibitors against SARS-CoV-2 and HCoV-229E resulted in identification of high proportion of active compounds with low toxicity to host cells. A select lead compound, T-1686568, showed dose-dependent activity against SARS-CoV-2 transcription, translation and viral particle release in multiple cell lines and primary organoids. A protein kinase substrate profiling assay combined with western blot analysis showed that SARS-CoV-2 nucleocapsid is phosphorylated by GSK3β on S180/S184, S190/S194 and T198 which have already been primed in the adjacent phospho-sites S188, T198 and S206 respectively. Inhibition by T-1686568 resulted in reduction of the S1 Spike protein levels, an accumulation of the Nucleocapsid (N) protein and maintenance of the non-structural (NSP2) level in infected Huh-7.5.1 cells, indicating that N phosphorylation might serve as a critical precursor for processing and release of mature viruses.

THE RECONSTRUCTION OF A DYNAMIC INVENTORY MODEL TOWARD SHARED HBIM LIBRARIES FOR VAULTED SYSTEMS

The complexity and variety of solutions in the domain of cultural heritage are the result of a heterogeneous network of social and historical factors generating them. Each individual architectural object, including not only notorious typologies but little-known construction systems such as brick vaults, are generated by a specific social and physical framework. The richness of vaulted systems stresses the need for an in-depth investigation supported by informative models connected in a single geo-spatial platform. The paper describes a methodological workflow starting from an abacus of vaults, based on different Levels of Detail and Information, to populate a geo-spatial database. Focusing on the acquisition of an open and shared database of historical elements, the definition of a HBIM targeted Library, integrating different types of data in one dynamic model, requires the management of different Levels of Detail and guidelines to establish a correct hierarchy of information. The proposed study can be considered a modern approach to the traditional practice of Répertoires, to set up a methodology that could be applied indifferently and widely, regardless of how the model is built. The diverse vaulted systems of Palazzo Magio Grasselli provide the possibility to build an inventory model as a result of multiple informative models coming from different geographical backgrounds to solve critical issues such as data loss and to enhance knowledge dissemination.

Performance of Targeted Library Preparation Solutions for SARS-CoV-2 Whole Genome Analysis

Single next-generation sequencing (NGS) proved to be an important tool for monitoring the SARS-CoV-2 outbreak at the global level Until today, thousands of SARS-CoV-2 genome sequences have been published at GISAID (Global Initiative on Sharing All Influenza Data) but only a portion are suitable for reliable variant analysis. Here we report on the comparison of three commercially available NGS library preparation kits. We discuss advantages and limitations from the perspective of required input sample quality and data quality for advanced SARS-CoV-2 genome analysis.

Targeting the water network in cyclin G associated kinase (GAK) with 4-anilino-quin(az)oline inhibitors

Water networks within kinase inhibitor design and more widely within drug discovery are generally poorly understood. The successful targeting of these networks prospectively has great promise for all facets of inhibitor design, including potency and selectivity on target. Here we describe the design and testing of a targeted library of 4-anilinoquinolines for use as inhibitors of cyclin G associated kinase (GAK). The GAK cellular target engagement assays, ATP binding site modelling and extensive water mapping provide a clear route to access potent inhibitors for GAK and beyond.

Making research visible: A library competition for graduate students

Graduate students have always been a core academic library user group. However, because undergraduates typically make up a larger percentage of the on-campus student body, the development of targeted library services and programming for graduate students has often taken a back seat. Recently, however, interest in academic library services for graduate students has increased, as demonstrated by conferences devoted to this topic and the formation of an ACRL interest group focused on this issue.

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

Systematic Engineering of a Protein Nanocage for High-Yield, Site-Specific Modification

<div> <p>Site-specific protein modification is a widely-used strategy to attach drugs, imaging agents, or other useful small molecules to protein carriers. N-terminal modification is particularly useful as a high-yielding, site-selective modification strategy that can be compatible with a wide array of proteins. However, this modification strategy is incompatible with proteins with buried or sterically-hindered N termini, such as virus-like particles like the well-studied MS2 bacteriophage coat protein. To assess VLPs with improved compatibility with these techniques, we generated a targeted library based on the MS2-derived protein cage with N-terminal proline residues followed by three variable positions. We subjected the library to assembly, heat, and chemical selections, and we identified variants that were modified in high yield with no reduction in thermostability. Positive charge adjacent to the native N terminus is surprisingly beneficial for successful extension, and over 50% of the highest performing variants contained positive charge at this position. Taken together, these studies described nonintuitive design rules governing N-terminal extensions and identified successful extensions with high modification potential.</p> </div>

A High-Throughput Enzyme-Coupled Assay for SAMHD1 dNTPase

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a recently discovered enzyme that plays a central role in nucleotide metabolism and innate immunity. SAMHD1 has deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase activity that depletes the dNTP substrates required for DNA synthesis in cells. The involvement of SAMHD1 in biological processes as varied as viral restriction, endogenous retroelement control, cancer, and modulation of anticancer/antiviral nucleoside drug efficacy makes it a valuable target for the development of small-molecule inhibitors. We report a high-throughput colorimetric assay for SAMHD1 dNTP hydrolase activity that takes advantage of Escherichia coli inorganic pyrophosphatase to convert PPPi to 3 Pi. The assay was validated by screening a library of 2653 clinically used compounds. Fifteen primary hits were obtained (0.57% hit rate); 80% of these were confirmed in a direct secondary assay for dNTP hydrolysis. The zinc salt of the antibiotic cephalosporin C was a potent inhibitor of SAMHD1 with an IC50 of 1.1 ± 0.1 µM, and this inhibition was largely attributable to the presence of zinc. The assay also screened a targeted library of nucleosides and their analogs, revealing that the antiviral drug acycloguanosine (acyclovir) is an inhibitor possessing excellent properties for future fragment-based drug development efforts.