Discovery of SARS-CoV-2 Nsp14 and Nsp16 Methyltransferase Inhibitors by High-Throughput Virtual Screening.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses mRNA capping to evade the human immune system. The cap formation is performed by the SARS-CoV-2 mRNA cap methyltransferases (MTases) nsp14 and nsp16, which are emerging targets for the development of broad-spectrum antiviral agents. Here, we report results from high-throughput virtual screening against these two enzymes. The docking of seven million commercially available drug-like compounds and S-adenosylmethionine (SAM) co-substrate analogues against both MTases resulted in 80 virtual screening hits (39 against nsp14 and 41 against nsp16), which were purchased and tested using an enzymatic homogeneous time-resolved fluorescent energy transfer (HTRF) assay. Nine compounds showed micromolar inhibition activity (IC50 < 200 µM). The selectivity of the identified inhibitors was evaluated by cross-checking their activity against human glycine N-methyltransferase. The majority of the compounds showed poor selectivity for a specific MTase, no cytotoxic effects, and rather poor cell permeability. Nevertheless, the identified compounds represent good starting points that have the potential to be developed into efficient viral MTase inhibitors.

Neutralization of MERS coronavirus through a scalable nanoparticle vaccine.

MERS-CoV continues to cause human outbreaks, so far in 27 countries worldwide following the first registered epidemic in Saudi Arabia in 2012. In this study, we produced a nanovaccine based on virus-like particles (VLPs). VLPs are safe vaccine platforms as they lack any replication-competent genetic material, and are used since many years against hepatitis B virus (HBV), hepatitis E virus (HEV) and human papilloma virus (HPV). In order to produce a vaccine that is readily scalable, we genetically fused the receptor-binding motif (RBM) of MERS-CoV spike protein into the surface of cucumber-mosaic virus VLPs. The employed CuMVTT-VLPs represent a new immunologically optimized vaccine platform incorporating a universal T cell epitope derived from tetanus toxin (TT). The resultant vaccine candidate (mCuMVTT-MERS) is a mosaic particle and consists of unmodified wild type monomers and genetically modified monomers displaying RBM, co-assembling within E. coli upon expression. mCuMVTT-MERS vaccine is self-adjuvanted with ssRNA, a TLR7/8 ligand which is spontaneously packaged during the bacterial expression process. The developed vaccine candidate induced high anti-RBD and anti-spike antibodies in a murine model, showing high binding avidity and an ability to completely neutralize MERS-CoV/EMC/2012 isolate, demonstrating the protective potential of the vaccine candidate for dromedaries and humans.

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.