McLellan, J. | bioRxiv Science

bioRxiv2024

Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody

SUMMARY/ABSTRACTThree coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

molecular biology↗

bioRxiv2022

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

Protein S-acylation is a reversible post-translational modification that modulates the localisation and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC domain-containing proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyse S-acylation in a two-step process involving "auto-acylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. In this study, we developed and implemented a novel FRET-based high throughput assay for the discovery of compounds that interfere with auto-acylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. A screen of >350,000 compounds identified two related tetrazole containing compounds (TTZ-1 and -2) that inhibited both zDHHC2 auto-acylation and substrate S-acylation in cell-free systems. Furthermore, these compounds were also active in HEK293T cells, where they inhibited substrate S-acylation mediated by different zDHHC enzymes, with some apparent isoform selectivity. Resynthesis of the hit compounds confirmed their activity, providing sufficient quantities of material for further investigations. The assays developed herein provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating the cellular activities of S-acylation and zDHHC enzymes.

cell biology↗