Friday, November 22, 2024
11/22/2024
Reagents to Chemically Tag Specific Coronavirus Spike Proteins Firebird Biomolecular Sciences, LLC. Steven A. Benner Bharat Gawande Abstract While vaccines and antivirals may be long term solutions to the current coronavirus pandemic, it is now widely appreciated that the pandemic can be managed before these emerge by rapid, point-of-sampling "public space entry tests" (PSETs), given appropriate FDA regulatory relief. PSETs will be even more important if a good vaccine never emerges, a possibility given experience with other coronaviruses. In the 2003 SARS crisis, we delivered a "best in class" PCR kit for SARS that targeted coronaviral RNA. This was possible due to our first generation nucleic acid innovations, which were also used in respiratory pathogen panels and cystic fibrosis tests, inter alia. Luminex acquired EraGen in 2011 for $34 million. Since then, the PI generated 2nd and 3rd generation innovations that supported tests for MERS, arboviruses, norovirus and, this year, CoV-19 itself. These include a PSET that gives 10 minute results using proprietary displaceable probe loop amplification. Intrinsic chemistry suggests, however, this is the fastest any RNA-targeted test can be. Here, we will use our 3rd-generation DNA innovations not to target coronaviral RNA, but the coronavirus itself. Firebird will create reagents (AEGISZymes) from an artificially expanded genetic information system (AEGIS) that catalyze covalent acylation of lysines on the surface of ~300 spike proteins per virus. Each acylation will generate ~300 redox active moieties on an electrode surface per virion. The test is conceptually advanced over antibody tests, as it uses stable covalent bond formation, rather than non-covalent antibody: antigen interactions, and exploits electrochemical detection rather than a lateral flow. Our Phase 2 partner, MightyGate, has shown with standard aptamers detection in less than a minute with such architectures. With >100x sensitivity over Abbott BinaxNOWTM, the assay should detect virus in any contagious individual at entrances to arenas, schools, and other spaces as fast has handbags and are checked. Specifically, we will: Aim 1. Under IRB 2020001, we will use AEGIS-LIVE to evolve AEGISZymes in saliva that acylate lysines in spike proteins and peptide loops of CoV-19, SARS, and MERS. Aim 2. Metric the AEGISZymes that emerge, measuring their binding affinities, acylation rates, and specificity among the 3 homologous surface loops. The metrics are: M1. Acylation with kcat ≈ 1 sec-1. This should be readily achieved based on pmolar affinities that are now routine with AEGIS-LIVE; similar rate constants are now routine in analogous catalysts. M2. Specificity with kcat/Kdiss ratios >103, to ensure that CoV-19 is distinguished from other coronaviruses. If the metrics are met, MightyGate has committed itself to participate in Phase 2 work that will incorporate AEGISZymes onto its kiosk-style instrument. The kiosk has been proven with standard aptamers to give electrochemical readouts in less than 1 minute. Firebird's AEGISZymes will immediately improve the sensitivity of that kiosk, and immediate commercial value. However, our approach is general to any virus target.
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