In a collaboration between researchers at the Universities of Oxford and Johannesburg; Instruct-ERIC; the Ineos Oxford Institute for Antimicrobial Research; Diamond Light Source; Exscientia, and Scripps Research, Martin Redhead (Head of Molecular Pharmacology at Exscientia) and his colleagues enlisted the help of the highly experienced peptide synthesis team at Cambridge Research Biochemicals to synthesise custom FRET peptide substrates and research-grade prodrug compounds to test as potential coronavirus treatments, including COVID-19. The team were able to identify two non-antiviral therapeutic agents that target essential proteases, Mpro and PLpro.
Throughout this pandemic, it has become increasingly clear that effective medicines to treat coronavirus infections are urgently needed. Not just to treat COVID-19 but also to treat future coronavirus outbreaks. To achieve this, targeting proteins highly conserved between coronaviruses is a key approach. Martin Redhead and his colleagues, therefore, chose to target the main protease (Mpro) and the papain-like protease (PLpro), both of which are essential during the coronavirus replication cycle and are conserved between known coronaviruses.
The peptide team at CRB synthesised two fluorescence resonance energy transfer (FRET) substrate peptides for use in screening assays for potential Mpro or PLpro inhibitors. Both of these COVID-19 peptides contain fluorescent dyes as well as fluorescent quenchers at either terminal:
Mpro substrate, [5-TAMRA]-AVLQSGFR-[Lys(BHQ-2)]-K-amide
PLpro substrate, [5-TAMRA]-VLRLRGG-[Lys(BHQ-2)]
*where 5-TAMRA is 5-carboxytetramethylrhodamine and BHQ-2 is black hole quencher 2.
When these peptides remain intact, the proximity of the quencher to the fluorophore means that no fluorescence is detected. However, upon cleavage of the substrate peptide by its corresponding protease, the fluorophore and quencher separate, and fluorescence can be detected.
Using these two peptides, the research team could screen the drug repurposing library ReFRAME, containing 12,000 therapeutic agents in an extensive screening programme and identify inhibitors to each of these two proteases.
These two non-antiviral therapeutic agents were a caspase-1 inhibitor (compound 4), a potent, non-reversible inhibitor of Mpro and Tarloxotinib (compound 1), a sub-micromolar inhibitor of PLpro. Both compounds have undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation. Compound 5, in contrast to its parent (compound 4) has limited potency for Mpro. CRB also synthesised compounds 4 and 5 which were used in follow-up anti-viral assays.
Identification of SARS-CoV-2 protease inhibitors can provide structural starting points for the design of more potent antiviral anti-inflammatory drugs. The generation of custom peptides by CRB for researchers can continue to push the frontiers of COVID-19 therapy.
Redhead et al., (2021). Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19. Sci Rep. 11: 13208. PMID: 34168183.