A cyclic peptide inhibitor of the SARS-CoV-2 main protease

Challenges of short substrate analogues as SARS-CoV-2 main protease inhibitors

“…Previously successful strategies, including cyclization and the use of unnatural amino acids, did not help to overcome these challenges. The lack of affinity of cyclic substrate analogues described here and previously by Kreutzer et al 40 is particularly noteworthy as computational work in both studies predict binding to the active site with poses very similar to ligands observed in crystal structures. Given that non-covalent inhibitors appear to be unusually ineffective against M pro , it is not surprising that the first generation of SARS-CoV-2 M pro inhibitors discovered at the beginning of the COVID-19 pandemic in 2020 were substrate derived covalent inhibitors bearing α -ketoamide, aldehyde, and Michael acceptor reactive groups.…”

“…We also tested compounds 12 – 14 , where the P 1 glutamine was replaced by glutamate or asparagine. Compound 15 was inspired by the low-affinity inhibitor reported by Kreutzer et al 40 Remarkably, none of the peptides 1 – 20 displayed IC 50 values below 100 µM in our well-validated M pro activity assay ( Table 1 ).…”

“… 42 , 43 , 44 It is possible that our specific cyclization linkers are the cause of this observation; however, Kreutzer et al, who used an unrelated cyclization chemistry, equally failed to produce cyclic substrates with sufficient affinity. 40 It is notable that peptide 16 , which is a long (13 amino acids) cyclic analogue of the assay substrate, did not display inhibition at 100 µM. Only its linear analogue 21 showed moderate inhibition with an IC 50 value of 71 ± 5 µM ( Figure 3 a ).…”

“… 39 Very recently, a cyclic peptide has been reported, which binds to SARS-CoV-2 M pro without forming a covalent bond. 40 With an IC 50 value of about 160 µM, however, the activity of this compound is many orders of magnitudes below those of substrate-based analogues with warheads.…”

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“…Progress in solid-phase synthesis has stimulated great interest in protein design and synthesis of structurally diverse peptide libraries that can be screened against biological targets. Kreutzer et al designed and synthesized a cyclic peptide containing a [4-(2aminoethyl)phenyl]-acetic acid (AEPA) linker, named UCI-1, by FMOC-based solid synthesis [31]. Treatment of the Fmoc-AEPA-OH intermediate with 2-chlorotrityl chloride resin followed by linear peptide synthesis and resin cleavage provided the desired cyclic peptide used by authors for in vitro and in silico studies.…”