# Structure‐Based Development of Ultra‐Broad‐Spectrum 3C‐Like Protease Inhibitors

## TL;DR

Researchers developed a new broad-spectrum antiviral drug that effectively inhibits coronavirus proteases across multiple species, including those resistant to existing treatments.

## Contribution

Structure-based optimization led to compound 8, an ultra-broad-spectrum 3CLpro inhibitor effective against 32 coronavirus proteases.

## Key findings

- Compound 8 inhibits 32 3CLpros across all coronavirus subgenera with IC50s ranging from 19–146 nm.
- Compound 8 effectively inhibits nirmatrelvir-resistant 3CLpro mutants and shows antiviral efficacy in cells.

## Abstract

Recurrence of coronavirus outbreaks and zoonotic origins of human coronaviruses underscore the importance of developing pan‐coronavirus antivirals. The highly conserved 3C‐like protease (3CLpro) in coronaviruses, together with the well‐established druggability, makes it an ideal target for broad‐spectrum antiviral therapeutics. Here, the inhibitory activity of approved 3CLpro inhibitors, including nirmatrelvir, ensitrelvir, and simnotrelvir, against fifteen 3CLpros is first reported by enzymatic assays. Despite their potent inhibition toward 3CLpros of β‐CoVs, these inhibitors show reduced potency against 3CLpros from the other three genera, particularly against two newly identified human coronaviruses (α‐CCoV‐HuPn‐2018 and δ‐PDCoV). In this context, continued efforts in structure‐based optimization of nirmatrelvir lead to the identification of compound 8 that potently inhibits a panel of 32 3CLpros across all subgenera (IC50s: 19–146 nm), with an IC50 value of 61 and 81 nm against α‐CCoV‐HuPn‐2018 and δ‐PDCoV 3CLpros, respectively. Moreover, it effectively inhibits nirmatrelvir‐resistant 3CLpro mutants and demonstrates broad‐spectrum antiviral efficacy in cells. These findings suggest an important rule that a small, non‐cyclic P2 segment and a P4 segment with a suitable size are preferred by the design of ultra‐broad‐spectrum 3CLpro inhibitors, and provide a proof‐of‐concept guide for developing broad‐spectrum antivirals as potential pan‐CoV therapeutics.

This study provides an in‐depth analysis of the substrate binding pocket of 3CLpros across all coronavirus species using bioinformatics and structural insights, revealing the critical impact of S2/S4 subsite diversity on the broad‐spectrum activity of approved therapeutics. Structure‐based optimization of nirmatrelvir led to the identification of compound 8, an ultra‐broad‐spectrum 3CLpro inhibitor that potently inhibits a panel of 32 3CLpros across all subgenera.