Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease (3CLpro) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and is a promising target for the development of drugs against coronaviral infection. We designed and synthesized four nitrile-based peptidomimetic inhibitors with different N-terminal protective groups and different peptide length, and examined their inhibitory effect on the in-vitro enzymatic activity of 3CL pro of severe-acute-respiratory-syndrome-coronavirus. The IC 50 values of the inhibitors were in the range of 4.6-49 ฮผM, demonstrating that the nitrile warhead can effectively inactivate the 3CL pro autocleavage process. The best inhibitor, Cbz-AVLQ-CN with an N-terminal carbobenzyloxy group, was โˆผ10x more potent than the other inhibitors tested. Crystal structures of the enzyme-inhibitor complexes showed that the nitrile warhead inhibits 3CLpro by forming a covalent bond with the catalytic Cys145 residue, while the AVLQ peptide forms a number of favourable interactions with the S1-S4 substrate-binding pockets. We have further showed that the peptidomimetic inhibitor, Cbz-AVLQ-CN, has broad-spectrum inhibition against 3CLpro from human coronavirus strains 229E, NL63, OC43, HKU1, and infectious bronchitis virus, with IC 50 values ranging from 1.3 to 3.7 ฮผM, but no detectable inhibition against caspase-3. In summary, we have shown that the nitrile-based peptidomimetic inhibitors are effective against 3CLpro, and they inhibit 3CLpro from a broad range of coronaviruses. Our results provide further insights into the future design of drugs that could serve as a first line defence against coronaviral infection.ยฉ 2012 Elsevier Masson SAS.