Recently, Dai W et al. published a study on Science,1 in which the two lead compounds 11a and 11b were designed and synthesized based on the features of a key enzyme Mpro of SARS-CoV-2 (Fig. 1). In particular, compound 11a is a potential drug candidate for coronavirus disease 2019 (COVID-19) with strong anti-SARS-CoV-2 infection activity, good pharmacokinetics characteristics, and low toxicity.

Fig. 1
figure 1

Design and discovery of drug candidates targeting Mpro against COVID-19

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Since December 2019, the outbreak of COVID-19 caused by SARS-CoV-22 has caused a serious global public health emergency. So far, the global epidemic is still in the outbreak stage, and the number of new confirmed cases every day has exceeded 100,000 for several days. At present, the main drugs used clinically include interferon-alpha, lopinavir/ritonavir, ribavirin, arbidol, etc. However, these drugs are facing huge controversy due to the large side effects or the lack of clinical verifications of the therapeutic effects.3 Therefore, clarifying the origin and pathogenesis of pneumonia and decoding the key targets against SARS-CoV-2 are the cornerstone to design and discover safe and effective antivirus drugs.

Previously, the crystal structure of the Mpro protein of SARS-CoV-2 was resolved in complex with an effective inhibitor N3, which was completed by the same group, laying an important foundation for this research.4 Mpro plays an irreplaceable role in the life cycle of the virus in the light of it could release a series of functional peptides by hydrolyzing the two proteins necessary for replication and transcription, pp1a and pp1ab.5 Notably, conservatism in coronavirus and lack of homolog in human also enhance the application of Mpro in antiviral drug design.5

The active sites of Mpro, which usually include S1’, S1, S2, and S4, are highly conserved in all coronaviruses. Accordingly, the rational design can be applied in the discovery of novel SARS-COV-2 inhibitors. Because SARS-CoV Mpro inhibitors usually have (S)-γ-lactam ring to occupy the S1 site, (S)-γ-lactam ring is introduced to interact with the S1 site. Furthermore, an aldehyde group is selected to form a covalent bond with the thiol of the Cys145 residue. The S2 region is capable to accommodate a large group, so a cyclohexyl or 3-fluorophenyl group with large spatial volume is introduced at the corresponding position. Afterwards, an indole group is placed in the S4 region to form intermolecular hydrogen bonds so as to improve drug-like properties. Finally, a synthetic route is developed to afford the lead compounds 11a and 11b.

Next, activity, pharmacokinetics properties and toxicity of 11a and 11b were evaluated. In a fluorescence resonance energy transfer (FRET)-based cleavage assay, both compounds 11a and 11b exhibited strong inhibitory activities, with the potency of IC50 = 0.053 ± 0.005 μM and 0.040 ± 0.002 μM, respectively. A further assay revealed that 11a and 11b also displayed good antiviral activities in Vero E6 cells (EC50 of 0.53 ± 0.01 μM and 0.72 ± 0.09 μM, respectively) with low cytotoxicity (CC50 is greater than 100 μM). Finally, pharmacokinetics and toxicity studies showed that 11a had better pharmacokinetics properties and no obvious toxicity in vivo.

To elucidate the inhibitory mechanism of 11a and 11b, the crystal structure of Mpro in complex with 11a (PDB code: 6LZE) and 11b (PDB code: 6M0K) was resolved, respectively, at a resolution of 1.5 Å. The structures show that 11a and 11b have a similar inhibitory binding mode. The aldehyde group forms a covalent force with Cys145 in the S1’ region, whereas (S)-γ-lactam ring and indole group form intermolecular hydrogen bonds with S1 and S4 regions, respectively. A subtle difference between 11a and 11b in the S2 site was most probably due to the stereostructure and electronegativity difference between cyclohexyl and 3-fluorophenyl groups. Notably, multiple water molecules also participated in the binding of protein—ligand complexes via hydrogen bonds. Overall, the binding modes of 11a and 11b with the Mpro are consistent with those of compounds N1, N3, and N9 that are reported as wide spectrum inhibitors targeting coronavirus Mpro.

Taken together, with no vaccine or proven effective drug against SARS-COV-2, Dai W et al. designed and synthesized effective inhibitors based on the structure of the specific target Mpro active site. Compound 11a is expected to become a promising clinical drug candidate. This research provides an effective strategy to design and discover anti-SARS-CoV-2 and even anti-coronavirus drugs targeting Mpro. Before the emergence of specific drugs, the comprehensive application of drug design, medicinal chemistry, multidisciplinary technologies such as structural biology will help accelerate the development of anti-COVID-19 drugs.