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. 2020 Apr 24;368(6489):409-412.
doi: 10.1126/science.abb3405. Epub 2020 Mar 20.

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

Linlin Zhang  1   2 , Daizong Lin  1   3 , Xinyuanyuan Sun  1   2 , Ute Curth  4 , Christian Drosten  5 , Lucie Sauerhering  6   7 , Stephan Becker  6   7 , Katharina Rox  8   9 , Rolf Hilgenfeld  10   2
Affiliations

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors

Linlin Zhang et al. Science. .

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a global health emergency. An attractive drug target among coronaviruses is the main protease (Mpro, also called 3CLpro) because of its essential role in processing the polyproteins that are translated from the viral RNA. We report the x-ray structures of the unliganded SARS-CoV-2 Mpro and its complex with an α-ketoamide inhibitor. This was derived from a previously designed inhibitor but with the P3-P2 amide bond incorporated into a pyridone ring to enhance the half-life of the compound in plasma. On the basis of the unliganded structure, we developed the lead compound into a potent inhibitor of the SARS-CoV-2 Mpro The pharmacokinetic characterization of the optimized inhibitor reveals a pronounced lung tropism and suitability for administration by the inhalative route.

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Figures

Fig. 1
Fig. 1. Chemical structures of α-ketoamide inhibitors 11r, 13a, 13b, and 14b.
Colored ovals and circles highlight the modifications from one development step to the next (see text).
Fig. 2
Fig. 2. Three-dimensional structure of SARS-CoV-2 Mpro in two different views.
One protomer of the dimer is shown in light blue, the other one in orange. Domains are labeled by Roman numerals. Amino acid residues of the catalytic site are indicated as yellow spheres for Cys145 and blue spheres for His41. Asterisks mark residues from protomer B (orange). Black spheres indicate the positions of Ala285 for each of the two domains III (see text). Chain termini are labeled N and C for molecule A (light blue) and N* and C* for molecule B (orange).
Fig. 3
Fig. 3. Compound 13b in the substrate-binding cleft located between domains I and II of the Mpro in the monoclinic crystal form (space group C2).
Fobs – Fcalc density is shown for the inhibitor (contouring level 3σ). Carbon atoms of the inhibitor are magenta, except in the pyridone ring, which is black; oxygen atoms are red, nitrogens blue, and sulfur yellow. Light blue symbols Sn (n = 1, 2, 3…) indicate the canonical binding pockets for moieties Pn (n = 1, 2, 3…) (red symbols) of the peptidomimetic inhibitor. Hydrogen bonds are indicated by dashed red lines. Note the interaction between Ser1*, the N-terminal residue of molecule B, and Glu166 of molecule A, which is essential for keeping the S1 pocket in the correct shape and the enzyme in the active conformation. Inset: Thiohemiketal formed by the nucleophilic attack of the catalytic cysteine onto the α-carbon of the inhibitor. The stereochemistry of the α-carbon is S. Fobs − Fcalc density (contoured at 3σ) is shown in blue. See fig. S9 for more details.
Fig. 4
Fig. 4. Compound 13b inhibits SARS-CoV-2 replication in human Calu-3 lung cells.
(A) Calu-3 cells were infected with SARS-CoV-2 using a multiplicity of infection (MOI) of 0.05. Varying amounts (5, 10, 20, or 40 μM) of 13b (blue bars) or 14b (orange bars) were added. DMSO was used as vehicle control (black bar). Total RNA was isolated from cell lysates, and viral RNA content was analyzed by quantitative polymerase chain reaction. Data are means ± SD of two biological experiments with two technical replicates each. (B) For the estimation of the EC50 value of compound 13b against SARS-CoV-2, a dose-response curve was prepared (GraphPad).
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