Small-Molecule Thioesters as SARS-CoV-2 Main Protease Inhibitors: Enzyme Inhibition, Structure-Activity Relationships, Antiviral Activity, and X-ray Structure Determination.
Pillaiyar T, Flury P, Krüger N, Su H, Schäkel L, Barbosa Da Silva E, Eppler O, Kronenberger T, Nie T, Luedtke S, Rocha C, Sylvester K, Petry MRI, McKerrow JH, Poso A, Pöhlmann S, Gütschow M, O'Donoghue AJ, Xu Y, Müller CE, Laufer SA.
The main protease (M<sup>pro</sup>, 3CL<sup>pro</sup>) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure-activity relationships of novel small-molecule thioesters as SARS-CoV-2 M<sup>pro</sup> inhibitors. Compounds <b>3w</b> and <b>3x</b> exhibited excellent SARS-CoV-2 M<sup>pro</sup> inhibition with <i>k</i><sub>inac</sub>/<i>K</i><sub>i</sub> of 58,700 M<sup>-1</sup> s<sup>-1</sup> (<i>K<sub>i</sub></i> = 0.0141 μM) and 27,200 M<sup>-1</sup> s<sup>-1</sup> (<i>K</i><sub>i</sub> = 0.0332 μM), respectively. In Calu-3 and Vero76 cells, compounds <b>3h</b>, <b>3i, 3l</b>, <b>3r</b>, <b>3v</b>, <b>3w</b>, and <b>3x</b> displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of <b>3w</b> and <b>3af</b> with SARS-CoV-2 M<sup>pro</sup> was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the M<sup>pro</sup> of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.