Targeting the Main Protease of SARS-CoV-2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors

Abstract

The main protease of SARS-CoV-2 (M^pro ), the causative agent of COVID-19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed M^pro . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in-depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of M^pro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M^pro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k_inac /K_i =37 500 m^-1 s^-1 , K_i =24.0 nm) and pyridyl ester 17 (k_inac /K_i =29 100 m^-1 s^-1 , K_i =10.0 nm), promising drug candidates for further development have been discovered.

Keywords: SARS-CoV-2 main protease; azapeptide nitriles; fluorogenic substrates; high throughput screening; pyridinyl 1H-indole-carboxylates.

Figure 1

Conversion of fluorogenic substrates by His‐tagged SARS‐CoV‐2 main protease (M^pro), lysate obtained from human embryonic kidney (HEK) cells, HEK cell lysate spiked with M^pro, human cathepsin L (cat L), human cathepsin B (cat B), bovine trypsin, and in the absence of enzymes (FU, fluorescence units). The product formation was monitored for 10 min at 37 °C with an initial substrate concentration of 50 μm in all cases. Each enzyme was used at the same concentration in all respective experiments.

Figure 2

Selected, confirmed SARS‐CoV‐2 M^pro inhibitors identified by HTS. Compounds 1 and 2 were identified in the library of in‐house chloroalkyl derivatives (entry 1 in Table 1), compound 9 in the Pathogen Box library (entry 6 in Table 1), and compound 10 in the virtually generated library (entry 8 in Table 1).

Scheme 1

Synthesis of the azapeptide nitriles 3–8.

Figure 3

M^pro‐catalyzed hydrolysis of 50 μm (=1.03×K _m) of Boc‐Abu‐Tle‐Leu‐Gln‐AMC in the absence (black) or presence of increasing concentrations of inhibitor 4 (from top to bottom: 0.6 μm, 1.2 μm, 1.8 μm, 2.4 μm, 3.0 μm). Inset: A plot of first‐order rate constants versus the inhibitor concentrations and non‐linear regression gave a k _inac/K _i value of 1150 m ⁻¹ s⁻¹.

Figure 4

Predicted binding pose of the azapeptide nitrile 8 (orange) in the active site of SARS‐CoV‐2 M^pro with relevant amino acids (green). The model was obtained based on a reported enzyme–inhibitor complex (PDB ID: 6LU7). The covalent bond between the sulfur of the active site Cys145 and the cyano carbon of the warhead generated an isothiosemicarbazide‐type enzyme‐inhibitor adduct. The P1 glutamine side chain resides in the S1 pocket. The aromatic ring of the P2 phenylalanine is positioned in the hydrophobic S2 pocket (His41, Met49, Met165) in proximity to the Gln189 side chain. The P3 tert‐leucine is oriented towards the solvent. The P4 aminobutyric acid and the N‐terminal, Cbz‐capped part of the inhibitor are oriented towards the S3/S4 region. Hydrogen bond interactions are shown in yellow dotted lines, for details see Figure S15. The binding mode of 8 is consonant with X‐ray crystal structures of other peptidic inhibitors in complex with M^pro.[ ⁴ , ¹⁰ , ¹⁴ ]