Identification of a dual acting SARS-CoV-2 proteases inhibitor through in silico design and step-by-step biological characterization

Abstract

COVID-19 pandemic, starting from the latest 2019, and caused by SARS-CoV-2 pathogen, led to the hardest health-socio-economic disaster in the last century. Despite the tremendous scientific efforts, mainly focused on the development of vaccines, identification of potent and efficient anti-SARS-CoV-2 therapeutics still represents an unmet need. Remdesivir, an anti-Ebola drug selected from a repurposing campaign, is the only drug approved, so far, for the treatment of the infection. Nevertheless, WHO in later 2020 has recommended against its use in COVID-19. In the present paper, we describe a step-by-step in silico design of a small library of compounds as main protease (M^pro) inhibitors. All the molecules were screened by an enzymatic assay on M^pro and, then, cellular activity was evaluated using Vero cells viral infection model. The cellular screening disclosed compounds 29 and 34 as in-vitro SARS-CoV-2 replication inhibitors at non-toxic concentrations (0.32 < EC₅₀ < 5.98 μM). To rationalize these results, additional in-vitro assays were performed, focusing on papain like protease (PL^pro) and spike protein (SP) as potential targets for the synthesized molecules. This pharmacological workflow allowed the identification of compound 29, as a dual acting SARS-CoV-2 proteases inhibitor featuring micromolar inhibitory potency versus M^pro (IC₅₀ = 1.72 μM) and submicromolar potency versus PL^pro (IC₅₀ = 0.67 μM), and of compound 34 as a selective SP inhibitor (IC₅₀ = 3.26 μM).

Keywords: Biophysical assays; Cellular characterization; Enzymatic assays; In-silico design; SARS-CoV-2 proteases dual inhibitor.

Graphical abstract

Fig. 1

General structures of synthesized compounds.

Scheme 1

Synthesis of final compounds 7, 22–29. Reagents and conditions: a) HOBt (1.2 eq), HBTU (1.2 eq), DIPEA (2.4 eq), amine (1.2 eq), DCM, 12h, RT b) TFA/DCM (1/3, v/v), triisopropylsilane (0.25 eq), 30–120 min, RT c) HOBt (1.0 eq), HBTU (1.0 eq), DIPEA (2.0 eq), L-Boc-aa-OH (0.83 eq), DCM, 12h, RT d) RCOH (1.2 eq), MeOH, N₂ stream, RT, 12 h. Then, NaBH₄ (3.0 eq), RT, 3h e) TEA (1.2 eq), chloro acetylchloride (1.2 eq), THF, RT, 20 min.

Scheme 2

Synthesis of final compounds 34 and 35. Reagents and conditions: a) NaH (1.5 eq), alkyl iodide (1.5 eq), DMF, 0 °C to RT, overnight. b) Formaldehyde (2.0 eq), TFA (2.0 eq), amine (2.0 eq), DCM, 12h, RT. c) Aldehyde (1.2 eq), MeOH, N₂ stream, RT, 12 h. Then, NaBH₄ (3 eq), RT, 3h d) HOBt (1.2 eq), HBTU (1.2 eq), DIPEA (2.4 eq), L-Boc-Pra-OH (1.2 eq), DCM, 12h, RT.

Fig. 2

A) SARS-CoV-2 M^pro binding site; S1’, S1, S2, and S4 sites and Cys145 key residue are highlighted. B) Chemical structures of compounds A1 and B1, with specified the chemical moieties interacting with the S1’, S1, S2, and S4 M^pro sites.

Fig. 3

A) mtrp (colored by atom type: C green, O red, N blue, polar H light grey), B) 25 (colored by atom type: C yellow, O red, N blue, polar H light grey); C) 23 (colored by atom type: C purple, O red, N blue, polar H light grey); D) 22 (colored by atom type: C pink, O red, N blue, polar H light grey); E) 7 (colored by atom type: C aqua green, O red, N blue, polar H light grey); F) 24 (colored by atom type: C light green, O red, N blue, polar H light grey); G) 27 (colored by atom type: C light brown, O red, N blue, polar H light grey); H) 26 (colored by atom type: C light violet, O red, N blue, polar H light grey); I) 28 (colored by atom type: C violet, O red, N blue, polar H light grey); J) 29 (colored by atom type: C light yellow, O red, N blue, polar H light grey); K) 34 (colored by atom type: C light blue, O red, N blue, polar H light grey); L) 35 (colored by atom type: C dark grey, O red, N blue, polar H light grey) in docking with SARS-CoV-2 M^pro (transparent molecular surface colored in grey and secondary structure colored in orange; key residues are reported as sticks and colored by atom type: C grey, O red, N blue, S yellow, polar H light grey).

Fig. 4

A) non-covalent and B) covalent molecular docking complexes between 29 (colored by atom type: C light yellow, O red, N blue, polar H light grey) and SARS-CoV-2 PL^pro (transparent molecular surface colored in grey and secondary structure colored in green; key residues are reported as sticks and colored by atom type: C grey, O red, N blue, S yellow, polar H light grey).∖

Fig. 5

Molecular docking complex between 34 (colored by atom type: C light blue, O red, N blue, polar H light grey) and SARS-CoV-2 SP (transparent molecular surface colored in grey and secondary structure colored in light yellow; key residues are reported as sticks and colored by atom type: C grey, O red, N blue, S yellow, polar H light grey).