Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis

Figures & data

Figure 1. Left. Reaction of the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore with thiol group at the catalytic site (showing Cys111 and His272) of SARS-CoV-2 PL^pro. Initially, a non-covalent enzyme-inhibitor association is formed (E:I, 1) leading to a covalent adduct. Right. Structure of BBC DU-UC15 (2c) with the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore in red.

Table 1. Bis(benzylidene)cyclohexanones studied in this work.

Inhibitor/Label	Structure	Inhibitor/Label	Structure
DU-UC15 (2c) T97		T121
T98		T122
T105		T123
T106		T124
T120		T125

Scheme 1. Synthesis of bis(benzylidene)cyclohexanone inhibitors.

Table 2. Chemical structures of the reference inhibitors of M^pro and PL^pro of SARS-CoV-2 or proteasome-associated deubiquitinating enzyme inhibitors included in this study.

Inhibitor/Label	Structure	Inhibitor/Label	Structure
PR-619 C4		Remdesivir C8
GRL0617 C9		GC376 C10
RA-9		RA-190

Figure 2. Left: 3D structure of the SARS-CoV-2 PL^pro-GRL0617 complex (PDB entry 7CMD⁶²). The inhibitor GRL0617 that occupies the S₄ - S₃ pockets of the substrate binding cleft is shown in the CPK representation. Catalytic triad of PL^pro: Cys111 - His272 - Asp286 coloured purple shows the location of the catalytic site. Right: 2D scheme of inhibitor - residue interactions. Binding of GRL0617 induces closure of the flexible blocking loop BL2 (Gly266 - Gly271) and narrows the substrate binding cleft. The residues Tyr268 and Gln269 in the BL2 loop shift towards the bound GRL0617.

Table 3. Determined IC₅₀ and EC₅₀ and cytotoxicity of selected reference inhibitors of cysteine proteases.

Label	Protease inhibitor	IC50 (µM)				CTE* (µM)	EC50 (µM)		(Ref.)
		Reference value M^pro/ PL^pro target	Enzyme assay			Cell viability MTT assay
			PA*		DUA*
			M^pro	PL^pro	PL^pro		Reference value	Plaque reduction assay
C4	PR-619	6.1 − 12.9*	0.7	0.1	0.2	>10.0	n/a	9.0	(a, b)
C8	Remdesivir	n/a	> 100	> 100	> 100	n/o	0.77 − 0.99^#	1.8	(c, d)
C9	GRL0617	1.37 − 2.10*	> 100	3.2	2.0	n/o	n/a	2.5^‡	(e, a)
C10	GC376	0.03 − 0.15	0.02	> 100	>100	n/o	3.37^#	1.0	(f, g)

* Abbreviations used: Deubiquitinase activity (DUA); protease activity (PA); compound concentration that cause >40% reduction in the number of viable cells (CTE) averaged on screened cell lines; no cytotoxic effect observed within the concentration range (n/o); reference value is not available (n/a) in literature or the data range not measured; literature reference (Ref); ^# measured in vitro on virus infected cells or cell-free assay; ^‡ value out of acceptable range of R² correlation coefficient.

References used: (a) ⁴⁶; (b) ⁷³; (c) ⁵⁴; (d) ⁷⁴; (e) ^48,62; (f) ⁵⁰; (g) ⁷⁵.

Table 4. Determined IC₅₀ and EC₅₀ values of bis(benzylidene)cyclohexanones T97 - T125 and reference inhibitors of proteasome-associated deubiquitinating enzymes RA-9 and RA-190.

Label	Molecular weight (Mw)	IC50 (µM)			CTE (µM)	EC50 (µM)
		Enzyme assay
		PA		DUA
		M^pro	PL^pro	PL^pro target	Cell viability MTT assay	Plaque reduction assay
T97	380.4	>100	>100	>100	>5.0	n/a
T98	481.5	>100	50	75	>15.0-30.0	6.7
T105	652.7	>100	>100	>100	n/o	n/o
T106	852.8	35	40	60	>30.0	n/o
T120	536.5	>100	90	85	>10.0	3.4
T121	570.2	>100	>100	>100	>10.0-15.0	8.4
T122	482.5	>100	60	75	>30.0	17.0
T123	382.4	>100	60	75	>30.0	n/o
T124	593.6	>100	>100	>100	>30.0	15.9
T125	382.4	>100	85	45	n/o	n/o
RA-9	365.3	>100	>100	>100	>30.0	12.1^‡
RA-190	596.8	>100	>100	>100	>5.0	3.0

Abbreviations used: Deubiquitinase activity (DUA); protease activity (PA); compound concentration that causes more than 40% reduction in number of viable cells (CTE) averaged over screened cell lines; no cytotoxic or antiviral effect observed within the concentration range (n/o); value not available (n/a) due to compound’s higher cytotoxicity, insolubility, or data range not measured; ^‡value out of acceptable range of R² correlation coefficient.

Figure 3. Evaluation of antiviral efficacy with calculation of the effective inhibitory concentration EC₅₀ for compounds: T98 (A1, B1), T120 (A2, B2), T121 (A3, B3), T122 (A4, B4) and T124 (A5, B5). Monitoring the trend of decreasing the SARS-CoV-2 virus titre (normalised to untreated control, presented as mean with SEM) with increasing compound concentration (A1-A5). Determination of half-maximal effective concentrations EC₅₀ [M] (B1-B5) from the sigmoidal model with the corresponding regression coefficient R² (the goodness of fit). Data (B1-B5) are presented as mean and error with SEM.

Figure 4. Left: Molecular surface of the binding site of GRL0617 that occupies the S₄ - S₃ pockets of the substrate binding cleft S₄ - S₃ - S₂ - S₁ of PL^pro of SARS-CoV-2. The flexible BL2 loop G266-NYQC-G271 stabilises the bound ligand in its position by closing on the inhibitor (PDB entry 7CMD) ⁶². Right: Binding site of the model of noncovalent complex PL^pro - T98. The partially transparent molecular surface of the enzyme illustrates the shape of the pockets of the substrate binding cleft. The amine tail is oriented downward and is not fully visible. Atom colouring scheme: H, white; C, yellow; N, blue; O, red. Only polar hydrogens are shown.

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