Tuaimenal A, a Meroterpene from the Irish Deep-Sea Soft Coral Duva florida, Displays Inhibition of the SARS-CoV-2 3CLpro Enzyme

Abstract

Cold water benthic environments are a prolific source of structurally diverse molecules with a range of bioactivities against human disease. Specimens of a previously chemically unexplored soft coral, Duva florida, were collected during a deep-sea cruise that sampled marine invertebrates along the Irish continental margin in 2018. Tuaimenal A (1), a cyclized merosesquiterpenoid representing a new carbon scaffold with a highly substituted chromene core, was discovered through exploration of the soft coral secondary metabolome via NMR-guided fractionation. The absolute configuration was determined through vibrational circular dichroism. Functional biochemical assays and in silico docking experiments found tuaimenal A selectively inhibits the viral main protease (3CLpro) of SARS-CoV-2.

Figure 1

Four specimens of Duva florida (Cnidaria: Anthozoa: Octocorallia: Alcyonacea: Alcyoniina: Nephtheidae) were collected at a depth of 823 m from a submarine canyon north of Porcupine Bank on the Irish Continental Margin by ROV Holland I, deployed from RV Celtic Explorer. For scaling, a laser light is used (red dots indicate a span of 10 cm). Subsea photographs are copyright Marine Institute, Oranmore, Ireland. Used with permission.

Figure 2

Proposed planar structure of tuaimenal A (1) based on NMR data. Key HMBC (→) and COSY (bold bonds) correlations are shown.

Figure 3

Absolute configuration at C-2 of tuaimenal A (1) was determined to be the R configuration based on VCD analysis. Top graph shows the congruence between the calculated VCD for the R-configured stereocenter (green) and the measured VCD (blue). In the lower graph, the calculated FTIR absorbance (green) also demonstrates congruence with the calculated data (blue).

Figure 4

Final pose of the flexible docking of tuaimenal A (1) into the main protease (3CLpro). Hydrogen bonds (in dashed yellow lines) can be seen between the ligand and side chains Asn28, Cys117, and Ser144.

Figure 5

(A, B) Inhibition of 3CLpro activity and evaluation of the specificity of tuaimenal A (1) as a protease inhibitor. (A) The activity of 3CLpro (500 nM) was measured in the presence of tuaimenal A (1) (20 μM) or the broad-spectrum serine protease inhibitor AEBSF (5 mM). (B) Determination of the IC₅₀ of 1 against 3CLpro. The activity of 3CLpro (500 nM) was assayed in the presence of a range of 1 concentrations (1.25 to 20 μM). Inhibition is presented relative to the activity of 3CLpro in the absence of inhibitors, and error bars indicate standard deviation of three separate experiments. (C) The ability of 1 to inhibit different proteases was evaluated using a panel of serine and cysteine proteases. The activity of the human proteases chymotrypsin (4.0 nM), trypsin (168 nM), thrombin (800 pM), and HsCL (0.2 nM) or of the unrelated parasite proteases FhCL1 (2.7 nM) and FhCL3 (5 nM) was tested in the presence of 20 μM 1 (dark bars). The broad-spectrum serine proteases inhibitor AEBSF (5 mM; white bars) or cysteine proteases inhibitor E64 (100 μM, gray bars) was used as a positive control. Inhibition is presented relative to the activity of each enzyme in the absence of inhibitors, and error bars indicate standard deviation of three separate experiments.