Translational suppression of SARS-COV-2 ORF8 protein mRNA as a Viable therapeutic target against COVID-19: Computational studies on potential roles of isolated compounds from Clerodendrum volubile leaves

Abstract

The open reading frame 8 (ORF8) protein of SARS-CoV-2 has been implicated in the onset of cytokine storms, which are responsible for the pathophysiology of COVID-19 infection. The present study investigated the potential of isolated compounds from Clerodendrum volubile leaves to stall oxidative bursts in vitro and interact with ORF8 mRNA segments of the SARS-CoV-2 whole genome using computational tools. Five compounds, namely, harpagide, 1-(3-methyl-2-butenoxy)-4-(1-propenyl)benzene, ajugoside, iridoid glycoside and erucic acid, were isolated from C. volubile leaves, and their structures were elucidated using conventional spectroscopy tools. Iridoid glycoside is being reported for the first time and is thus regarded as a new compound. The ORF8 mRNA sequences of the translation initiation sites (TIS) and translation termination sites (TTSs) encoding ORF8 amino acids were retrieved from the full genome of SARS-CoV-2. Molecular docking studies revealed strong molecular interactions of the isolated compounds with the TIS and TTS of ORF8 mRNA. Harpagide showed the strongest binding affinity for TIS, while erucic acid was the strongest for TTS. The immunomodulatory potentials of the isolated compounds were investigated on neutrophil phagocytic respiratory bursts using luminol-amplified chemiluminescence technique. The compounds significantly inhibited oxidative burst, with 1-(3-methyl-2-butenoxy)-4-(1-propenyl)benzene having the best activity. Ajugoside and erucic acid showed significant inhibitory activity on T-cell proliferation. These results indicate the potential of C. volubile compounds as immunomodulators and can be utilized to curb cytokine storms implicated in COVID-19 infection. These potentials are further corroborated by the strong interactions of the compounds with the TIS and TTS of ORF8 mRNA from the SARS-CoV-2 whole genome.

Keywords: And SARS-CoV-2; COVID-19; Clerodendrum volubile; Cytokine storm; Iridoid glycoside; ORF8.

Fig. 1

Evolutionary relationship between ORF8 mRNAs of genomes of SAR-CoV-2 variants from China, Brazil, India, Nigeria, South Africa, USA and the UK.

Fig. 2

Isolated compounds from the ethyl acetate and butanol fractions of C. volubile methanolic extract. (1) Harpagide, (2) 1-(3-Methyl-2-butenoxy)-4-(1-propenyl)benzene, (3) Ajugoside, (4) 4,5-dihydroxy-6-(hydroxymethyl)-5-methoxy-3-(1,3,4,5-tetrahydroxypentan-2-yloxy)tetrahydro-2H-pyran-2-yloxy)-5-hydroxy-7-methyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran-7-yl acetate, and (5) Erucic acid.

Fig. 3

(A) Significant HMBC (blue lines) and NOESY (red lines); and (B) Significant HSQC (blue lines) and ¹H–¹HCOSY (red lines) of compound 4.

Fig. 4

A) Modeled single-stranded mRNA 3D-structure of TIS, the atoms are shown in Ribbons and Sticks and the structure is colored in Salmon B) Modeled single-stranded mRNA 3D-structures of TTS atoms are depicted in Ribbons and Sticks and the structure is colored in sea-green. The images were rendered using UCSF Chimera software.

Fig. 5

Representation of global binding sites of various ligands bound at the various sites of TIS mRNA 3D-structure colored in Salmon. The surface of the TIS is shaded in white and the various binding sites are labeled as Site A, Site B, Site C.

Fig. 6

The mRNA structure of TIS is colored in Salmon. The binding site nucleotides of TIS are labeled. Compounds 2, 5, and 4 are shaded in cyan, yellow, and magenta respectively. The H-bonds are shown in black dashed lines.

Fig. 7

The mRNA structure of TIS is colored in Salmon. The binding site nucleotides of TIS are labeled. Compounds 1 (A) and 3 (B) are shaded in white and green colors, respectuvely. The H-bonds are shown in black dashed lines.

Fig. 8

Representation of global binding sites of various ligands bound at the various sites of TTS mRNA 3D-structure colored in sea-green. The surface of the TTS is shaded in white and the various binding sites are labeled as Site A, Site B, Site C.

Fig. 9

The mRNA structure of TTS is colored in Sea-green. The binding site nucleotides of TTS are labeled. Compound 2, 5, and 4 are shaded in cyan, yellow, and magenta colors respectively. The H-bonds are shown in black dashed lines.

Fig. 10

The mRNA structure of TTS is colored in Sea-green. The binding site nucleotides of TTS are labeled. Compounds 1 (A) and 3 (B) are shaded in white and green colors, respectively. The H-bonds are shown in black dashed lines.

Fig. 11

Effect of compounds isolated from C. volubile leaves on (A) oxidative burst of neutrophils; (B) and (C) T-cell proliferation. Values = mean ± SD; n = 3. +C = positive control; –C = negative control; RLUs = relative light units; TCP = T-cell proliferation; CPM = counts per minute. *Statistically significant (p < 0.05) compared to positive control; #Statistically significant (p < 0.05) compared to negative control. ^abcValues with different letter above the bars are significantly (p < 0.05) different from each other.