Phytotherapy Research | 2021
In silico and in vitro evaluation of kaempferol as a potential inhibitor of the SARS‐CoV‐2 main protease (3CLpro)
Abstract
To the Editor, The recent SARs-CoV-2 created focal news when the paradoxical spread was reported in the Wuhan province of China, further jeopardizing the existence of the human race on the planet earth. Due to its rapid expansion throughout the world, the COVID-19 outbreak was declared as a global pandemic by the World Health Organization (WHO) on March 20, 2020. The updates of November 13, 2020, have been reported 53,429,139 infections and 1,304,470 deaths. Fever, shortness of breath, coughing, myalgia, dyspnea, and radiological indications of ground-glass lung opacity compatible with atypical pneumonia are the signs exhibited by most patients with COVID-19. However, some patients have also been reported to have asymptomatic or mildly symptomatic (Chen et al., 2020; Huang et al., 2020; Lu et al., 2020). The whole proteome of the SARS-CoV-2 is encoded by ~30 kb a genome. The whole-genome encodes three major components, including non-structural (NS), structural, and accessory proteins (Durojaiye, Clarke, Stamatiades, & Wang, 2020). The genes found on the 30-terminus encode the four structural proteins constituting the main envelope of the virus and eight accessory proteins. Among the structural proteins small envelope protein (E), spike surface glycoprotein (S), nucleocapsid protein (N), and the membrane protein (M) while the eight accessory proteins codes for 3a, 3b, p6, 7a, 7b, 8b, 9b and ORF14 (Wu et al., 2020). In contrast, the two overlapping genes, ORFIa and ORF1ab, encode the non-structural proteins (polypeptides pp1a and pp1ab) and form a replication/transcription complex (RTC). These pp1a and pp1ab polypeptides are translated and then proteolytically cleaved by two main viral proteases, (papain-like protease) PLpro and (3-chymotrypsin-like protease) 3CLpro or main proteases (Mpro) (Perlman & Netland, 2009). PLpro is accountable for the cleavage of non-structural proteins (nsp 1–3). In contrast, the 3CLpro (Figure 1A) cleaves the polyprotein at 11 discrete sites downstream of nsp4 to yield different non-structural proteins that play a crucial role in the viral life cycle. Previous studies have indicated that 3CLpro (Mpro) plays an important role in cell proliferation and maturation. So, the inhibition of this target would significantly contribute to controlling the COVID-19 (Needle, Lountos, & Waugh, 2015). Because of these multi-faceted aspects, 3CLpro has been deemed as a promising drug development target for anti-coronaviruses (Hatada et al., 2020). In this study, we have used both in silico and in vitro approaches to confirm the activity of an active compound Kaempferol (Figure 1B), which was reported in our previous study to potentially interact with the SARs-CoV-2 main protease 3CLpro (Khan et al., 2020). The compound found through molecular search from the Traditional Chinese Medicine (TCM) was re-docked here against the active site of the main protease. AutoDock Vina (Trott & Olson, 2010) with exhaustiveness set as 64 was used to dock kaempferol against the main protease (3CLpro). The docking predictions revealed the docking scores for the 10 conformations. Among the 10 conformations, the docking score for the first three conformations was −6.4 kcal/mol. Previously, these residues His41, Met49, Tyr54, Phe140, Leu141, Asn142, Cys145, His163, Met165, Glu166, Leu167, Phe185, Asn187, Arg188, and Gln192 comprised the active site. Therefore, the first three conformations were analyzed for potential interactions with these residues. Conformation 1 (Figure 1C) formed six hydrogen bonds with the active site residues, including Phe140, Leu141, Asn142, His163, Glu166, and Arg188. A pie-sulfur interaction was formed by Cys145, while the Met165 formed a pie-alkyl interaction. The second conformation (Figure 1D) with the docking score (−6.4 kcal/mol) also formed six hydrogen bonds with the key active site residues. Among the key interactions, five were formed with Phe140, Leu141, Asn142, and Cys145, while one hydrogen bond was formed with Arg188. The conformation 3 formed only three hydrogen bonds with the key residues. As shown in (Figure 1E) Met49, Phe140, and His163 are involved in the interaction with Kaempferol. Hence, the docking predictions significantly confirm that kaempferol potentially interacts with the same active site residue even in different conformations, thus verify its activity against the 3CLpro. To further validate the potential of kaempferol as an active drug, a biophysical investigation was performed. Using Amber20 (SalomonFerrer, Case, & Walker, 2013), a 250 ns simulation for each conformation was performed to reveal the dynamic behavior of Kaempferol-3CLpro complexes. The parameters were set as used in the previous study (Khan et al., 2020). To demonstrate the structural stability of the complexes, root mean square deviation (RMSD) of each complex was calculated. As given in Figure 1F–H, all the complexes reached the stability at 1.8 Å and reached the equilibrium state at 15 ns. For conformation 1 the average RMSD was reported to be 2.0 Å. The system remained stable during the 250 ns simulation time. No significant convergence was reported. The stability graph of the conformation 2 followed the same pattern as conformation 1 except the average RMSD remained relatively higher as 2.2 Å. The system faced a little convergence between 90 and 110 ns; however, the system remained stable during the simulation time. On the other hand, conformation 3 was relatively unstable, and the RMSD converged after 150 ns but then decreased afterward. The average RMSD for this conformation was reported to be 2.8 Å. Their Received: 25 November 2020 Revised: 9 December 2020 Accepted: 11 December 2020