Introduction

The current outbreak of corona virus disease 2019 (COVID-19) caused by a novel coronavirus SARS-CoV-2 was first reported from Wuhan, China, in late December 2019 [1], which has subsequently affected the entire world, reporting nearly 26 million of confirmed cases of COVID-19 along with ~ 9.0-lakh deaths as per data recorded in September 1st week, 2020, posing a global threat for human health and economy. With so many novel studies and findings surfaced, since its inception, we are still lagging behind in development of an effective treatment strategy to control the virus spread and prevent the disease [2,3,4,5,6,7].

SARS-CoV-2 is an enveloped non-segmented large positive sense, single-stranded RNA virus (~ 30 kb) with 5′-cap structure and 3′-poly-A tail belonging to β-CoV category [8, 9]. Its RNA genome contains 29,891 nucleotides and encoding for ~ 9860 amino acids [9]. The genome codes for both structural proteins like spike (S), envelope (E), membrane (M), and nucleocapsid (N), along with many non-structural proteins (NSPs 1–16) [10]. While these NSPs linked to RNA replication and processing of subgenomic RNAs, the functions of some of the NSPs are not known. A key component, NSP6, is a membrane protein of approximately 34 kDa with eight transmembrane helices and a highly conserved C-terminus. Together with NSP3 and NSP4, NSP6 is involved in the formation of replication-transcription complexes (RTCs) or replication organelles (RO) by stimulating the rearrangement of host cell membranes [11]. These replication complexes serve many important functions during the virus life cycle and play an important role in infection [12, S1). The drug binding thus increases the number of highly frustrated residues and thus increases the local frustration and flexibility of the protein, especially in dextromethorphan binding.

Moreover, the residues involved in drug binding were dominantly neutral to minimal frustration. However, some of the dextromethorphan-binding residues like Ser32 and Leu276 gain frustration upon binding, whereas the frustration of Tyr132, Tyr136, Gly177, Arg236, and Lys270 decreased upon binding. Also, haloperidol binding increases the frustration of Leu231, Leu237, and Thr238 upon binding, and at the same time, it also decreased the frustration of residues like Tyr136, Phe184, and Phe269 (Table S1).

Moreover, to find out how drug binding induced the local frustration changes, we performed a comparative analysis of the spatial distribution of local frustrations mapped onto the secondary structure of the protein (Fig. 9). In particular, highly frustrated residues in the NSP6-dextromethorphan complex increased in the α-helix H1, H7, and H13, while the increase is seen in H11 for the NSP-haloperidol complex (Fig. 9a). The decrease in frustration was also seen for both the complexes mainly in C-terminal H11, H12, S1, and H14 for the NSP6-dextromethorphan complex, and for NSP6-haloperidol, a decrease is observed in H5, H8, and S1 (Fig. 9a). Additionally, the drug binding decreased the minimal frustration in H5, H7, H9, and H11 helices, and also increased the minimal frustration in H2 and H12 helices (Fig. 9b).

Fig. 9
figure 9

Frustration analysis in NSP6-drug complexes. The changes in residual frustration are distributed along the structural regions of NSP6. a The changes in minimal frustration values in NSP6-drug complex. b The changes in highly frustration values in NSP6-drug complex. The secondary structural regions are represented as: H-α-helix and S-β-strands

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Thus, the coupling between structurally rigid C-terminal helices H10 and H11, and conformationally flexible helices, H5 and H13, is important for drug binding, and also the frustration index of the regions close to the binding site changes upon association.

Discussion

The all-atom MD simulation shows significant differences in the tertiary structure of the NSP6-drug complexes. The structural snapshots of the protein-drug complex were used to analyze the differences in tertiary structure caused by each drug (Figure S8).

Overall comparison of the binding conformations of haloperidol and dextromethorphan in NSP6

The NSP6-drug complexes show the significant differences in H2, H5, H7, and C-terminal regions comprising H12, H13, β1, and β2 (Figure S8). The haloperidol and dextromethorphan complexes have a kink in H2, H5, and H7 at midway of simulation (50 ns) that causes a change in orientation when the drug binds (Figure S8 A, C). The dextromethorphan binding induces much larger kink at H2 and H5 with a much larger deviation also (RMSD = 3.59) (Figure S8C). At the end of the simulation, the haloperidol and dextromethorphan complexes showed an increase in twist of H12 and H13, and both the strands of the C-termini along with the kinks present in H2, H5, and H7 (Figure S8B, D). Similar to what was observed at ~ 50 ns, dextromethorphan binding induces much larger disruption (RMSD = 3.24). Thus, although both drugs lead to bending of the helices, dextromethorphan binding induces larger disruption of the helices and thus more destabilization observed in the protein.

The protein-drug binding analysis shows that dextromethorphan and haloperidol interact with many key residues and are shared between both the drugs. The residues interacting with each drug during the simulation were compared and visual 2D representations are shown in Figures S9 and S10.

Figure 2 a shows the initial pose of the dextromethorphan molecule in the MD simulation, which is also the best pose from the docking study. As can be seen, the dextromethorphan molecule formed hydrogen bonds with residue Lys 61, and several van der Waals interactions with His62, Asn232, Arg233, Arg236, Thr238, Asp243, Leu245, and Pro282. Figure 2 b shows the haloperidol binding sites in NSP6 which is comprised of helix H7, and H9 residues (i.e., H7: Tyr132, Asp134, Ala136, Arg137, Trp140; H9: Asn174, Tyr175, Ser176, and Val178) and C-terminal positively charged residues Leu231, Leu237, Leu239, and Ser265.

The structural snapshots of the drug-protein complexes have been shown in Fig. 10. It has been seen during simulation that dextromethorphan has drifted to the different locations inside the NSP6 during ~ 20–100 ns of simulation. In the first ∼ 20 ns, dextromethorphan stayed in its initial location (Fig. 10a). After that, it drifted away from its initial location and moved into the water above the binding pocket and the interaction becomes minimal, only with Tyr234, Phe235, and Leu276 (Fig. 10b). Without drifting further into the water, the dextromethorphan molecule re-entered to the enlarged binding pocket, where it interacts mainly with H12 residues (Phe225, Leu230, Arg233, Tyr234, and Arg236) through the hydrophobic interaction (Fig. 10c).

Fig. 10
figure 10

The structural snapshots of NSP6-drug complex observed during the MD simulation (0 ns, 25 ns, 50 ns, 100 ns) for the most abundant structure of a–d NSP6-dextromethorphan and e–h NSP6-haloperidol

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During the second half of the MD simulation (~ 50–100 ns), the dextromethorphan molecule stably remained at a new binding pose with much stronger interactions. At this new location, dextromethorphan coordinates with Ser32 through H-bonds, and hydrophobic interactions by Trp31, Ile189, Val190, Met192, Cys193, Phe200, and Phe201 (H9-H10) (Fig. 10d).

In contrast, the drug haloperidol was stably bound inside the NSP6 pocket and explored two binding poses during the simulation. For the first 50 ns, it remained at its initial docking pose where it forms H-bonds to Ser 176 and Ser265, and several hydrophobic interactions mediated by Arg137, Trp140, Asn174, Tyr175, Val178, and Thr238 (Fig. 10e, f). After that, it drifted to another binding site and remained stable there, until the end of the simulation. At this new site, haloperidol interacts mainly with H7-H9 residues (Trp140, Asn144, Trp165, Ala166, Ile169, Ser170, Ser176, Val178, and Phe184), Leu237, Leu239, and Ile266, Phe269 (H14) (Fig. 10g, h).

Conclusion

To better understand the mechanism of biased activity of SARS-CoV-2 in the presence of two sigma-R1 binding drugs to NSP6, molecular dynamics simulation studies were employed. Our data suggests dextromethorphan binds to C-terminal helices while haloperidol binding sites are in the middle of the protein domain in helices H7 and H9. The disruption of the alpha helix H7 and H8 is significant for the dextromethorphan complex along with a large kink in H5 and H7. The NSP6-haloperidol complex showed a less significant change in the tertiary structure despite major disruption of the H12 helix.

Furthermore, the analyses of RMSD, RMSF, PCA, FEL, and dynamic cross-correlation matrix (DCCM) indicated that dextromethorphan binding leads to destabilization of the protein with the loss of correlated motions and residual frustrations. In contrast, haloperidol binding brings milder alterations in these order parameters and thus showed minimal changes in stability compared with dextromethorphan system. Besides, intermolecular hydrogen bonds were constantly formed in haloperidol with high occupation indicating the more stabilization of the NSP6-haloperidol system. In conclusion, the study elucidated the detailed interaction mechanism of dextromethorphan and haloperidol to NSP6 protein and the associated structural and dynamical changes upon drug binding. These results will significantly enhance our understanding of the working mode of these drugs at the molecular and structural level and will contribute to the future rational drug design for COVID-19.