Antiviral activity of curcumin and its analogs selected by an artificial intelligence-supported activity prediction system in SARS-CoV-2-infected VeroE6 cells

Abstract Curcumin has been reported to exert its anti-SARS-CoV-2 activity by inhibiting the binding of spike receptor-binding domain (RBD) to angiotensin-converting enzyme-2 (ACE2). To identify more potent compounds, we evaluated the antiviral activities of curcumin and its analogs in SARS-CoV-2-infected cells. An artificial intelligence-supported activity prediction system was used to select the compounds, and 116 of the 334 curcumin analogs were proposed to have spike RBD-ACE2 binding inhibitory activity. These compounds were narrowed down to eight compounds for confirmatory studies. Six out of the eight compounds showed antiviral activity with EC50 values of less than 30 µM and binding inhibitory activity with IC20 values of less than 30 µM. Structure-activity relationship analyses revealed that the double bonds in the carbon chain connecting the two phenolic groups were essential for both activities. X-ray co-crystallography studies are needed to clarify the true binding pose and design more potent derivatives. Graphical Abstract


Introduction
Various vaccines and therapeutics have been developed and approved worldwide to end the COVID-19 pandemic.However, the pandemic has not been sufficiently controlled, owing to the emergence of more transmittable and toxic variants.Consequently, increasing attention is being paid to the supplementary effects of natural products (Demeke et al. 2021;Malabadi et al. 2021, Ribaudo et al. 2022;Coghi et al. 2022).One of the most representative natural products is curcumin, because many studies have shown its therapeutic and prophylactic effects on COVID-19 through its multiple pharmacological profiles (Rattis et al. 2021;Vahedian-Azimi et al. 2022).
Recent research has focused on the binding of curcumin to the spike receptor-binding domain (RBD) and inhibiting spike RBD-angiotensin-converting enzyme-2 (ACE2) binding.Several in silico studies have been conducted to demonstrate the potential binding of curcumin to spike RBD (Coghi et al. 2021;Jena et al. 2021;Nag et al. 2021).Although experiments demonstrating that the binding of curcumin to spike RBD and the inhibition of spike RBD-ACE2 binding are limited, Goc et al. clarified these effects (Goc et al. 2021).Marín-Palma et al. demonstrated that curcumin inhibits in vitro SARS-CoV-2 infection (Marín-Palma et al. 2021).In silico studies were not restricted to curcumin itself and were expanded to its analogs, including curcuminoids (Shanmugarajan et al. 2020;Patel et al. 2022).However, the inhibitory effects of curcumin and its analogs on spike RBD-ACE2 binding and antiviral activities remain to be fully investigated.Therefore, we examined the relationship between spike RBD-ACE2 binding inhibition and antiviral activities of curcumin and its analogs.The affinity of curcumin analogs to spike RBD was predicted using an artificial intelligence (AI)supported activity prediction system, the AI-guided INTerprotein's Engine for New Drug Design (INTENDD®; Komatsu et al. 2022).Compounds that exhibited a potential affinity for spike RBD were selected and subjected to assays for spike RBD-ACE2 binding inhibition, and antiviral activity.

Selection of spike RBD-ACE2 binding inhibitor candidates from curcumin analogs
First, we examined whether curcumin orthosterically inhibits protein-protein interaction (PPI) between spike RBD and ACE2 using the X-ray crystal structure of the complex of spike RBD and ACE2, 6M0J.pdb(Figure S1_A).When we observed the PPI interface, many ACE2 atoms were detected within 6 Å of the spike RBD, at the left-side cavity of Tyr505, suggesting that this cavity is a hotspot that plays a crucial role in PPI (Figure S1_B).As the size of this cavity was not sufficient for binding compounds with a certain level of potency, we selected another cavity on the right side of Tyr505; thus, we proposed a target pocket consisting of these two cavities (Figure S1_C).Docking simulations revealed that curcumin potentially binds to this target pocket with a score of −6.2 kcal/mol (Figure S1_D).Based on the study outline (Figure S2), we selected 334 curcumin analogs with a Tanimoto coefficient exceeding 0.9 from PubChem compounds; these compounds were subjected to docking simulation with the target pocket all compounds were found to have the potential to bind to the target pocket; therefore, the 3D structures of the compounds and the pocket were inputted into AI-guided INTENDD.As a result, AI-guided INTENDD proposed 116 compounds whose Good Prediction Class (a single predicted class ± 1 digit error) was expected to be less than 1 µM as the output.The docking score of these compounds was in a range −8.7 to −4.3 kcal/mol (Figure S1_E).These compounds were further narrowed down to 13 compounds according to the AI score (prediction probability).Purchasability (delivery time and cost) was also considered in the selection process.The purchased compounds were subjected to spike RBD-ACE2 binding inhibition and antiviral activity assays.

Spike RBD-ACE2 binding inhibition and antiviral effect on SARS-CoV-2 replication
Primary screening of the 13 selected compounds was performed using a spike RBD-ACE2 binding assay kit.Based on the fact that eight compounds showed inhibitory effects with inhibition rates exceeding 5% at any of the tested concentrations, we examined the effects of these compounds on spike RBD-ACE2 binding and SARS-CoV-2 replication.Table 1 shows the names, chemical structures, docking scores and pharmacological activities of the eight selected compounds.As the maximum spike RBD-ACE2 binding inhibition rate was 40.3%, the IC 20 value was calculated as the spike RBD-ACE2 binding inhibitory activity.Curcumin and its five analogs, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), curcumin sulfate (CSULF),  monodemethycurcumin (MDC), and bisdemethycurcumin (BDC), but not tetrahydrocurcumin (THC) or hexahydrocurcumin (HHC), showed spike RBD-ACE2 binding inhibitory activity with IC 20 less than 30 µM and antiviral activity with EC 50 less than 30 µM.The cytotoxicity of these compounds was also tested using SARS-CoV-2-infected VeroE6 cells, and no cytotoxicity was observed up to 30 µM, except for MDC, which had a CC 50 of 25.2 µM.

Relationship between antiviral activity and spike RBD-ACE2 binding inhibition
To determine whether the antiviral effects of curcumin and its analogs were caused by spike RBD-ACE2 binding inhibition, we examined the relationship between EC 50 and IC 20 values of the eight compounds tested.A scatter plot of the spike RBD-ACE2 binding inhibitory activity versus antiviral activity of the compounds is shown in Figure S3.Because the EC 50 and IC 20 of the two compounds (THC and HHC) exceeded 30 µM, we used 31 µM as the value for these compounds.Although Spearman's rank correlation test revealed no significant correlation, a correlation trend was observed (r s = 0.642, p = 0.086).These results suggest that at least part of the antiviral activity of curcumin and its analogs is mediated by inhibition of spike RBD-ACE2 binding.

Structure-activity relationships of curcumin and its analogs
Two curcumin metabolites, THC and HHC, which have no double bonds in the carbon chain connecting the two phenolic groups, showed neither antiviral nor spike RBD-ACE2 binding inhibitory activities up to 30 µM.This finding indicates that the double bonds in the carbon chain connecting the two phenolic groups are essential for both activities.An L-shaped bent structure is probably necessary for curcumin and its analogs to bind tightly to the two target pockets of the spike RBD that we proposed (Figure S1_C).The binding free energy of curcumin to the spike protein might be lower than that of THC and HHC, which is probably due to the loss of conformational flexibility related to the restricted rotation around the double bonds.

Estimated binding site and clinical significance
The estimated key amino acid residues for the interaction with curcumin and the seven selected analogs are summarized in Figure S4.We proposed a curcumin-binding site that includes a cavity towards which the atoms of ACE2 approach, implying that curcumin and its analogs are potential orthosteric modulators of spike RBD-ACE2 interaction.Consequently, the estimated interaction of curcumin and its analogs with the spike RBD differed from that proposed by Patel et al. (Patel et al. 2022).Patel et al. suggested that interactions between BDMC and the spike RBD occur through hydrogen bonds (Arg355, Thr430, and Leu517) and π-stacking (Phe464), whereas we considered that key interactions occur through hydrogen bonds (Tyr453 and Asn501) and π-stacking (Tyr505) (Figure S4).Further research using X-ray co-crystal structure information is required to clarify the true binding sites and poses.
The FDA is currently providing an Emergency Use Authorization for two anti-spike RBD antibody products.The epitopes of these antibodies appear to differ from the estimated binding site of curcumin and its analogs, suggesting that these small-molecule compounds might have an additive effect in the treatment of SARS-CoV-2 with antibody products (Figure S5_A and B).However, we cannot rule out the possibility that the small-molecule compounds are less effective against Omicron variants because the compounds were estimated to also interact with key amino acid residues observed in some of the mutated Omicron variants (BA. 1, 1.1, 2, or 3; Figure S5_C).

Experimental section
See Supplemental online material.

Conclusions
Curcumin and its five analogs showed anti-SARS-CoV-2 effects with EC 50 values less than 30 µM, which were at least in part mediated by spike RBD-ACE2 binding inhibition.Structure-activity relationship analysis revealed that the double bonds in the carbon chain connecting the two phenolic groups are essential for both spike RBD-ACE2 binding inhibitory and antiviral activities against SARS-CoV-2.Further research using X-ray co-crystallography is needed to clarify the true binding pose and to design more potent derivatives of curcumin.

Table 1 .
summary of docking score, spike rBd-ace2 binding inhibition and antiviral activity of curcumin and its analogs.