key: cord-0826278-wa55zpsv
authors: Caruso, Francesco; Rossi, Miriam; Pedersen, Jens Z.; Incerpi, Sandra
title: Computational studies reveal mechanism by which quinone derivatives can inhibit SARS-CoV-2. Study of embelin and two therapeutic compounds of interest, methyl prednisolone and dexamethasone
date: 2020-10-14
journal: J Infect Public Health
DOI: 10.1016/j.jiph.2020.09.015
sha: 68e2b3dbd6ce5ac89f6efd8156f119dd8445ead9
doc_id: 826278
cord_uid: wa55zpsv

BACKGROUND: Quinones are reactive to proteins containing cysteine residues and the main protease in Covid-19 contains an active site that includes Cys145. Embelin, a quinone natural product, is known to have antiviral activity against influenza and hepatitis B. Preliminary studies by our group also indicate its ability to inhibit HSV-1 in cultured cells. METHODS: Docking and DFT methods applied to the protease target. RESULTS: a mechanism for this inhibition of the SARS-CoV-2 Mpro protease is described, specifically due to formation of a covalent bond between S(Cys145) and an embelin C(carbonyl). This is assisted by two protein amino acids (1) N(imidazole-His41) which is able to capture H[S(Cys145)] and (2) HN(Met165), which donates a proton to embelin O(carbonyl) forming an OH moiety that results in inhibition of the viral protease. A similar process is also seen with the anti-inflammatory drugs methyl prednisolone and dexamethasone, used for Covid-19 patients. Methyl prednisolone and dexamethasone are methide quinones, and possess only one carbonyl moiety, instead of two for embelin. Additional consideration was given to another natural product, emodin, recently patented against Covid-19, as well as some therapeutic quinones, vitamin K, suspected to be involved in Covid-19 action, and coenzyme Q10. All show structural similarities with embelin, dexamethasone and methyl prednisolone results. CONCLUSIONS: Our data on embelin and related quinones indicate that these natural compounds may represent a feasible, strategic tool against Covid-19.

December 2019 was the beginning of an ongoing global pandemic caused by an unknown coronavirus named Covid-19. This initiated a plethora of infections and deaths around the world [1, 2] and to limit the spread of this pandemic biosafety and hygiene measures are now applied.

Unfortunately, there are currently no medications or vaccines proven to be effective for the treatment or prevention of Covid-19. Several antiviral drugs are promising for COVID-19, among them remdesivir is under clinical trial [3] . The antimalarial drug chloroquine has been proposed but its use is controversial in the scientific community [4, 5] , whereas hydroxychloroquine is a more soluble and less toxic metabolite of chloroquine, which causes less side effects [6] . Nonantiviral drugs as methyl prednisolone and dexamethasone show a positive effect for treating Covid patients [7, 8] .

The recent uncertainties of a future vaccine (for example, AstraZeneca is re-evaluating its clinical trials) suggests the need and urgency for other pharmaceutical options and is also encouraging research into not-yet-approved medicinal compounds. In this context, even if an effective vaccine may become available, effective drugs are currently needed for infected people [9] .

A rational based drug design can reduce the time needed in the drug discovery process.

Molecular docking can identify lead molecules against the target proteins [10, 11] . Plant based compounds have provided the scaffold for many effective pharmaceutical compounds and the J o u r n a l P r e -p r o o f natural products are sources of promising antiviral drugs [12] . In fact, many approved antiviral drugs are derived from natural sources [13] . Examples of plant-derived proven antiviral activities include blocking HIV replication [14] , inhibiting Dengue virus type-2 (DENV-2) [15] and against Hepatitis C virus (HCV) [16] .

The main protease in SARS-CoV-2 is known as 3C-like protease (3CL pro ), and it consists of a highly conserved catalytic domain from the SARS virus. It is essential for controlling several functions of Covid-19, hence targeting the 3CL pro would prevent the virus from building its proteins and help in inhibiting viral replication. In fact, its mutation is often lethal to the virus and drugs targeting the enzyme significantly reduce the risk of mutation-mediated drug resistance and display broad-spectrum antiviral activity [17] . The three dimensional X-ray crystal structure of this enzyme in complex with ketoamide inhibitors was recently determined [18] , suggesting further investigation for other potential drugs. Due to the rise in the number of infected and death cases from COVID-19 and the lack of effective therapeutic drugs and vaccines, computer-aided drug design may be an important and fast way to find useful drugs.

Embelin, a plant natural product found in Lysimachia punctata (Primulaceae), and Embelia ribes Burm (Myrsinaceae) fruit, possesses interesting biological and pharmacological properties.

The fruits contain alkyl-benzoquinones, including embelin [19] , embelinol, and embeliol [20] . E.

ribes is cardio-protective [21] , antibacterial [22] and has anti-inflammatory [23] and antidiabetic activity [24] . Embelin is a cell-permeable, small-molecular weight inhibitor of XIAP and was the first non-peptide XIAP inhibitor [25] which induced additional anti-carcinogenic research [26] .

Embelin suppresses paraquat-induced oxidative stress in lung [27] , an organ particularly affected by Covid-19. It interacts with several proteins in mammalian cells: as a signal transducer and activator of transcription 3 [28] , peroxisome proliferator-activated receptor-γ [29] , and plasminogen activator inhibitor-1 [30] . Importantly, embelin is not toxic when given orally to rodents. Daily oral administration of embelin (25 and 50 mg/kg/day) for three weeks causes no side effects in rats [31] . The binding properties of embelin to proteins shows it is bound to human serum albumin through interactions with hydrophobic albumin amino acids [32] . Embelin is a unique chemical species as it includes both quinone and hydroquinone functional groups plus a long hydrophobic tail. Chemical and biological aspects of embelin have been reviewed [33] and recently, embelin was defined as the second solid gold of India, after curcumin [34] . [35] , the highest antiviral activity was IC50 of 0.2 μg/mL. The pure compound, embelin, was isolated and tested against the same virus with an IC50 of 0.3 μM. Embelin also proved to be an inhibitor of hepatitis B virus when tested along with twelve non-cytotoxic natural compounds and was among those showing high inhibition of HBV replication [36] . That quinones show antiviral activity is already known [37] [38] [39] . Quinones react with several functional groups of proteins or amino acids. Potential reactive targets for embelin of particular interest are lysine and cysteine, which react through the ε-amino and thiol groups, respectively [40] .

The current Covid-19 pandemic has elicited enormous interest in enveloped viruses.

Knowing that quinones show antiviral activity on viruses such as influenza, hepatitis B [35] [36] , together with our preliminary results showing embelin inhibition of the Herpes simplex virus-1 (HSV-1) enveloped DNA virus [41] were stimuli to explore its potential action on Covid-19. In this study we explore the potential of embelin against Covid-19 virus. We describe the mechanism behind embelin action by using results of DFT computational studies after docking embelin, methyl prednisolone and other related interesting compounds, scheme 1, at known sites which the viral protease is susceptible to mild quinones. 

Calculations were performed using programs from Biovia (San Diego, CA, USA). Density functional theory (DFT) code DMol 3 was applied to calculate energy, geometry, and frequencies implemented in Materials Studio 7.0 [42] . We employed the double numerical polarized (DNP) basis set that included all the occupied atomic orbitals plus a second set of valence atomic orbitals, and polarized d-valence orbitals [43] ; the correlation generalized gradient approximation (GGA) was applied including Becke exchange [44] , plus Perdew correlation (PBE) [45] . All electrons were treated explicitly and the real space cutoff of 5 Å was imposed for numerical integration of the Hamiltonian matrix elements. The self-consistent field convergence criterion was set to the root mean square change in the electronic density to be less than 10 −6 electron/Å 3 . The convergence criteria applied during geometry optimization were 2.72 × 10 −4 eV for energy and 0.054 eV/Å for J o u r n a l P r e -p r o o f force. All calculations did not include solvent effect. Docking studies were performed with the CDOCKER package in Discovery Studio 2020 version [46] .

The importance of the two catalytic residues His41 and Cys145 for the design of αketoamide inhibitors against SARS-CoV-2 Mpro has been recently described [18] . In addition, structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease has been suggested [47] . In a recent preliminary report, several anthraquinones were placed using docking procedures into the same important protease protein, one of the most critical drug targets for the blockage of viral replication [48] . Antiviral activity is known to be an important property of anthraquinones [49] [50] . The receptor [51] was PDB 6Y84 and comparison was done with remdesivir the first approved drug for Covid-19. Ranking immediately after remdesivir, the quinone emodin's ΔG value differed from the drug only by 0.51 kcal/ mol.

We have recently described the antioxidant activity of emodin, another quinone plant natural product [52] . Emodin and embelin possess similar redox properties, and their capture of an electron by the quinone ring are closely related [53] . The existence of the Cys145 residue at the protease attracted our attention because of a potential strong bond between Cys and the quinone ring. Therefore, we envisioned embelin as a potential inhibitor of this important viral enzyme and describe docking and DFT procedures for embelin into the same target protein.

Protein PDB 6Y84 crystal structure contains a conserved non-canonical His41-Cys145 dyad located within the cleft between domains I and II [54] . Using Discovery Studio version 2020 we followed the standard procedure of "preparing" the protein, which includes assignment of H atoms and force field (CHARMm), and selected the Cys145-His41 area for a docking sphere, 9.5 Å radius, to perform the docking of embelin, Figure 1 . Of 10 possible poses, numbers 6 and 9

showed a potential sulfur quinone-centroid approach, resembling embelin interaction with the superoxide anion [53] . CDocker interaction energy is -36.0 kcal/mol. 

Embelin docked pose 9 was treated with DFT methods. The essential molecular components of this pose, Cys145, His41 and model embelin with its C11 tail substituted by an ethyl group, are shown in Figure 7 , containing 3 molecules as found after docking procedure. For simplification, the peptide chain is replaced by 2 H atoms, which were kept fixed during the geometry optimization. This is imposed to conserve the protein environment; Figure 8 shows the best refined structure after DFT minimization and Figure 9 its converged structure after adding an H atom to the embelin O(carbonyl) and performing geometry optimization. We added a proton to the system shown in Figure 8 , separated at van der Waals distance from the embelin O(carbonyl) located farther away from the C2H5 group, indicated as stick style in Figure 9 . The rational of this insertion is based on the H-bond contact shown in Figure 5 , Figure 10 . Right: After minimization of the same system, but not imposing any restraint.

We were interested to see if there is some energy barrier in the process of capturing H(Cys145) by N(imidazole-His41). Since we mimicked the polypeptide role by replacing the amino acid chain linked to Cys145 and His41 with 2 H atoms each, these 4 H were kept fixed to conserve the protein environment. However, fixing atoms both in the reagent and the product does not allow a canonical search for transition state to be performed. Basically, our quantum mechanical program does not permit atoms to be in equivalent positions in both the reagent and the product; in this case the "H" atoms fixed during minimizations that replaced the polypeptide chain. We therefore decided to perform geometrical minimization without any constraints in both systems, corresponding to Figures 9 (reagent) and 11 left (product). From these geometry optimizations the product showed minimal atomic variation, that is, the product is confirmed. In contrast, for the reagent the obtained structure was just the product, Figure 11 right, which ensures that no barrier exists, and the system can evolve spontaneously towards the product. We conclude that the docking of pose 9 provides solid elements to establish an important role for embelin, as it is able to inhibit the protease by establishing a strong S(Cys145) bond to the quinone ring.

We already mentioned that after docking of embelin into the protease site, pose 9 provided two potential interactions for H(Cys145), one having N(imidazole-His41), explained above. The other was O(carbonyl-His164), at the peptide chain, which was confirmed after a standard dynamic cascade, Figure 6 . We proceeded with this configuration along the same lines shown in and quinone has been described [40, 56] . CDocker interaction energy is -29.6 kcal/mol. Therefore, we performed a dynamic cascade on one pose and the S atom became close to the quinone center suggesting a potential role for emodin as an inhibitor of this SARS-CoV-2 3CL pro , also known as Mpro protease, Figure 13 . We plan to do further work to completely elucidate this reactivity of emodin.

J o u r n a l P r e -p r o o f 

The present study shows that for embelin some amino acid residues at the Covid-19

protease active site, including Cys145, His41 and His163, are important factors to establish interaction with the protease. The associated mechanism is indicated in the following scheme 2. The most important result is the covalent bond formed between Cys145, after losing its H atom, with one carbonyl of embelin, thus inhibiting the protease virus. The other potential aminoacid residue able to establish an interaction similar to His41, [His164 through its O(carbonyl)], is shown to be not effective. Emodin seems to act similarly, and progress in its potential mechanism is underway by our group. Interestingly, it was only after we applied molecular dynamics that the interaction between S(Cys145) and the quinone centroid was established. That is, the docking procedure showed, instead, S(Cys145) interacting with one aromatic ring centroid adjacent to the quinone ring.

(1) The anti-inflammatory drug methyl prednisolone is being used to treat Covid-19 patients [7] .

Our interest was stimulated by its p-methide-quinone moiety, resembling quinone embelin. We (2) Recently, doctors at the University Medical Center, Maastricht, The Netherlands noted severely reduced vitamin K levels in Covid-19 patients [57] , and suggested it was due to a pathological condition. There is marked structural similarity between embelin and vitamin K, including their long alkyl tails. Docking of Vitamin K on the same PDB 6Y84 Covid-19 protease receptor shows CDocker interaction energy of -32.8 kcal/mol. Although the separation between S(Cys145) and the quinone center is slightly longer than in methyl prednisolone, Figure 15 , the pattern is similar to that of embelin. We conclude that vitamin K decrease in Covid patients may be due to S(Cys145)-vitamin K bond formation. Vitamin K, embelin and coenzyme Q10, are all quinones having long alkyl tails that allow insertion in the host cell membrane, a property recently described for embelin [53] . Therefore, we also explored coenzyme Q10 to verify its insertion at the protease active site. Again, correlation J o u r n a l P r e -p r o o f with the earlier described embelin mechanism was observed, Figure 16 , and its CDocker increases the negative charge on the S atom, inducing it to point to the quinone center, Figure 5 .

The final result is bond formation between S(Cys145) and the same carbon also attacked by the proton through the HN(His163) donation: a Michael addition [56] . to the quinone (or quinone methide for methyl prednisolone and dexamethasone) and which inhibits the virus protease. Emodin, a quinone present in goji berry, has recently received approval for a patent against Covid-19 [59] and we plan to further explore emodin interaction with Covid-Besides the recent important study of the main protease Covid-19 crystal structure containing α-ketoamide inhibitors [18] , the interaction between potential drugs and this active site is subject of current interest and intense studies using molecular mechanics techniques. This is shown in very recent literature [60] [61] [62] [63] for several compounds docked at the site. However, to our knowledge, specific detailed chemical mechanisms regarding molecular interaction are not described. In contrast, our work on the action of quinones, as described here, suggests inhibition of the protease by a well-defined chemical mechanism. Thus, there is a specific covalent bond formation between a quinone carbonyl and the Cys(145) thiolate, helped by 2 proton transfers from neighboring residues. One residue, His41, allows cleavage of the H-S(Cys145) bond by capturing the H-(Cys145), and the second residue releases a proton to the same C(carbonyl) quinone engaged with S(Cys145) thiolate. The second amino acid residue may differ when comparing embelin with dexamethasone, methyl prednisolone, coenzyme Q10 and Vitamin K docking into the protease target, but the mechanism appears to be similar.

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