Introduction

The COVID-19 outbreak originated from Wuhan during December 2019, affects patients in three levels. The first level is patients with asymptomatic, the second level is patients showing the symptoms of fever, cough and the third level is patients suffer very severe conditions like difficulty in breathing and other respiratory illness or cytokine storm, which means the body’s immune system cause damage to the host organs (COVID-19, 2020). To help the global researchers to hunt the COVID-19 infection, on April 9, 2020, China research laboratory issued the three-dimensional structure of coronavirus

main protease (Jin, Du, & Xu, 2020). This helped the researchers around the world to use different molecular modeling and virtual screening methods to identify the existing drug that could inhibit this enzyme possibly.

There are several drugs have been tested against this COVID-19 infection, includes Japanese influenza drug favipiravir, anti epileptic drug valproic acid, anti-malarial drug-hydroxy chloroquine along with anti-biotic azithromycin (Gautret et al., 2020), anti-HIV drugs lopinavir and ritonavir, and anti-viral drug remdesivir. However, the problems in these drugs have their unique side-effects like heart palpitation, permanent blindness, migraine, and in some cases, even death (Zeitlinger & Koch, 2020).

The proteins/enzymes involved in the budding and exocytosis processes which are essential for viral replication may be considered as the important targets for the development of novel anti-viral drugs. Anti-viral drugs most commonly target the proteins, which is essential for the virus to synthesize the offense protein from human cells. In novel coronavirus, there are four proteins, namely, M (membrane protein), required for virus budding; S (viral spike glycoprotein), that has receptor binding and membrane fusion activities; E (small membrane protein), used for the assembly of coronavirus; N (nucleo capsid phosphoprotein), the viral RNA has been connected with this protein inside the virion (Holmes, 2003).

The WHO declared that the outbreak of COVID-19 has been considered as a pandemic, and on the 13th of May, it has officially declared the statistics on outbreak has reached to 4139794 confirmed cases, 285328 confirmed deaths, and 215 countries are affected with this virus (WHO, 2019). Whereas, in India, the statistics on COVID-19 Dashboard as on: 13th of May 2020, 08:00 GMT+5:30 articulate that, there were 47480 Active Cases, 24385 Cured/Discharged cases, and 2415 deaths (COVID-19, 2020). This pandemic is still enduring and hence there is an emerging call to find out the novel preventive measures to combat this novel pandemic situation. However, until now, there is no specific treatment was found to cure this disease. With response to this statement by WHO, there is an emerging need to find curative measures against this virus through diverse techniques like adjusting the immune system through various immunomodulators, by the development of diagnostic, vaccines, re-purposing of drugs, etc. Furthermore, in agreement with the WHO report, the development of anti-coronavirus vaccines or anti-coronavirus drugs might take another 18 months and this COVID-19 is considered a seasonal disease shortly.

We have chosen the co drug or mutual pro drug approach, which means conjugating two or more therapeutically active compounds through covalent chemical linkage or a cleavable spacer. The co drug consists of two different scaffolds have different therapeutic actions molded together in a single entity through a specified bio cleavable linker to release the parent drugs at their site of action (Das, Dhanawat, Dash, Nagarwal, & Shrivastava, 2010). Co drugs are made through the combination of two scaffolds/drugs with similar or different pharmacological activities into a single hybrid form to complement each other to elicit the therapeutic effect. Co drug strategy is employed to ameliorate physicochemical, biopharmaceutical, and drug delivery/targeting of the therapeutic actives (Aljuffali, Lin, Chen, & Fang, 2016; Das et al., 2010) Co drug-based therapy is reported to treat various diseases such as neurodegenerative disorders, cardiovascular problems, tumors, glaucoma, retinal neovascularization, alcohol addiction, virus infection, and bacterial infection (Das et al., 2010).

Chloroquine and hydroxychloroquine have been found to be efficient on SARS-CoV-2 and reported to be efficient in Chinese COVID-19 patients. Hydroxychloroquine is widely used worldwide for the prophylaxis is derived from available evidence of benefit as treatment and supported by preclinical data. On the other hand, the immune system is a very important and dynamic one to regulate the body’s inadequate immune response which could cause many diseases, while excessive and unregulated immune responses produce autoimmunity. Interleukin plays a vital role in maintaining this immune in a balanced condition (Waters, Perry, Han, Bielekova, & Gedeon, 2018).

Cytokines play a vital role in host cell normal immune responses. When a severe immune reaction occurs, the body could synthesize and release too many cytokines into the bloodstream quickly. However, having overproduction of this immune cell (cytokines) in the bloodstream could be very harmful and as a result, a cytokines storm may occur, which leads to infection, organ failure, high fever, an autoimmune condition, and other associated disease (NIH, 2020). These conditions may occur during the flu infection, lung inflammation, and fluid buildup, causing pneumonia and might cause mortality in patients (The Scripps Research Institute , 2014).

However, not only the allopathic treatment has been considered for treating the COVID-19. China has been using traditional medicine along with allopathic drugs for prophylactic and treatment of this COVID-19. Besides, our Indian traditional system does not just boost immunity but also have antiviral properties (Kumar et al., 2012). Curcuminoids, a mixture of three different curcumins are isolated from the spice herb of Curcuma longa, Indian saffron, showed the effectiveness in several preclinical and clinical study data and used in the prophylaxis and treatment of diverse diseases such as cancer, cardiovascular disorders, inflammatory responses, metabolic and neurological disorders, and skin problems (Kunnumakkara et al., 2017). The immunomodulatory effect of this active ingredient might be due to the interaction with assorted immunomodulators like cellular components, macrophages, lymphocytes, cytokines, and other transcription factors (Momtazi-Borojeni et al., 2018).

Keeping all the above facts in our minds, we have designed eight co-drugs by combining the Curcumin, a well-known plant-based immuno booster along with hydroxychloroquine markedly available prophylactic treatment drug for COVID-19. In this present scenario with this life-threatening virus, failure to find the targeted therapy and treatment options to this situation is inadequate, unfortunately. However, to accelerate the discovery of the novel leads, we move forward to think of an alternate remedy for prophylaxis and treating this situation. In order to achieve this strategy, we have chosen the in-silico molecular modeling approach to design the novel co drugs to combat this COVID-19.

Methods

Computational workup

Computer-assisted simulated docking experiments were carried out under an MMFF94X force field on COVID-19 main protease (PDB Id-6LU7, Figure 1 a)

and IL-1β (PDB Id- 6Y8M, Figure 1 b) using Chemical Computing Group’s Molecular Operating Environment (moe-dock 2005) software, Montre´al, Canada.

Design of co drugs

The proposed scheme consists of three steps reaction as follows; In step 1, an equimolar mixture of curcumin is treated with 2-pyridyl acetic acid in the presence of pyridine, chloromethane to give the Product A. In step 2, animation process, an equimolar concentration of hydroxychloroquine is treated with amine derivatives (R) in the presence of specialized catalysts like ruthenium or iridium type of catalyst to get the Product B. In step 3 process, by merging step 1 and step 2 products together with the diverse linkers such as, primary amines, hydrazine, and carbazide compounds in the presence of the suitable solvents like ethanol and glacial acetic acid to obtain the proposed co-drugs 3a-3h (Figure 7; Figure 6; Figure 5).

Preparation of ligand files

The ligand files of eight proposed co drugs were constructed using the builder module of MOE-DOCK and the energies were minimized. The molecular geometries were drawn; correct 3D structures were ensured and followed by energy optimization at a standard MMFF94 force field level, with a 0.0001 kcal/mol energy gradient convergence criterion. The molecule builder of the (MOE, 2009). 10 program was used for this purpose and after building the molecule, the energy was minimized, potential and partial energy were corrected and then saved as a molecular database (.mdb) file in a local directory for the further process.

Selection of targets

The prime target, coronavirus main protease (Mpro), which is a chief culprit enzyme, guides in mediating the transcription and replication process of the virus (Anand, 2002; Yang et al., 2003) has been assigned for our study. There are 30,000 nucleotides have compressed together in the virus genome of novel COVID-19, its gene may perhaps encode with two polyproteins namely, pp1a and pp1ab, which are desirable for the viral replication and transcription (Wu et al., 2020; Zhou et al., 2020). The main proteolytic enzyme, Mpro, through the proteolytic process, could release the polypeptides from these polyproteins. Besides, this Mpro enzyme is the main protein in the viral cell life cycle together with the absence of closely related homologs of human protein, recognizes the Mpro as a promising target for the anti-viral drug discovery (Pillaiyar, Manickam, Namasivayam, Hayashi, & Jung, 2016). Therefore, the inhibition of Mpro may perhaps block the biosynthesis viral polyproteins, resulting in viral death. Interleukin-1β (IL-1β), signaling protein, a type of cytokines regulates and promotes the development of white blood cells mostly lymphocytes, which is responsible for immunity. IL-1 regulates the body’s natural response towards any microbial and viral pathogenic infection (Smith & Humphries, 2009). When a coronavirus enters into the body system, the immune capacity of the COVID-19 infected patients is suppressed that is characterized by a decrease in the T cell counts and the increase in immunosuppressive cytokines. However, in COVID-19 disease, the overproduction of IL-1 (immune-stimulatory cytokines) is thought to regulated by this co drug approach and thereby it could decrease the immune response towards this COVID-19 infection (Gao, Zhao, Wan, & Zhu, 2015; Guimond et al., 2009; Pedersen et al., 2014; Verdecchia, Cavallini, Spanevello, & Angeli, 2020).

Preparation of macromolecules

The coordinates of the crystal structure of COVID-19 main protease in complex with an inhibitor N3 (PDB Id: 6LU7; Figure 1 a) (Jin et al., 2020) and Interleukin-1 beta bound with bikinin (PDB Id: 6Y8M; Figure 1 b) (Nicola & Nichols, 2020), were retrieved from RCSB Protein Data Bank. These PDB files were imported into the MOE suite in which the receptor preparation module was performed to prepare the proteins. All the bound water molecules and hetero atoms were removed from the complex by using the sequence editor (SEQ) window, which is a default program MOE. Both the polar and non-polar hydrogens added and 3D structures were corrected. The binding pocket was identified via using MOE-Alpha Site Finder. In order to visualize the binding pocket, the alpha spheres were created followed by the generation of dummy atoms on the centers of these spheres (Figure 2). The pockets were found to be deep small canyons lined with the key residues, including both hydrophobic and hydrophilic amino acids.

Docking methodology

With an objective to explore the potential binding affinities of the proposed co drugs towards the validated COVID-19 Mpro enzyme and IL-1 beta, docking was performed. (Jin et al., 2020; Nicola et al., 2020). For docking simulations, the placement was set as a triangular matcher, rescoring was set as London dG, the number of retaining was set as 10, and the refinement was set as a force field to generate the 10 poses of each target ligand conformations (Figure 3). As a result of the docking run, the mdb.output files were created with scoring and multiple conformations of each compound. All the docked conformations were analyzed and the best-scored pose for each compound had been selected for further ligand interaction studies. Besides, the ligand-receptor interaction, followed by the surface analysis of the selected best pose ligand molecule, was generated and viewed for an interpretation.

Results and Discussion

The new series of proposed co-drugs are depicted in Figure 4. By combining hydroxychloroquine and curcumin scaffolds into one chemical signature through the hydrolysable linker, it is assumed that both drugs may act together synergistically for the treatment of the COVID-19 situation. When the pathogen enters the host cell, the inflammatory response mediates the host immune cell system and thereby eliminates the pathogen which causes the tissue repair. On the other hand, the highly pathogenic virus-like CoVs induces the prolonged cytokines response which could cause morbidity and mortality in CoVs affected patients. Owing to this excessive cytokines responses in CoVs infection guides to the viral sepsis, where the viral cell replicates further and experience the excessive uncontrolled systemic inflammation which could cause pneumonia, acute respiratory symptoms, respiratory and organ failure, shock, and eventually death (Aljuffali et al., 2016; Mesaik et al., 2012).

It is assumed and reported that coronavirus enters into the host cell through interaction with angiotensin-converting enzyme-2 (ACE-2) (Magrone, Magrone, & Jirillo, 2020). Many researchers showed that the high expression of ACE-2 may help to bind the cell membrane of the coronavirus to use its spike (S) glycoprotein to enter, replicate, and to infect the human cells, Many human cell enzymes like furin (Braun & Sauter, 2019), which is present in tissues, lungs, and small intestines, could help to synthesize this coronavirus S protein. During the infection, this S protein has cleaved into its subunit like S2 and S2 proteins (Kirchdoerfer et al., 2016). This S2 protein has the receptor-binding domain, whereas; the S2 protein plays a role in membrane fusion. This S1 protein helps the coronavirus to directly bind to the peptidase domain of the ACE-2 through the help of a host cell enzyme. Further, this enzyme helps to allow the virus-cell into the host and thereby it uses the host cell machinery for its replication. The ACE-2 is not only the receptor to accept the S protein of the coronavirus and also involved in post-infection regulation such as immune response (Verdecchia et al., 2020), B cell-mediated immunity regulation, and the regulation of cytokines secretion like IL-1, IL-10, IL-6, and IL-8 (Li et al., 2020).

At present, there is restricted information is available on host immune response towards CoVs. Zhoe et al., in 2020 reported that, in the investigation of 99 CoVs infected patients in Wuhan city, it was found that there is a considerable increase in total neutrophils, decrease in lymphocytes, and increase on serum IL-6 levels which in turn modulates the innate immune system. This report prompted us to investigate how the host immune system responds or recognize the invaded virus.

Herbal plants have rich in flavonoids, carotenoids, and Vitamin C, which could help to increase the host immunity and also promotes the activity of lymphocytes, increase the phagocytosis and induce the interferon production (Khodadadi, 2015). By understanding the pathophysiology of CoVs, i.e. an RNA virus, one might come to know that, the recognition of the virus by endosomal RNA leads to the activation of nuclear translocation. At this stage, the nuclei by the help of transcription factors induce the pro-inflammatory cytokines and this response is considered as the first level of defence mechanism to the entry of the virus into the host cell (Wit, Doremalen, Falzarano, & Munster, 2016). In CoVs infection, interestingly the transmission of the virus could occur even the infected individual does not show any such type of symptoms. This may be considered as the delayed early response of the immune system. Thus, the innate immune system plays a crucial role in protective or destructive responses. Based on the diverse studies provide evidence that this inflammatory cytokine plays an important role in the pathogenic condition. Consequently, the therapeutic intervention to target such type of pro-inflammatory cytokines could prove the undesirable inflammatory response in CoVs infection (Prompetchara, Ketloy, & Palaga, 2020). Interleukin-1 is a signaling protein necessary to activate the T cells by promoting the production of IL-2 (Berkenbosch, Rooijen, & Tilders, 1993). However, the Interleukin-1 (IL-1), which is a drug target for various immunological responses, plays a vital role in the activation and maintenance of an immune response and lymphocyte development (Sobia, Zaheer-Ul-Haq, & Sauleha, 2018).

Whereas, ACE-2 has been related to the CoVs infection, and mediates the cytokines and as well related to innate and adaptive immune responses, β-cell regulation, and enhanced the inflammatory responses by IL-1, IL-10, IL-6 and IL-8 (Huang et al., 2020). Hence, the viral infection causes tissue injury, and death is related to the pro-inflammatory process and this might be treated by the use of IL-1 β blockers, which may have some benefit in COVID-19 patients.

The co drug strategy is also beneficial to promote the targeting of drugs to specific proteins by the co drug approach. This strategy can be achieved by modifying the chemical structure and developing new carriers for co-drug delivery. With this aim, we have designed eight co drugs by combining the naturally available plant secondary metabolite curcumin along with presently used prophylactic drug for treating COVID-19 infection, i.e. hydroxychloroquine through the hydrolysable linker. All the docked conformations of the designed eight co drugs were found with the favourable docking poses. This fact was evidenced by the maximum number of amino acid interactions and ranked by the highest score based on the least binding energy (Table 1). The most favourable docking poses of the ten docked conformations for each molecule were analyzed for the further investigation of the ligand interactions within the protein active sites. All the co drug ligands showed a similar binding pattern and anchored tightly inside the active site canyon (Site I) of each protein. The 2D ligand-protein interactions have visualized for all the analogues. The identified compounds had a promising binding affinity with the Mpro receptors, in the narrow range of binding energy for the protein PDB Id: 6LU7 and the score range was between -28.2498 to -35.8648 kcal/mol: London dG was -8.4560 to -11.3205 kcal/mol when compared to the standard drug Remdesivir which had the binding score of -21.8600 kcal/mol. Similarly, all the designed co drugs showed the binding affinity with interleukin -1β in the narrow range of binding energy for the protein PDB Id: 6Y8M and the score range was 25.8032 to -34.6973 kcal/mol: London dG was -6.8749 to -9.7877 kcal/mol when compared to the standard co-crystal which showed the binding score of -17.3274 kcal/mol.

Further, the top docked confirmation of these designed co-drugs depicted a greater alignment with the native ligand pose. The information detailed in the Table 2 showed clearly that, how well the all active ligand molecules were bound and interacted with the amino acid residues through various pharmacophoric features such as side chain acceptor, side-chain donor, backbone acceptor, backbone donor, arene-cation, and arene-arene interaction, etc. with the receptor site (Table 2). The number of conformations generated by these entire molecules is in the range between 6 to 10, which indicates that the flexibility is an important parameter for the ligands to dock deeply within the binding pocket of COVID-19 and as well Interleukin-1β receptor site. Further, a careful calculation of surface analysis of the binding pocket of these molecules indicated that the entire compounds had adopted the position in a hydrophobic cage surrounded by the diverse amino acid residue and these have approached closely to the ligand molecule for the stronger interactions. Among the eight co drugs designed, the co drugs 3d and 3e were identified as topmost HITS. The two co drugs have shown a significant binding affinity towards both proteins when compared to the standards. Among the diverse hydrolysable linkers used in our study, viz. various primary amines, hydrazines, and carbazides; the phenylhydrazine and 2,4-dinitro phenyl hydrazine linkers have shown the increased binding affinity proving these linkers can serve as a building block in the preparation of further compounds.

Conclusions

The drug design by this co drug approach is one of the versatile and potent strategies that can be applied in a wide range of administration routes. Through this co drug approach, quite a number of issues are resolved like solubility, stability, drug resistance, and biomembrane permeability, etc. we hypothesized that there is a significant correlation between host immune system and the viral load. Base on the hypothesis, eight co-drugs were designed by merging the two pharmacophores, namely hydroxychloroquine (antiviral) and curcumin (immunomodulator) with a high probability of directly inhibiting the novel COVID-19 targets. To support our hypothesis, an in -silico docking study was performed. The docking results revealed that the eight designed co drug molecules (3a-3h) have shown significant docking scores with Mpro and as IL-1β protein when compared to the standard drugs. These designed co drugs could serve as useful probes for COVID-19 drug discovery. Of course, further in-vitro studies may require to prove the therapeutic action of these designed co-drugs on COVID-19 disease.