Inhibition of Viral Macrodomain of COVID-19 and Human TRPM2 by losartan

Background: The SARS-CoV-2 virus is a new highly contagious Coronavirus with a positive-sense RNA encoding 16 non-structural proteins (nsps16, nsp15, nsp3). In this study, the coronavirus pathogenicity and the losartan functional ligand for inhibiting TRPM2 and macrodomain have been molecularly evaluated.Material and method: In this study, the structures of macrodomain binding ADP ribose in CoVs and human Transient Receptor Potential Cation Channel Subfamily M Member 2 (TRPM2) protein were downloaded from protein data bank. Then, a virtual screening was done to recognize the hit compounds from GalaXi_2019-10, KnowledgeSpace_2019-05, and REALspace_2019-12 databases. This collection, then, was imported to the ligandScout software, on the base of Adenosine Diphosphate Ribose (ADPR) pharmacophore model. Result: Among seven compounds, five compounds were finally evaluated as the structural analogs of ADPR or other nucleotides, from which one compound was a non-FDA-approved sulphonamide and was removed. The other compound, losartan, was finally selected for molecular docking and molecular dynamic simulation. According to the virtual screening and docking, losartan was candidate as an effective ligand for TRPM2 and macrodomain. Conclusion: In the current study losartan earned a proper dock score and binding affinity to create the complexes with TRPM2 and macrodomain. The inhibitory effect of losartan on PARP has been shown and it could interfere positively in several points (PARP, PARG- macrodomain and TRPM2) and decreases oxidative stress and apoptosis in COVID-19.


Introduction
At the end of 2019, the emergence of the novel coronavirus infection in Wuhan, China, it has rapidly spread across China and many other countries. The International Committee on Taxonomy of Viruses has been named SARS-CoV-2 and the disease it causes has been named coronavirus disease 2019 . The SARS-CoV-2 virus is a new pathogen that is highly contagious, which can spread quickly (1). This virus must be considered capable of causing enormous health, economic and societal impacts with their setting The SARS-CoV-2 virus like SARS-CoV and MERS-CoV, is a beta-coronavirus which all of them have originated from bats.
On 30 January 2020, the World Health Organization (WHO) declared that the SARS-CoV-2 outbreak constituted a Public Health Emergency of International Concern (2). The outbreak has rapidly evolved, and more than 180 countries worldwide have reported laboratory-confirmed cases of SARS-CoV-2 (3).
Coronaviruses (COVs) are positive-sense enveloped RNA viruses which are larger than the other RNA viruses, encoding 16 non-structural proteins (nsps). They have different proteins that are important for preventing and blocking the innate immune response, including enzymes such as an endoribonuclease (nsp15), an O-methyl transferase (nsp16), and an macrodomain with ADPribosyl hydrolase (nsp3) (4). The macrodomain of nsp3 harbors several conserved domains in which some enzymatic activities have been attributed. COVs macrodomain has a close functional association with poly (ADP-ribose) polymerases (PARPs), which is known as the regulator of apoptosis in eukaryotic cells. PARPs regulate important cellular processes such as cell-cycle progression, cell division, host-virus interaction, DNA damage repair, genome maintenance, transcription regulation, protein degradation, ageing, and cell death. PolyADP-ribosylation can be removed by the action of two divergent enzymes, poly (ADP-ribose) glycol hydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3) (5,6). PARG is a catabolic enzyme that cleaves ADP-ribose (ADPR) polymers that synthesized by members of the PARPs family of enzymes.
The Viral macrodomains involve in cellular signaling consist of; ADP-ribose-generating orconsuming pathways and roles in determining RNA synthesis, replication, and virulence. This protein hydrolase ADPR polymer from ADP-ribosylated proteins. ADPR-binding to the CoVs nsp3 macrodomain that is necessary for the initiation of virus replication, whereas the hydrolase activity facilitates to amplify the replication complex. Therefore, macrodomains can remove ADP-ribosy polymer from modified proteins (7).
The antiviral properties of PARPs are regulated by interferon (IFN), and under evolutionary selection, it has been given that primary function of viral macrodomain is counter the host response viral infection. Accordingly, macrodomain by removing ADP-ribosylation from modified host proteins, prevent host responses to viral infection (8).
On the other hand, PARG activity of MD leads to increase the level of free ADPR in the cell, which activates the Transient Receptor Potential 2 (TRPM2). TRPM2, a subtype of TRPmelastatin subfamily (TRPM), is a multifunctional Ca 2+ permeable and nonselective cation channel. Exposure to hydrogen peroxide (H2O2), tumor necrosis factor α (TNFα), reactive oxygen and nitrogen species is generally accepted to promote channel activation through the generation of ADPR, an agonist for TRPM2 (9). It performs as an important Ca 2+ signaling regulator in a variety of cells, contributing to cellular functions including cytokine production, insulin release, cell motility, oxidative stress and cell death. TRPM2 activation cause to induce Ca 2+ influx, and providing positive feedback for channel activation, led in necrosis and cell death (9). The Severe inflammatory response, cytokine storms, and necrosis in the patient's lungs with COVID-19, can explain the activation of TRPM2 signaling pathway, which can result the action of macrodomain or stress conditions that lead to accumulation of ADPR. Therefore, both macrodomain and TRPM2 inhibitors blocked calcium influx through TRPM2, inhibiting the cytokine storms, tissue necrosis, protecting cells from plasma membrane damage and cell death (9,10).
Losartan is an angiotensin-converting enzyme (ACE) inhibitors that used to treat hypertension. ACE inhibitors are used for a similar indication but associated with a cough (11). Here, we showed that Losartan is a functional ligand for inhibition of the TRPM2 and macrodomain and has a high affinity to them. The comparative data have shown Losartan is effective agent for inhibition of the TRPM2 and macrodomain.

Protein and ligand preparation
High-resolution X-ray diffraction SARS CoV-2 of macrodomain binding ADP ribose phosphate and human TRPM2 protein structures were downloaded from protein data bank (https://www.rcsb.org/pdb). The exported files included 6W02 consists of two COVID-19 macrodomain molecules complexes with ADP ribose phosphate as the main ligand. Because macrodomain has only one monomer, we elected, chain A of the PDB structure, and other additional parts were omitted. Another exported file, 6PUR contains four TRPM2 molecules complexes with ADP ribose phosphate. Among the four molecules, chain A was selected, and extra parts were removed. In chain A, sequence between residue 1234 to 1534 was selected because it forms biding site of TRPM2, and this sequence which has been located in the cytoplasmic part involved in the cell cascade (12,13).
All hydrogen atoms were added by the Discovery Studio software 2.5 (DS, Accerlys Inc, San Diego) and the proteins were prepared for the docking and virtual screening. Energy minimization was performed through a simulation module of Discovery Studio by conjugate gradient method and CHARMm force field (12,13). This process was done with energy gradient of 0.1 calÅ −1 . Ligands' 3D structures were prepared and minimized by the Marvin Suite software (14).

Chemical library
The procedure of this study shown the Figure 1. The virtual screening was done to recognition of the hit compounds from the GalaXi_2019-10, KnowledgeSpace_2019-05, and REALspace_2019-12 databases in biosolveit (https://www. biosolveit.de/) with infinisee software (15), for finding similar compounds to ADP ribose phosphate. The all current databases collections include up to 2.9×10 14 compounds, which consist of every compound on the world, but in this study on the base of the necessity and urgency of investigation about coronavirus treatment was more focused on the GalaXi_2019-10 database that has drug-like and all drug library (other databases is not removed).
Primary virtual screening was performed over all databases by the infiniSee software (ligand base, similarity 30-100), resulted in to obtain 20 compounds. This collection was imported to the ligandScout software and converted to ligandScout library, then, on the base of the ADPR pharmacophore model (two hydrogen donors and six hydrogen acceptors) (figure2) secondary virtual screening was done. After that, seven compounds were obtained by high pharmacophorefit score. Figure 2 Pharmacophore model of molecules that are similar to ADPR and pharmacophore of ADPR Five of seven obtained compounds, were a structural analog of ADPR or other nucleotides, so these compounds were removed due to the potential toxic effects on human DNA and RNA polymerase. One compound was sulphonamide which was removed due to not being FDAapproved drug. The final compound, i.e. Losartan was finalized for the molecular docking step.

Molecular docking
In this step, we used SeeSAR 9.2, AutoDock Vina module of ligandScout 4.3, and Molegro Virtual Docker v6.0 to investigate the conformational of Losartan in interaction with macrodomain and TRPM2 proteins (16,17). At first SeeSAR 9.2 was used for a primary docking of Losartan. The applied parameters were used the genetic algorithm. The population size, selection pressure, and number of run were 100, 1.2 and 100, respectively. Conformations that were obtained in this step were used for final docking with Molegro Virtual Docker and AutoDock Vina, and the best affinity score was estimated between our load conformations (18,19). The population size and the number of run in this step were 500 and 200, respectively.

Profiles of interactions between Losartan and proteins were obtained via Discovery Studio
Visualizer and LIGPLOT software (20).

Molecular dynamic simulation
The molecular dynamic simulation was performed by AMBER18 package (21), and LEAP module of AMBER18 that was used for adding hydrogen to atoms. The ligand charges were generated by AM1-BCC method of Antechamber module. The topology of macromolecules was created by AMBER99SB-ILDN force field and tleap module of AMBER tools (22,23). The generalized AMBER force field (GAFF2) was used for the ligand topology building with Antechamber module of AMBER18. Then the complexes were solvated in a truncated octahedral box of TIP3P. The water molecules with a buffer size of 10 Å, were neutralized by Na + or Clions for each complexes. The default protonation of Amber18 was used for titratable residues in protein. The energy minimization and MD simulations were used for each system with the sander module. Ten thousands cycles of minimization were performed with the steepest descent; and conjugate gradients for all parts of the systems were done to remove the bad steric interactions and get better minimum energy. Then, the position restraints at the constant volume (NVT) for 100 ps, with a restraint force of 10 kcal/mol at temperature of 100 K, and then at constant pressure (NPT) for 100 ps, with a restraint force of 1 kcal/mol at temperature of 300 K, were performed. Afterward, an equilibration step at NPT was performed for reaching to equilibrium in density for 100 ps, while the restraint force was removed. This method allows the water to equilibrate around the complexes. In the position restraints and equilibration steps, Langevin dynamics were used to control the temperature. In final step, 20 ns (nanosecond) MD simulation was used for each protein-ligand complex. For analyses of MD simulations , CPPTRAJ of AMBER18 was used (24). If the enthalpy change (ΔH) was negative numbers, the reactions between ligands and proteins would be exothermic.

The virtual screening for macrodomain and TRPM2
After the primary virtual screening on the database by the infiniSee software, a collection containing of 20 ligands was obtained table1. Then, seven compounds were screened after pharmacophore modeling by ligandScout with pharmacophore-fit score 86.16 -85.56 (table2 and fig2). Losartan (with pharmacophore-fit score 85.26) was between of them, and other compounds because were nucleotides or similar to nucleotides (such as guanosine ribose phosphate and other nucleotides or their analog) were removed, Losartan was selected as a hit compound.

ADP ribose and Losartan in the TRPM2
It was shown that ADP ribose phosphate, and losartan docking scores in the Molegro software were -149 and -157, respectively (table3). These ligands had a similar arrangement of score in both proteins in the Seesar and Autodock vina software tools. Losartan was exhibited four hydrogen binds with Asn1487, Arg1433 and Ser1382 residues within the range of 1.8 -2.8 Å. A total of 13 hydrophobic bonds were found with Pro1388, Val1377, Leu1379, Ser1382, Ala1386, His1348, and Tyr1349 in the range of 3.1-3.9 Å. The highest hydrophobic interactions were observed in Ala1386 with three contacts ( figure 4 and table 4).

Molecular dynamic simulation
For checking the stability of the protein-ligand complexes, 20 ns molecular dynamic simulations were used to all complexes. Figure   These results also are similar to RMSD and RMSF results.

Discussion
The worldwide spread of the coronavirus became a severe and significant threat to human health.
World health organization have announced that the coronavirus becomes a pandemic disease (1)(2)(3). As yet, there is not any precise treatment for coronavirus. In some articles have suggested that some of the protease inhibitors, remdisivir, and chloroquine are effective on the COVID-19, but have not shown an efficient and potent therapeutic effect on this virus. This limitation and threat made us work on some drug databases that are FDA approve and some drug-like databases. In the primary virtual screening by infinisee, the new bank had created by some molecules with relative similarity to ADPR. Next, pharmacophore modeling was shown seven molecules that have similar pharmacophore. Between them, because five compounds were analog of nucleotide, they were omitted. Nucleotides analog have toxicity for human cells and bodies, so were removed.
Criteria for selection of the ligand was six terms consist of: Scalaradial is not an available compound and, more importantly, is not as an FDA approved drug. Then there is an urgent necessity for drug and inhibition of the TRPM2 and macrodomain for treatment of any person affected by COVID-19 (28).
Our knowledge Until now, there was not any study for the inhibition of the macrodomain and its function. This study is for TRPM2 and macrodomain inhibition by a bioinformatics approach with the utilization of the drug and drug-like database. Particularly as now, there is no study for inhibition any coronavirus's macrodomain (that has high affinity to bind ADPR). It seems the activity of MD protein of COVID19 runs a lethal cycle between PARP, MD and TRPM2 Zeinalian, M., et al explained this cycle and suggested that PARP, MD (NSP3 of virus) and TRPM2 are critical therapeutic targets in COVID19 (29).
In this study, it has been focused on the coronavirus pathogenicity and mechanisms that how the human cell can combat them. It was concluded that viral macrodomain has the essential roles for the initiation of the cell infection and the virus-cell cycle. Macrodomain with poly ADPR degradation and increase of the free ADPR's levels can provide activation of the TRPM2 and prevent of the produce interferon-gamma so that cell would be susceptible to COVID-19 infection; therefore, it has been suggested as the target protein for efficient therapeutic approaches against the coronavirus infection.
Some different studies have shown that ACE inhibitors such as Losartan could have a protective effect on some tissue injury due to the systemic inflammatory diseases. Losartan has antiaggregative platelet, anti-diabetic, and prevent end organ injuries and fibrosis. Also in the disease that affected with chronic kidney disorder, and blood pressure, prescription losartan led to decrease from kidney damage. In another study has shown that Losartan was able to prevent

Conclusion
It seems Losartan can inhibit the viral macrodomain and TRPM2 so that it can interrupts the cell cycle of the virus and apoptosis, necrosis, fibrillation, and cytokine storm consequence of the COVID-19 infection. Therefore, Losartan, with preventing the induction of overexpression of the inflammatory cytokine, may decrease the severity of the disease and time of hospitalization.
Presumably, Losartan with inhibition of these proteins activate the production of the interferongamma and intracellular defense against the COVID-19 virus, so enhancement resistance of person to the coronavirus. Docking score in Molegro Virtual Docker and docking affinity in the AutoDock Vina software tools. Docking score and binding affinity of the Losartan is very close to ADPR, even higher than the ADPR.       B-after the interring to the cell, virus RNA convert to the viral nsps proteins. One of these nsps is the macrodomain which degrade poly-ADPR from the protein, so prevent the IFNγ signaling in the cell, and increase ADPR volume in the cell. A-Red arrows show that increasing of ADPR in the cell active the TRPM2 and its signaling cascade consequence of its activating, so cytokine storm, and apoptosis signaling cascade will initiate. D-losartan can bind to the viral macrodomain, and human TRPM2 and prevent their function, therefore cytokine storm will halt, and human cellular immune response will increase