Triple Targeting of Human IMPDH and Both RdRps of SARS-CoV-2 and Rhizopus Oryzae: An in Silico Perspective

Mucormycosis has been reported in many regions associated with SARS-CoV-2 infections during the past few months. The viral RNA-dependent RNA polymerase (RdRp) is a crucial protein target in viral and fungal pathogens. Molecular docking combined with molecular dynamics simulation (MDS) is utilized to test nucleotide-based inhibitors against the RdRps of SARS-CoV-2 solved structure and Rhizopus oryzae RdRp model built in silico. Additionally, the human Inosine monophosphate dehydrogenase (IMPDH) was targeted by the same inhibitors. The results reveal a comparable binding a�nity of four nucleotide derivatives compared to remdesivir and sofosbuvir against both IMPDH and the RdRps of SARS-CoV-2 and Rhizopus oryzae, the main causing agent of mucormycosis. The binding a�nities are calculated using different conformations of the RdRps after 100 ns MDS and trajectories clustering. The present study suggests the triple inhibition potential of four nucleotide inhibitors against SARS-CoV-2 & R. oryzae RdRps and the human IMPDH, while experimental validation is yet to be performed.


Introduction:
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and its associated pneumonia (COVID- 19) caused health and economic burdens during the last two years.The currently developed pandemic of Mucormycosis in COVID-19 patients and COVID-19 recovered people is causing a debate about the therapeutics approved against COVID-19 (1-3).Several in silico studies are able to suggest possible medicines against COVID-19 even before the deposition of any solved structures for the viral proteins (4)(5)(6).Some of the recommended drugs are now approved by the Food and Drugs Administration (FDA) and save lives (6-8).
The viral RNA-dependent RNA polymerase (RdRp) is one of the most targeted viral proteins in many diseases caused by RNA viruses (6, [9][10][11][12].The active site aspartates of the RdRp is very conserved even in fungal species of Mucorales (13).On the other hand, the human Inosine monophosphate dehydrogenase (IMPDH) has been identi ed as a key enzyme in cell proliferation and differentiation regulation (14,15).IMPDH has recently emerged as an important therapeutic target in the expedition to discover drugs belonging to the immunosuppressive, antiviral, antimicrobial, and anticancer therapeutic era.IMPDH catalyzes the de novo synthesis of purine (guanine) nucleotides, the nicotinamide adenine dinucleotide (NAD)-dependent oxidation of inosine 5-monophosphate (IMP) to xanthosine 5monophosphate (XMP).The rst step of IMPDH reaction involves the attack of catalytic (Cys331) on substrate IMP followed by hydride transfer to NAD+, forming the covalent intermediate E-XMP*.E-XMP* is hydrolyzed during the second step, yielding the product XMP (16,17).
Inhibition of IMPDH produces an overall reduction in guanine nucleotide pools.Subsequent interruption of DNA and RNA synthesis results in cytotoxicity inhibition of IMPDH is also used as a strategy in immuno-suppressive therapy.The growth and differentiation of human lymphocytes are particularly dependent on the IMPDH-catalyzed de novo pathway for purine nucleotide synthesis (18).Inhibition of IMPDH leads to suppression of both T and B lymphocyte proliferation.For compounds targeting the IMP-binding site, development has focused on the nucleotide analog.The competitive IMPDH inhibitors for IMP, such as ribavirin (19) and mizoribine (20), are nucleoside analogs that undergo phosphorylation to the active inhibitor (triphosphate) in vivo.
Thuringiensin (Thu), also known as β-exotoxin, is a thermostable secondary metabolite secreted by Bacillus thuringiensis.It has insecticidal activity against many insects, including species belonging to the orders Diptera, Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Isoptera, and several nematode species.The insecticidal mechanism of Thu is not fully understood.However, it is known to be an ATP analog (21).The chemical formula of Thu is C 22 H 32 O 19 N 5 P, and it is composed of adenosine, glucose, phosphoric acid, and gluconic diacid.Thu inhibits RNA synthesis by competing with ATP on binding sites, affecting insect molting & pupation, and causing teratological effects at sublethal doses (22)(23)(24).
In this study, our approach is to test four nucleotide inhibitors based on Thu against the RdRps of SARS-CoV-2 and Rhizopus oryzae in addition to the human IMPDH.Molecular docking combined with dynamics simulation emphasizes the binding effectiveness using computational tools that are yet to be validated experimentally.
On the other hand, the three-dimensional structures of the receptors were retrieved from the Protein Data Bank (http://www.rcsb.org//pdb)(28).The selected PDB les for the SARS-CoV-2 RdRp and the human IMPDH were 7BTF and 1NF7, respectively (29).No solved structure was deposited yet for the R. oryzae RdRp in the protein data bank, so we used the previously in silico generated all-atoms 3D-model by the Swiss Model webserver (13,30).This model was subjected to 100 ns MDS in a previous study and clustered through UCSF Chimera software (31).Seven different conformations of the R. oryzae model representing the most populated clusters are tested against the inhibitors.The structure 7BTF was subjected in a previous study to 100 ns MDS and then clustered into ten clusters (see Figure S1).We use the representative structure from each cluster to test the binding of the nucleotide inhibitors.

Molecular docking
The receptors were stored in PDB format after removing the water molecules and ligands with PyMOL software (32).Then polar hydrogen atoms and Kollman charges were added to the receptors using Autodock Tools 1.5.6 then saved in PDBQT format for the docking experiments.Autodock Vina was used for the docking of the ligands to the receptor using exible ligands and a exible active site protocol (33).The grid box was centered at the active residues (D760 & D761 for SARS-CoV-2 RdRp and D193 & D194 for R. oryzae RdRp), de ned by the native co-crystallized ligands, while box size was set to (40 × 40 × 40) Å 3 .On the other hand, the grid box size for the human IMPDH was set to be (40 × 60 × 64) Å 3 and centered at (76.7, 83.8, 53.9).The active site for the human IMPDH was S68, N303, R322, S388, Y411, M414, and Q441.Other docking parameters were set to default while the exhaustiveness value was adjusted to 8 in all the docking trials.Discovery studio software is utilized to analyze the docking complexes (34).

Results And Discussion
In the current study, four nucleotide inhibitors are tested against both host-cell (Inosine monophosphate dehydrogenase) and pathogen's proteins (RNA-dependent RNA polymerase).These proteins proved their effectiveness as drug targets (6, 13,35,36).Figure 1 shows the 2D structures of the four nucleotide inhibitors which are Thuringiensin derivatives (CID: 99213, 194147, 20056441, and 102074352) and the positive control drugs (Sofosbuvir and Remdesivir).The FDA approves these drugs against RNA viruses such as Hepatitis C Virus, Ebola virus, and SARS-CoV-2 (37)(38)(39)(40)(41).The four Thuringiensin derivatives are based on the nucleotide adenosine (A), where the added moieties are present at the 5' position of the ribose ring.On the other hand, the positive controls drugs (remdesivir and Sofosbuvir) are adenosine triphosphate modi ed at position 1' of the ribose (C ≡ N) and uridine derivative modi ed at position 1' of the ribose (F, methyl), respectively.

Binding a nities of the nucleotide inhibitors against the pathogenic proteins
As shown in Fig. 2, the average binding a nities of the four nucleotide inhibitors (blue columns) (CID: 99213, 194147, 20056441, and 102074352) against SARS-CoV-2 RdRp (A), Rhizopus oryzae RdRp (B), and the human IMPDH (C) are in the same range of the positive control drugs.In Fig. 2, the error bars represent the standard deviations.Red columns in Fig. 2 represent the positive control drugs (Sofosbuvir and Remdesivir).The docking trials are performed on different conformations of the RdRp proteins generated from the 100 ns MDS runs, while Each conformation resembles a cluster from the trajectories.The MDS's role was to equilibrate the protein systems and ensure different possible conformations during the simulation period.
The average binding a nity for the four nucleotide inhibitors lies between − 6.84 kcal/mol (194147 versus SARS-CoV-2 RdRp) and − 9.8 kcal/mol (102074352 versus human IMPDH).For the SARS-CoV-2 RdRp, the average binding a nity for the four nucleotide inhibitors is -7.24 kcal/mol, while the average binding a nities of sofosbuvir and remdesivir against SARS-CoV-2 RdRp are − 7.4 and − 7.3 kcal/mol, respectively.Additionally, the average binding a nity of the four nucleotide inhibitors against R. oryzae RdRp is -7.63 kcal/mol, while it is -8.06 and − 7.8 kcal/mol for the sofosbuvir and remdesivir, respectively.These values indicate that the Thuringiensin derivatives have comparable average binding a nities to that of Sofosbuvir and Remdesivir against the viral and fungal RdRps.
On the other hand, the average binding a nity of the four nucleotide inhibitors against the human IMPDH is -8.71 kcal/mol, which is lower (better) than the average binding a nity of the positive controls (-8.3 and − 7.85 kcal/mol for sofosbuvir and remdesivir, respectively).The best compound from the Thuringiensin derivatives in binding all the proteins is the Thuringiensin 6,3-lactone (CID: 102074352).In order to check the binding mode of each ligand to the three proteins, the interaction map of the complexes is mined by the Discovery studio software.Based on the analysis, the compounds' binding modes against SARS-CoV-2 RdRp, R. oryzae RdRp, and human IMPDH are explored and tabulated.
Table 1 The interactions that were established after docking the four adenosine derivatives and positive controls (Sofosbuvir and Remdesivir) into the SARS-CoV-2 RdRp, Rhizopus oryzae RdRp, and the human IMPDH.
Red-colored residues are residues that interact with salt bridges, while green-colored residues are that interact through halogen bonds.Bold residues are the active residues in each protein.Figure 3 shows the interactions that established between Thuringiensin 6,3-lactone (CID: 102074352), and the SARS-CoV-2 RdRp (top left), R. oryzae RdRp (top write), and the human IMPDH (down).This compound shows the best average binding a nities against the three proteins (see Fig. 2) compared to other nucleotide inhibitors.The current work reveals the favorable binding a nity of four adenosine derivatives against the SARS-CoV-2 RdRp, Rhizopus oryzae RdRp, and the human IMPDH.Thuringiensin 6,3-lactone has the best binding a nity to the three proteins, hence can be a potential inhibitor against COVID-19/Mucormycosis coinfection, while experimental validation is yet to be performed.

Supplementary Files
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Figure 2 The
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