Isolation of Thioinosine and Butenolides from a Terrestrial Actinomycetes sp. GSCW‐51 and Their in Silico Studies for Potential against SARS‐CoV‐2

Abstract In our continuous screening for bioactive microbial natural products, the culture extracts of a terrestrial Actinomycetes sp. GSCW‐51 yielded two new metabolites, i. e., 5‐hydroxymethyl‐3‐(1‐hydroxy‐6‐methyl‐7‐oxooctyl)dihydrofuran‐2(3H)‐one (1), 5‐hydroxymethyl‐3‐(1,7‐dihydroxy‐6‐methyloctyl)dihydrofuran‐2(3H)‐one (2), and two known compounds; 5′‐methylthioinosine (3), and 5′‐methylthioinosine sulfoxide (4), which are isolated first time from any natural source, along with four known compounds (5–8). The structures of the new compounds were deduced by HR‐ESI‐MS, 1D and 2D NMR data, and in comparison with related compounds from the literature. Additionally, owing to the current COVID‐19 pandemic situation, we also computationally explored the therapeutic potential of our isolated compounds against SARS‐CoV‐2. Compound 4 showed the best binding energies of −6.2 and −6.6 kcal/mol for Mpro and spike proteins, respectively. The intermolecular interactions were also studied using 2‐D and 3‐D imagery, which also supported the binding energies as well as put several insights under the spotlight. Furthermore, Lipinski's rule of 5 was used to predict the drug likeness of compounds 1–4, which indicated all compounds obey Lipinski's rule of 5. The study of bioavailability radars of the compounds 1–4 also confirmed their drug likeness properties where all the five crucial drug likeness parameters are in color area, which is safe to be used as drugs. Our isolation and computational findings highly encourage the scientific community to do further in vitro and in vivo studies of compounds 1–4.


Introduction
The increasing resistance of microbes to antibiotics has become a major global issue. Infections caused by multidrug resistant organisms, such as bacteria, fungi, and viruses, require therapeutics with novel chemical scaffolds and/or modes of action. The discovery of new natural compounds is still crucial for the development of new antimicrobial agents. The majority of antimicrobial medicines currently in use are of natural origin. [1] Actinobacteria remain a significant source of new secondary metabolites for drug development among natural sources of bioactive compounds. The most common group of microorganisms that produces the bioactive compounds is actinobacteria. They produce two-thirds of all naturally derived antibiotics used in medicine, veterinary medicine, and agriculture today. The majority of these compounds are from the genus Streptomyces. [1 -3] Varied microorganisms isolated from different environments produced variety of novel bioactive compounds, particularly those who have unique chemical structures and biological importance. [4] Actinomycetes, the soil Gram-positive bacteria, are producing wide variety of bioactive secondary metabolites. More than 140 Actinomycete genera have been discovered so far, however, only few of these are studied for their bioactive chemicals. [5] Most common antibiotics provided by this group include tetracyclines, streptomycin, dactinomycin and streptothricin, erythromycin and its derivatives, chloramphenicol and rifamycins etc. [6] Ahmpatinin i Bu, pentapeptide 4862F are recognized as the strongest antiviral agents from Actinomycetes [1] besides, antimycin A1a is also a potent antiviral compound from Actinomyecets. [7] In the present study, a terrestrial Actinomycete sp. GSCW-51 was investigated for its bioactive secondary metabolites. Two new butenolides (1 and 2), two new natural thioinosine derivatives 3 and 4 ( Figure 1) along with known compounds 3-methoxy-4,5-dihydroxybenzoic acid (5) [8] 3-methoxy-4-hydroxybenzoic acid (6), [9] tyrosol (7), [10] 4-(4'-hydroxyphenyl)butan-2-one (8) [11] were separated from the culture broth of this microorgan-ism. Further, in recent global pandemic of COVID-19 caused by SARS-CoV-2 is proving destructive to the world communities without boundaries. There is dire need to check every possibility to find a much-needed therapeutic drug candidate for SARS-CoV-2. As describe above the compounds isolated from Actinomycetes have antiviral properties and due to recent scenario of covid-19 pendemic, the isolated metabolites from the Actinomycetes GSCW-51 were studies in silico against SARS-CoV-2. Along the above stated lines, the activity of selected Actinomycetes was also checked against the main protease M pro and spike proteins of SARS-CoV-2 by using the molecular docking techniques. The M pro and spike proteins play crucial roles in protein replication and attachment of virus to the host cells, respectively. So, the study of intermolecular interactions of Actinomycetes with these two crucial proteins can bring several novel insights the therapeutic effects of our isolated Actinomycetes. The aim and objectives of the current investigation are twofold; isolation and characterization of metabolites from the Actinomycetes GSCW-51 and to perform molecular docking analysis for studying antiviral potential against SARS-CoV-2 which has caused deadly pandemic of COVID-19.
Pseudo-molecular ion peak in ( 1H), 2.88 (dd, J = 14.1, 5.9, HbÀ C(5'), 1H) and a methyl group at δ 2.10 (s, HÀ C(1''), CH 3 ). Consecutive COSY interactions from H-1' to H-5' disclosed a furanoside moiety in 3, which could further be substantiated through 13 C-NMR spectrum ( Table 2). Downfield carbon resonances at δ 158.9, 150.0, 146.8, 140.8 and 125.8 were in agreement with chemical shifts of hypoxanthine in ostrerine A [21] and the shimofuridins, [22] however, difference was found in C-chemical shifts of furanoside moiety where the up-field shift (δ 37.6) of the methylene signal pointed to a connection with sulfur instead of nitrogen or oxygen, respectively. The methylene protons showed HMBCs (Figure 4) with the methyl carbon resonance at δ 16.5 and vice versa indicating that sulfur is present between these groups; further HMBCs are shown in Figure 4.
These data and assignments made on the basis of HSQC finally disclosed compound 3 as 5'-methyl-5'thioinosine, which is new as natural product, however, was synthesized previously; [23] the reported 1 H-NMR data were identical with our data within narrow limits. In biosynthetic pathway, compound 3 is proposed, as deaminated version of 5-methylthioadenosine, a known metabolite in the methionine salvage pathway. The structural variety of naturally occurring thiomethylpentosides is very low however, the closely related 5'-S-methyl-5'-thioadenosine has been isolated from various sources. [24] Compound 4 was found to be sulfoxide analog of 3, since the HR-ESI-MS in positive mode displayed pseudo-molecular ion peak at m/z 315. S, with one oxygen atom more than in 3. The 1 H and 13 C-NMR spectra ( Table 2) were nearly identical to those of 3, with exception of the downfield shifted signals for the C-5'-methylene and the Smethyl group, and a surprising doubling of most 1 H and 13 C-NMR signals ( Table 2). Compound 4 is there-  Chem. Biodiversity 2022, 19, e202100843 www.cb.wiley.com (4 of 14) e202100843 fore 5'-methylthioinosine sulfoxide and occurs as a mixture of diastereoisomers due to the chiral sulfenyl group ( Figure 5).
It an unusual metabolite, which was thought to be an artifact during isolation procedure, however, the possibility of such a non-specific oxidation is ruled out because of the use of non-oxidative isolation protocol. Further several sulfoxides are reported as natural products, [25] especially from Allium species. [26] In addition, Streptomyces (a genus of Actinomycetes) are used for enantioselective sulfoxidation of sulfides, [27] which authenticate compound 4 as a new natural product. As for 3, the adenine derivative corresponding to 4 was described previously, [25] it is an inhibitor of platelet aggregation. Both 3 and 4 were inactive in cellular HIV tests. Due to the isolation of lesser amount we were not able to perform biological activities of these compounds, but the butenolides have gained special attention as α-glucosidase, cytotoxic and antiinflammatory agents. [28][29][30] Previous research has identified them as distinguishing characteristics of αglucosidase inhibitory actions. Recently butenolides showed the promising activity against the influenza viruses. A number of compounds were tested in vitro and in vivo against the influenza A virus H1N1 and was found that influenza A virus H1N1 could be inhibited by butenolides, by targeting the Neuraminidase   enzyme. [31] Further a natural butenolide (4R,6S)-2dihydromenisdaurilide (DHMD), as an efficient inhibitor of Hepatitis C virus (HCV). [32] On the other hands, sulfur-containing purine nucleoside analogs were considering important agents, which inhibit the RNA viruses. Further sofosbuvir thio-analogs and 2'-deoxy-4'-thioribo purine nucleosides derivatives also showed good antiviral activity. [33 -35] So, computationally study was also performed to explore the therapeutic potential of our isolated compounds against SARS-CoV-2 due to COVID-19 pandemic situation.

Computational Study Binding Energy and Inhibition Constants of Compounds 1-4 against SARS-CoV-2
The two most crucial parameters in molecular docking are binding interaction energy and inhibition constants for ligands docked with a macromolecule of target protein. The binding energies and inhibition constants have an inverse relationship where larger negative value of binding energy and lower value of inhibition constant indicates a good interaction between ligand and protein molecules. [36] The binding energies and inhibition constants for compounds 1 -4 with M Pro and spike proteins were shown in Table 3. A careful analysis of Table 3 reveals that binding energy of compounds 1 -4 with M Pro ranged from À 5.1 to À 6.2 kcal/mol and the increasing order (where negative sign indicates favorable interactions) of binding energies is 4 > 2 > 3 > 1 for M pro protein. A graphical comparison among the binding energies of the all compounds for M pro is shown in Figure 6 where the lowest and the most favorable energy is seen for compound 4 which is À 6.2 kcal/mol. The binding energy of compounds 1 -4 with spike protein ranged from À 5.8 to À 6.6 kcal/mol and their increasing order (where negative sign indicates favorable interactions) of binding energies is 4 > 3 > 2 > 1 for spike protein.
For more comprehensive understanding, a graphical comparison among the binding energies of the all compounds for spike protein is also shown in Figure 6 where the lowest and most favorable energy is also seen for compound 4. Among all compounds, compound 4 show the highest binding tendency to M pro and spike proteins with its binding energies which are À 6.2 and À 6.6 kcal/mol, respectively. The inhibition constants of the best dock compound 4 are found to be 24.46 and 13.94 μmol for M pro , and spike proteins, respectively. These results showed that among all the compounds, compound 4 exhibits reasonably good binding energies for both proteins of SARS-CoV-2, which are docked to the catalytic sites of M PRO and spike proteins. Additionally, we have performed docking studies for two positive control compounds including Remdesivir and Nirmatrelvir which are well-known COVID-19 drug compounds. [37] Nirmatrelvir is very recently approved oral drug as developed by Pfizer, which also an M pro enzyme inhibitor. [38] For comparative purpose, we have performed docking of Remdesivir and Nirmatrelvir under the similar calculation conditions. The binding energy for M pro enzyme with Remdesivir and Nirmatrelvir are found to be À 5.7 and À 6.4 kcal/mol while for spike protein with Remdesivir and Nirmatrelvir these are found to be À 7.6 and À 6.5 kcal/mol (see Table 3). A semi-quantitative comparative analysis indicates that our compound 4 possesses comparable binding energies to Remdesivir and Nirmatrelvir drug compounds. A detail docking analysis including 3-D and 2-D diagrams of Remdesivir and Nirmatrelvir complexed with both M pro and spike proteins were given in Figure S1 of Supporting Information of the article. Furthermore, it will be also interesting to compare the calculated binding energies of above entitled compounds with those of other similar studies. For instance, thiazole-based heterocyclic compounds as synthesized and docked against Mpor enzyme showed binding energies ranging from À 4.1 (compound 1) to À 8.5 kcal/mol (compound 15). [39] The binding energy of our best docked complex in terms of E-score is also comparable to azo imidazole derivative (L3 = À 6.7 kcal/mol), [40] Chloroquine (À 5.3 kcal/mol), 6-Gingerol (À 5.8 kcal/mol) and Myristicin (À 5.3 kcal/mol) etc. [41] It is important to pen down that this semi-quantitative comparison based only on binding energies as calculated through similar protocols using grid based docking techniques. The results should be carefully read considering many other drug-designing factors.

Visualization of Protein-Ligand Interactions
Though the binding energy and inhibition constants are important, it will also very useful to visualize the Chem. Biodiversity 2022, 19, e202100843 www.cb.wiley.com (7 of 14) e202100843 intermolecular interactions among ligands and protein macromolecules. Based on the binding energy of compound 4, it was selected to study the interaction of ligand-receptor protein complex. The data in the Table 3 shows that binding energy of compound 4 with the spike protein is better than that of M Pro . The M pro protein chain is composed of 306 residues. The active site of protease is activated by a protonation reaction in the catalytic site. The interactions of compound 4 with M pro showed the highest interactions with six residues of the M Pro including three Hbonds with ASN142, SER144, CYS145 and two hydrophobic interactions CYS145 and ASN124. While on the other hands, spike protein consists of 1281 amino acid residues and compound 4 reacts with six residues of the spike protein including four H-bonds which consists of SERL28, AGR408, SERL96 and SERL97. The protein-ligand interactions in 3D and 2D representations for compound 4 with M pro and spike proteins are shown in Figure 7. Furthermore, we have also drawn the total density surfaces of both M pro and spike proteins while interacting with ligand as shown in Figure 8. The Figure 8 also gives an overview about the orientation of ligand and its H-bonding and ionizability characteristics with over the density surface of both studied proteins.

Pharmacokinetic Properties and Lipinski's Rule of 5
The well-known Lipinski's rule of 5 was used to predict the drug likeness through pkCSM server. [42] According to this rule, any ligand is considered as a drug-like if it meets certain requirements including molecular weight < 500 g/mol, number of H-bond acceptor < 10, number of H-bond donors < 5 and lipophilicity represented as log P < 5 (Lipinski 2004). [43] In fact, all these molecules under investigation are said to have good absorption, low toxicity level good oral bioavailable permeable properties. The isolated all compounds obey Lipinski's rule 5. The bioavailability radar enables  a first glance at the drug likeness of a molecule. [44] The pink area represents the optimal range for each propriety of drug likeness with normal range. The bioavailability radar (Figure 9) of the four selected molecules under investigation shown to further confirm their drug likeness properties where all the five crucial drug likeness parameters are in color area which is safe to use as drugs.

Conclusion
The present study result in isolation of four new metabolites (1 -4) from a terrestrial Actinomycetes sp. GSCW-51 which belong to thioinosine and butenolides classes. The structure of the all the compounds were established through detail spectroscopic data. Furthermore, computational docking studies have shown a decent potential of our isolated compounds for inhibition of SARS-CoV-2 by interacting with its two very crucial M pro and spike proteins. All the four compounds have shown the different values of favorable binding energies with M pro and spike proteins. The binding energy of compounds 1 -4 with M pro protein are found to be À 5.3, À 5.7, À 5.4 and À 6.2 kcal/mol, respectively. The increasing order (where negative sign indicates favorable interactions) of binding energies is 4 > 3 > 2 > 1 for M pro protein.
While on the other hands, the binding energy of compounds 1-4 with spike protein are found to be Chem. Biodiversity 2022, 19, e202100843 www.cb.wiley.com (9 of 14) e202100843 À 5.8, À 5.9, À 6.2 and À 6.6 kcal/mol, respectively. A careful analysis of results indicated that the compound 4 possessed the best binding energies of À 6.2 and À 6.6 kcal/mol for M pro and spike proteins, respectively. Additionally, a renowned Lipinski's rule of 5 was used to predict the drug likeness of compounds 1 -4, which

Isolation, Identification, Cultivation of Bacteria and Extraction
The sandy soil sample was collected from the top 5 cm of earth surface and packed in sterile plastic polythene bags. The collected sample was air dried for one week and further heat treatment was done in a hot air oven at 120°C for 1 h in order to prevent growth of other bacteria. [45] Microscopic morphological, physiological study and biochemical analysis was carried out according to the reported method [46][47][48] to identify the isolated Actinomycete sp. GSCW-51.
A 15.0 L of culture media (200 g malt extract, 80.0 gm yeast extract and 80 gm glucose in tap water, pH 7.8) was prepared and transferred to 1.0 L Erlenmeyer flasks (250 ml each). The media was autoclaved at 120°C for 15 min and was infected by the precultured terrestrial Actinomycetes sp. GSCW-51. The flasks were then incubated on a linear shaker (110 rpm) at 28°C for 8 days until the broth attained dark brown color. The culture broth was harvested after 8 days, filtered under vacuum and was extracted with AcOEt to get dark brown gummy mass. The bacterial mass was also extracted with methanol, and both the extract were mixed to get total crude extract (4.0 g).