Anti-ageing and Anti-lung Carcinoma Effects of Vulpinic Acid and Usnic Acid Compounds and Biological Investigations with Molecular Modeling Study

norin, usnic acid on human cancer cells were investigated, and usnic acid was found to be a more effective anti-cancer agent than others. The effects of acids such as usnic acid, which are secondary metabolites of lichens, on the activities of topoisomerase I and II enzymes and the effects of this compound on DNA binding properties in cell-free media were investigated 4, 5 ） . The effects of usnic acid and atronin against human prostate and melanoma cancer cells were investigated and it was observed that both compounds had strong inhibitory effects against cancer cells. Additionally, vulpinic acid was first described in 1925. It is clear yellow in color and relatively toxic. It has been reported to show activity against Gram-positive bacteria. Wound healing and antiproliferative effect of tumor cells were investigated in different lichen compounds （ vulpinic acid ） . The anti-inflammatory effects of aromatic cyclic vul-Abstract: Disorganization and breakdown of extracellular matrix proteins like fibronectin, collagen, and elastin are key characteristics of skin aging due to the increased activation of important proteolytic enzymes like elastases and collagenase enzymes. Also, inhibition of their enzymatic activities by natural molecules might be a promising factor to prevent extrinsic skin aging. All chemicals were obtained from Sigma-Aldrich unless otherwise stated. The assay employed was based on spectrophotometric methods reported in the literature. The collagenase and elastase inhibition assays of some phenolic compounds were performed according to the previous studies. These compounds showed excellent to good inhibitory activities of vulpinic acid against studied these enzymes with IC50 values of 195.36 µM for collagenase and 25.24 µM for elastase. The molecular docking calculations were conducted to investigate the chemical and biological activity of vulpinic acid and usnic acid against collagenase and elastase. The results indicated that these two compounds can interact with the essential residues of the enzymes and affect their activities. The calculations of binding free energies were also performed to obtain more details about the characteristics and free energies of the ligand-enzyme complexes. Additionally, both compounds exhibited the most potent inhibition in the three lung cancer cells, with an IC50 value of 21–68 µM, indicating that vulpinic acid is more potent than Doxorubicin, which exhibited an IC50 value of 21–29 µM.


Introduction
Usnic acid occurs naturally from dibenzofuran derivatives found in various lichen species. This compound is one of the most popular and most abundant secondary metabolites. It has antiviral, antimitotic, anti-inflammatory and analgesic effects 1 . Usnic acid under normal conditions; It is bitter, yellow and solid. In addition to being a racemic mixture, it is known that there are D and L forms. Lichen extracts containing usnic acid have also been used in medical, cosmetics, perfumery, and ecology. It has strong antibiotic effect against gram positive bacteria such as Streptococcus, Staphylococcus, and Pneumococcus and other bacteria such as Mycobacterium tuberculosis and some pathogenic fungi 2, 3 . It has properties such as UV absorption, protective properties, growth inhibitory, and insecticide. The effects of lichen compounds Parietin, atra-norin, usnic acid on human cancer cells were investigated, and usnic acid was found to be a more effective anti-cancer agent than others. The effects of acids such as usnic acid, which are secondary metabolites of lichens, on the activities of topoisomerase I and II enzymes and the effects of this compound on DNA binding properties in cell-free media were investigated 4, 5 . The effects of usnic acid and atronin against human prostate and melanoma cancer cells were investigated and it was observed that both compounds had strong inhibitory effects against cancer cells. Additionally, vulpinic acid was first described in 1925. It is clear yellow in color and relatively toxic. It has been reported to show activity against Gram-positive bacteria. Wound healing and antiproliferative effect of tumor cells were investigated in different lichen compounds vulpinic acid . The anti-inflammatory effects of aromatic cyclic vul-pinic acid on rheumatoid arthritis in rabbits were investigated 6, 7 .
Elastase and collagenase are matrix metalloproteinase enzymes and comprise a family of ECM-degrading enzymes. These enzymes play a key role in the repair and remodeling of tissues. They have a significant role in the regulation of extracellular matrix degradation which is necessary for wound re-epithelialization. Indeed, they are involved in the pathogenesis of various diseases, like cardiovascular diseases, cancer, fibrosis, bone destruction, inflammation, as well as in the growth and healing of wounds 8,9 .
Considering the improvement of computational technologies and a growing number of biological and chemical databases, the importance of computational methods and modeling, in silico approaches, and artificial intelligence is undebatable 10 12 . Molecular docking as a versatile theoretical approach has attracted considerable attention among biologists in recent years. This method allows the user to investigate the interactions between small molecules and biomolecules at an atomic level 13 . The molecular docking study can determine the behavior of the ligands near binding sites and evaluate the characteristics of the interactions 14 . This is an in silico approach that could enhance the process of drug discovery in a computational manner 15 . Furthermore, the data obtained from the molecular docking calculations can be used as complementary information for experimental studies. The results can provide some essential characteristics of the ligand-biomolecule complex.
Cancer is the most important public health problem in the world, and lung cancer has the highest mortality rate among cancers. Most lung carcinomas 57 are not diagnosed at an early stage as they are typically asymptomatic during this period. Most patients with small cell lung cancer and stage IIIB/IV non-small cell lung cancer receive chemotherapy 16 . However, chemotherapy factor is not curative for metastatic lung cancer, but it can relieve symptoms or prolong life by weeks. Chemotherapy remains undesirable due to deficiencies in tumor tissues, including inadequate intracellular uptake, non-specific target site concentrations, and severe systemic toxicity of chemotherapeutic agents and even emerging drug-resistant cell lines 17 .

Biological activities
0.05 mL was taken from the prepared sample solutions. On top of it, 0.05 mL of elastase enzyme 0.16 U/mL was added. Then, 0.9 mL of tris hydrochloride Tris-HCl buffer solution of 0.2 M pH 7.8 was added to the sample solutions. It was prepared by adding 0.2 M pH 7.8 0.9 mL Tris-HCl buffer solution to 0.1 mL of elastase enzyme solution as a control solution 18 . The blank solution was prepared by adding 0.2 M pH 7.8 0.9 mL Tris-HCl buffer solution to 0.1 mL distilled water. Blank, control and sample solutions were incubated at 37 for 15 minutes. After incubation, 5 mM 0.05 mL N-Succinyl-Ala-Ala-Ala-p-nitroanilide substrate was added to the blank, control and sample solutions and incubated at 37 for 30 minutes. The absorbance values of the sample and control solutions against the blank were read at 410 nm 19 . In the study, the antielastase inhibition activity values of the samples prepared at different concentrations were calculated. Experiments were repeated 3 times and averaged. The inhibition values on elastase enzyme of eperezolide-like compounds synthesized for the first time were calculated. The IC50 value the concentration required to inhibit 50 of the activity was calculated from the regression equation obtained from the linear segment of the curve drawn by applying the concentration to abscess, elastase enzyme inhibition data to the ordinate 20 .
Modified inhibitory effect on collagenase enzyme Thring et al. 2009 21 was determined spectrophotometrically using the method. 50 µL of the solution containing 0.8 U/ mL collagenase was taken, 50 µL of plant extracts and chemical substance solutions at different concentrations prepared on it were added. This method was performed according to previous studies. The absorbance values of the sample solutions and control solution were read at 340 nm in the UV spectrophotometer against the blank. Experiments were repeated 2 times 22 . The IC50 value, which is the amount of substance required for the collagenase enzyme to have a 50 inhibition effect, was calculated with the regression equation obtained from the linear section of the curve drawn by applying the concentration to the abscess in the graph and the enzyme inhibition data to the ordinate 23 .
For lung cancer part, the MTT method was performed as explained previously. Cells were seeded onto a 96-well plate at a concentration of 104 cells/well and allowed to adhere overnight. Five replicates were prepared for each therapy and cultured for 48 or 72 h. After 20 mL of MTT 5 mg/mL was added to each well, the cells were cultured for another 4 h. The supernatant was discarded. After 150 mL of DMSO was added to each well, the samples were incubated at 37 for 30 min and then swirled for 10 min. The absorbance at 570 nm was measured using a microplate reader. Experiments were repeated three times 24 .

Molecular docking study
Theoretical approaches such as molecular docking calculations can provide beneficial information about the details and characteristics of the interactions between inhibitors and biological compounds. The enzymes used in this study were human neutrophil elastase PDB ID: 2Z7F 25 and Collagenase H from Clostridium histolyticum PDB ID: 4AR1 26 . The biological activities of the vulpinic acid and usnic acid were studied against these enzymes. The PDB format of the enzymes was obtained from the PDB database http://www.rcsb.org/pdb , and the protein preparation module of the Schrödinger Suite 27 was utilized for their preparation. By this, the hydrogen atoms were added, the water molecules were removed, and a network of H-bond was constructed. Finally, the structures were minimized using the OPLS3e force field. In the next step, the active sites of the enzymes were determined employing the SiteMap of Schrödinger 28 . The SDF files of the vulpinic acid and usnic acid were taken from the PubChem database, and the LigPrep module of Schrödinger 29 was used for the ligand preparation. Eventually, the docking estimations were performed employing the Glide of Schrödinger suites.

Binding free energy calculations
Another crucial calculation for understanding the characteristics of the ligand-enzyme complex is binding free energy calculations. Here, the MM/GBSA method has been used for these predictions. The calculations were conducted employing the prime module of Schrödinger. The solvation model of VSGB and OPLS-2005 force field were used for the calculations 30 . The equation of binding free energy is: Where ∆G bind symbolizes the binding free energy, G complex is the binding free energy of the complex. The G protein and G ligand show the binding free energy of the protein and ligand, respectively.

Biological activities
Inhibiting the activity of extracellular matrix-degrading proteins like elastases and collagenases may be a useful approach to prevent premature skin aging and UV-induced skin alterations. Scavenging of ROS by natural antioxidant compounds might be one option to inhibit such skin deteriorative enzymes, as ROS play a key role in the activation of these enzymes. Phenolic molecules are a significant class of natural antioxidant compounds 31 . They belong to various subclasses of secondary plant metabolites classified as flavonoids, phenolic acids, stilbenes, and lignans and are ubiquitously recorded in the plant kingdom. Also, white and red grapes contain high amounts of flavonoids and phenolic acids like catechin and gallic acid 32 . The collagenase and elastase inhibition assays of some phenolic compounds were performed according to the previous studies. These compounds showed excellent to good inhibitory ac-tivities of vulpinic acid against studied these enzymes with IC50 values of 195.36 µM for collagenase and 25.24 µM for elastase. The Usnic acid had IC50 values of 94.05 µM against collagenase and 7.38 µM against elastase. The most potent compounds against collagenase and elastase were compound usnic acid with IC50 values of 94.05 µM against collagenase and IC50 values of 7.38 µM against elastase, respectively.

Molecular docking
The biological and chemical activities of vulpinic acid  and usnic acid in the presence of collagenase and elastase were investigated using the molecular docking study. The results indicated the target residues for these two chemical compounds. The docking pose of vulpinic acid among collagenase residues is presented in Fig. 1, and Fig. 2 presents the interactions of the vulpinic acid with collagenase. Figure 2 indicates that vulpinic acid has formed three hydrogen bonds with the residues of collagenase. These residues are Lys375, which is from the catalytic subdomain, Lys641, and Arg652, which are from the helper subdomain of the enzyme 33 . There are also thirteen hydrophobic contacts that are created by the molecules of vulpinic acid. Ten residues of these thirteen amino acids are from the catalytic subdomain. These residues are Lys353, Val354, Asn378, Phe382, Asp388, Asp397, Asp398, Leu400, Tyr460, and Arg464. Figure 3 presents the docking pose of vulpinic acid among the residues of the elastase. Figure 4 shows the contacts created by vulpinic acid with the enzyme. As could be seen, there are six hydrophobic contacts. Phe215 and Arg217 are among these residues, which are two of the critical residues of elastase 34 . These residues have a   crucial impact on the catalytic activity of the enzyme. Since vulpinic acid has constructed considerable interactions with the key residues of collagenase and elastase, this compound could be considered as a potential inhibitor for these enzymes. The docking pose of usnic acid among the residues of collagenase and their interactions are presented in Figs. 5 and 6, respectively. Figure 6 indicates that usnic acid has constructed two hydrogen bonds and eleven hydrophobic contacts with collagenase. Lys375 is one of the residues with a hydrogen bond, which is from the catalytic subdomain of the enzyme 35 . There are also some other residues from the catalytic subdomain of collagenase with hydrophobic contacts. These residues are Lys353, Asn378, Lys382, Lys389, Tyr460, Arg464, and Tyr465. These interactions are crucial since they are in contact with the residues of the catalytic subdomain. Therefore, usnic acid can inhibit the activity of collagenase efficiently. The docking pose of this compound among the residues of elastase is presented in Fig. 7. Figure 8 shows that this chemical agent has constructed one hydrogen bond with Arg217. This H-bond has been created between the NH of the peptide backbone of Arg217 and an oxygen molecule of the ligand, which is presented in Fig. 8. There are also four hydrophobic contacts with Asn99, Arg177, Phe215, and Val216. Residues 215-217 are critical amino acids of the elastase. They have a direct impact on the catalytic activity of the enzyme 36 .
These interactions can lead to a considerable inhibitory activity of usnic acid in the presence of elastase and collagenase. Hence, this compound could be considered as a potential inhibitor for these two enzymes. There are also some other parameters that can describe the characteristics of the interactions between the chemical compound and biomolecules. Table 1 shows some of the parameters. One of these parameters is the docking score, which is one of the most essential parameters 37 and shows the binding affinity of the ligand to the biomolecule. This score is 3.6 kcal/mol for vulpinic acid against collagenase and 2.8 kcal/mol against elastase. Usnic acid has a docking score of 4.9 and 3.3 kcal/mol against collagenase and elastase, respectively. Another parameter is Glide Ligand Efficiency. This value indicates the efficiency of the molecules. Two of these parameters are interaction-related parameters Glide Evdw and Glide Ecoul . The following parameter is Glide energy, which shows the energy of the interaction, and the last parameter, Glide Emodel, indicates the value of interaction pose 38 .

Binding free energy
The parameters obtained from the calculations of binding free energy are presented in Table 2. ∆G bind indicates the total free binding energy between ligand and enzyme. This value is 22.35 kcal/mol for vulpinic acid against collagenase and 41.30 kcal/mol against elastase. These values have a positive correlation with the results of IC50 for vulpinic acid, which are 195.36 µM and 25.24 µM against collagenase and elastase, respectively. There are other parameters in Table 2, such as ∆G bind Coulomb ∆G bind H-bond ∆G bind Lipo ∆G bind vdW, which are free binding energies of coulomb energy, hydrogen bond energy, lipophilic energy, and van der Waals energy, respectively. Based on the data presented in Table 2, it is apparently obvious that coulomb energy has a key role in the interactions between vulpinic acid and enzymes. ∆G bind for usnic acid is 19.73 kcal/mol against collagenase and 40.50 kcal/mol against elastase. Considering the other free binding energies presented in Table 2, coulomb energy has  an essential role in the interactions between usnic acid and these two enzymes. That is because of the remarkable difference between the Coulomb energy of usnic acid against these two enzymes. The values of Coulomb energies are 19.28 kcal/mol and 81.55 kcal/mol for collagenase and elastase, respectively.

Anti-cancer results
The compounds of vulpinic acid and usnic acid were determined in vitro for their anticancer activities against SPC-A-1, 95D, and SK-LU-1 lung cancer cell lines, with the anticancer drug Doxorubicin, used as a control compound. Indeed, in vitro anticancer screening methods were conducted at various molecule concentrations. All of the tests were performed out in triplicate. Additionally, the IC50 values were calculated from the percentage of anticancer effect by nonlinear curve fitting and are presented in Table  3. In this part, both compounds exhibited the most potent growth inhibition in the three lung cancer cells, with an IC50 value of 21-68 µM, indicating that vulpinic acid is more potent than Doxorubicin, which exhibited an IC50 value of 21-29 µM. Also, other molecules exhibited significantly weaker activity, because the anticancer was lower than that of Doxorubicin in the three cell lines, with an IC50 value of 62-68 µM Table 3 .
Lung cancer arises from normal lung epithelial cells that undergo numerous genetic damages and eventually turn into cells that proliferate uncontrollably with abnormal growth and aggressive behavior in the airways of the lungs 39 . Lung cancer includes two main types: small cell non-cell lung cancer and small cell lung cancer. Based on histological classification, non-small cell lung cancer can be divided into three main subtypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. The importance of classification is evident in the treatment strategy and prediction of cancer outcomes. Today, surgery, radiation, chemotherapy and targeted therapy in the treatment of lung cancer; However, although many methods have been used to treat lung cancer, the clinical results of current treatments are still unsatisfactory. Many commonly prescribed chemotherapeutic drugs have been discovered by searching for possible compounds from plants, marine or-   ganisms, microorganisms, and animals, or by making natural product-derived lead compounds. According to this concept, some natural product-derived compounds have already been evaluated and their anti-cancer effect has been focused on newly discovered mechanisms in the hope that they can be used, or at least better strategies against cancer 40 .

Conclusions
In this study, both natural compounds showed inhibitory effects on both collagenase and elastase enzymes. Also, disorganization of extracellular matrix proteins like fibronectin, collagen, and elastin are major characteristics of skin aging due to the enhanced activation of metabolic enzymes like elastases and collagenases. Indeed, inhibition of their enzymatic activities by natural molecules might be a promising approach to prevent extrinsic skin aging. Theoretical approaches have provided a beneficial way to obtain pragmatic data that could be used as completing information for experimental results. Molecular docking is one of these approaches that has attracted considerable attention in recent years. In this study, molecular docking calculations were performed to evaluate the biological activities of vulpinic acid and usnic acid against collagenase and elastase. The calculations of free binding energy were also performed and contributed valuable information about the ligand-enzyme complex. The outcomes indicated that these compounds have a considerable potential to inhibit the activities of the mentioned enzymes. Lung cancer is popular malignancy and gives rise to around one-quarter of all cancer deaths. Great advances have been achieved in the therapy of lung cancer with new anticancer factors and also can improve technology. Indeed, mortality and morbidity rates remain extremely high, calling for an urgent need to develop new anti-lung cancer factors. Indeed, both natural substances of this study showed anti-lung cancer inhibition and the results were calculated in the form of IC50.