In silico investigation of the anti-leishmanial role of algae and corals active substances using molecular dynamic simulation and Molecular docking methods

doi:10.21203/rs.3.rs-4934620/v1

Materials and Methods

From the PubChem database, three-dimensional structures of Leishmania major proteins zinc leishmanolysin Glycoprotein 63 (GP63), Farnesyl diphosphate synthase (FPPS), and N-myristoyltransferase (NMT), as well as repressors and 389 coral compounds and 624 algal compounds, were obtained. Using PyRx and AutoDock vina software, molecular docking analysis was performed for each of the three Leishmania proteins using individual protein combinations and blockers. The activity, daily carcinogenicity and properties of ADMET are derived from the Swiss drug ADME, Lazar and Way 2. Using the GROMACS program, the coral and algal compounds with the highest binding scores for each protein were selected for molecular dynamics simulations.

Results

According to the results of molecular docking experiments, Alga-utd-01 and Coral-utd-01 have strong affinity for GP63 protein. Additionally, Alga-utd-05 and Coral-utd-02 showed the highest binding affinity to FPPS, while the top compounds for NMT were Alga-utd-14 and Coral-utd-03. In addition, Alga-utd-05, Alga-utd-22 and Alga-utd-16 are common algal compounds among the three proteins, and Coral-utd-01, Coral-utd-02, Coral-utd-03, Coral -utd-06 and Coral-utd-12 are common compounds of corals. The MD results confirm relatively stable interactions between the proposed compounds and three important Leishmania proteins. Also, according to the mentioned important medicinal sites, the mentioned compounds have the least interference and the most anti-parasitic properties.

Conclusion

According to information collected from pharmaceutical databases, the substances in question may have anti-inflammatory and therapeutic properties in addition to protein blocking. Therefore, experimental examination of these algae, corals and compounds may provide valuable clues for the control and treatment of leishmaniasis.

Leishmaniasis

Molecular dynamics

GP63

FPPS

NMT

The host species of Leishmania are generally humans and animals and transmitted by insects. An estimated 350 million people are at risk of leishmaniasis. There are more than 20 Leishmania species that cause different diseases, cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL). World Health Organization (WHO) has announced about 95% of all CL cases in the Americas, the Middle East, the Mediterranean basin, Syrian Arab Republic, and central Asia. It is apprised that 600,000 to 1 million people worldwide are affected annually (1). Leishmania has two forms: amastigote forms inside vertebrate macrophages, non-flagellar and non-motile, and promastigote forms: intracellular, extracellular, motile, and flagellate mosquitoes (2). Infected/Affected mosquitoes parasitize macrophages and cause symptoms ranging from self-healing ulcers, deformed mucosal lesions (spendia), to fatal systemic infections involving the spleen and bone marrow (Kala Azar) (3). Leishmania contains several proteins that can be effectively targeted to eliminate parasites, including leishmanolysin zinc metalloprotease, also known as GP63, promastigote surface proteinase (PSP), and major surface protease (MSP), which are expressed genetically and biochemically as surface antigens. In promasigotes of the Leishmania species, it is described with a variety of substrates, including gelatin, fibrinogen, albumin, casein and hemoglobin. GP63 is the most abundant surface protein in promastigotes, and its expression is significantly increased in amastigotes (4). GP63 interacts with the fibronectin receptor and thus plays an important role in parasite attachment to macrophages. From these different results, it is clear that GP63 can significantly alter macrophage activity, thereby contributing to the survival of Leishmania by sequestering or degrading various proteins (5). The GP63 protein contains 478 amino acids, the molecular weight of 63 kilodaltons and a β-sheet secondary structure, which plays a special role in the uptake of Leishmania by protein receptors, as well as in the resistance to promastigote lysis via complement mediator. Additionally, GP63-releasing promastigotes act through intracellular GP63 secretion or glycophosphatidylinositol GPI-bound GP63 autolysis, and its inhibition is a therapeutic target because it prevents the binding of the promastigote form to macrophage and its transition to the amastigote state in macrophages, inhibiting Leishmania. It is a potential vaccine candidate against Leishmania (6).

Farnesyl diphosphate synthase (FPPS) is a branching enzyme of the isoprenoid biosynthetic pathway, a widespread metabolic pathway from prokaryotes to eukaryotes and responsible for the synthesis of essential isoprenoids such as diholicols, carotenoids, chlorophyll, ubiholicones, and sterols (7). Protein important in the regulation of Leishmania and is one of the therapeutic targets. More than 30,000 individual isoprenoids have been identified, making these compounds the most diverse class of natural compounds. They perform several biochemical functions: are membrane structural components (prenyl lipids), photosynthetic pigments (carotenoids and chlorophyll), intracellular targeting of signaling proteins (prenyl protein synthesis), plant protection compounds (mono, di and sesquiterpenes)., in the electron transport chain (quinone) and plant growth regulatory hormones (gibberellin and abscisic acid) (8). FPPS participates in the post-translational modification of proteins mediated by farnesyltransferases, key GTP-binding factors, by nitrogen-containing bisphosphonates (N-BPs), such as risdronate, as well as their protein prenylation Ras, and is an important intermediate of sterols metabolism (9). Therefore, bisphosphonates could be potential targets for antiparasitic drug development due to their importance for L. major survival. Because most Leishmania strains share more than 90% of the FPPS sequence, there are great opportunities to develop new, more potent drugs to combat Leishmania parasites, and it may be possible to develop drugs that specifically target this enzyme (10). Another important Leishmania protein is N-myristoyltransferase (NMT), which was identified in previous research as potentially suitable for drug production against harmful protozoan parasites (11, 12). In eukaryotic cells, NMT catalyzes the transfer of the saturated fatty acid myristate from myristoyl-CoA (MyrCoA) to the amino-terminal glycine of proteins, which, in addition to facilitating protein-protein interactions, also helps target substrate proteins to membrane domains (13). Antifungal drug development activity in the pharmaceutical industry focuses on NMT. These factors suggest that NMT is a good candidate for the treatment of infectious and parasitic diseases, including malaria (caused by Plasmodium spp.), leishmaniasis (caused by Leishmania spp.), and sleeping sickness Africa (caused by Trypanosoma brucei) (14, 15). For infections caused by parasitic worms, NMT has also been suggested as a potential therapeutic target. More recently, Plasmodium NMT inhibitors have been created and shown to block the growth of important subcellular structures, thereby leading to rapid parasite cell death (16). These three proteins are essential for parasite survival and virulence and can be inhibited to kill the parasite. Meglumine antimoniate (Glucantim), sodium stibogluconate (Pentostam), and (Pentacrinate), which are treatments for leishmaniasis, have toxicity and side effects that make them ineffective when taken orally and require long-term injections (14, 17). Other drugs include the highly toxic amphotericin B and pentamidine, as well as the teratogenic miltefosine (Miltex), an alkylphospholipid used in cancer treatment. To combat multidrug resistance, scientists are currently working around the world to find new defenses (18). According to many studies, different plant species inhibit most parasites. Leishmaniasis can be treated with various vegetable oils because they regulate the immune system. In this context, the use of various plant extracts with leishmanicidal activity has been proposed, including alkaloids, chalcones, phenolic compounds, and terpenes (19). Additionally, in many regions of world yet there is currently no complete vaccine and effective. Therefore, it is urgent to create new, more powerful drugs with improved quality to replace or supplement drugs already on the market. Effective drugs made from plant, algae, and coral derivatives or extracts are expected to become an important resource. Furthermore, less than 1% of herbal medicines have been tested in clinical studies for the treatment of leishmaniasis. In the field of drug development, bioinformatics studies are extremely useful, especially because laboratory research is time-consuming, expensive, and is very dangerous(20, 21). With the subsequent emphasis of the WHO on the improvement of anti-leishmania agents from natural products, there has been an urgent search for potent drug candidates derived from marine life forms, and plants (1), on which numerous studies have led to the identification of substances with potential pharmacological activity (22–24).

Therefore, in this study, a bioinformatics approach was performed to inhibit three important proteins of the parasite L major. Active ingredients from corals and algae were compared with L. major's NMT, GP63 and FPPS proteins to find the best compound that inhibits them. Furthermore, molecular dynamics simulation analysis was performed to analyze the stability of the selected compounds and then the pharmacological properties of the best performing compounds were verified to include them as achievable goals for empirical research. Therefore, these results must be verified experimentally to ensure their effectiveness.

2.1. Finding proteins structures and characterization

The L. major proteins structures, ligand binding sites, and PDB ID were gained from previous articles. PDB ID of important target proteins GP63, FPPS, NMT in L. major, important amino acids involved in interaction with inhibitors and their resolution are presented in Table 1. Proteins PDB structure were received from the RCSB PDB database (https://www.rcsb.org) (25).

Table 1

PDB ID and important amino acids involved in the interaction of GP63, FPPS, and NMT proteins with compounds.
Protein Name	Code PDB	Resolution	Amine acids	Referenes
GP63	1LML	1.86 Å	HIS264, HIS268, HIS334, MET345, ALA348, ALA227, SER273, GLY222, THR228, GLU265, ALA350, PRO347, VAL223, GLY329, GLU220 LEU224, ALA225, SER418, GLN341, GLY222, SER465, VAL223, HIS264, LEU257, PRO460, ALA350 HIS264, ASP342, HIS268, HIS334, MET345, ALA348, PHE272, SER333	(Shaukat et al 2013)(26) (Mercado-Camargo et al 2020)(5) (Schlagenhauf et al 1998)(4)
FPPS	4K10	2.30 Å	LYS207, LYS264, ARG107, ASP102, ASP98, ASP99, ASP250, PHE94, LEU95, HIS93, MET101, GLN167 ARG107, GLN167, LYS264, ASP268, THR267, ASP170, LYS207 LYS207, LYS264, ARG107, PHE94, GLN167, THR163, ASP102, ASP98, ASP250, ASP254 ASP98, ILE100, MET101, ASP102, ARG107, ARG108, HIS93, PHE94	(Aripirala et al 2013)(10) (Mattos Oliveira et al 2018)(8) (Ferrer-Casal et al 2014)(14) (Gadelha et al 2020)(7)
NMT	4CGO	1.30 Å	PHE90, VAL81, PHE232, GLY397, PHE88, HIS219, ASP83, GLY205, TYR80, TYR217, LEU421, TYR345, LEU341, TYR92, ASN167, HIS398, ALA204, VAL374, ALA343, ASN376 TYR92, PHE90, ASN167, PHE88, THR203, HIS219, TYR217, PHE232, TYR326, VAL81, TYR80, GLU82, ILE328, SER330, LEU341, MET420, LEU421, ASP396, LEU399, ASP396, ASN376, VAL374, TYR345, ALA343 ALA204, TYR202, PHE90, MET420, TYR326, TYR345, LEU421, ASN167, VAL81, ASN383, TYR80, VAL378, LYS173, GLU82	(Brannigan et al 2014)(17) (Brannigan et al 2010)(11) (Orabi et al 2023)(19)

2.2. Collecting the herbal phytochemicals

All the articles related to algae, hard and soft corals that used validation methods for phytochemical screening including GC/MS, HPLC and chemical tests were studied and the effective substances of each part of each alga, and coral were get (27–30)and the cases. Their duplicates were removed and finally 389 coral compounds and 624 algae compounds were obtained. The composition of each alga and coral was reviewed by all available articles(27, 31, 32). The 3D structure of compounds and that of each protein's probable blockers were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) (33).

2.3. Preparation and molecular docking analysis

3D structures of protein, algae, and hard and soft coral compounds were prepared for docking by removing all water and optimizing H-bonding, and then Gasteiger charges were added. Proteins and ligands were converted to PDBQT format (a format that includes charges and types of atoms) and finally, a space was chosen for the dock that contains all the amino acids mentioned in Table 1 and according to the active site of the protein. The site coordinates of the interactions (grid size) used for GP63 are Center: 20.2471, 40.6108, and 18.2018, and Size: 16.7421, 20.6414, and 19.9465, for FPPS Center: 16.9245, 41.478, and 125.318, and Size: 20.9211, 29.8813, and 25.1407, and for NMT Center: 4.63281, 49.5063, and 62.6636, and Size: 25.3756, 23.6199, and 25.1636. After that, molecular docking analysis was performed for each of the three Leishmania proteins separately with corals and algae compounds and protein blockers using PyRx software version 0.8, and then those with a score − 8 kCal/mol or less were also docked with the corresponding protein by AutoDock vina chimera software version 1.16. Figures of proteins and compounds were made by Chimera software version 1.14. (34), and Discovery Studio 2021(35).

2.4. Analysis of complexes interactions

The compounds with the highest docking scores for each of the target proteins of Leishmania major were selected and then their interactions were fully analyzed. In addition, the interactions of the proteins with the proposed compounds and their known blockers were carefully compared.

2.5. Determination of pharmacokinetic features of compounds with a high docking score

Activity, carcinogenicity, and ADMET properties of coral and algae compounds with high score caught from way 2 drug(36) (www.pharmaexpert.ru/passonline/predict.php). Lazar(37)(lazar.in-silico.ch/predict) and the free and accessible SwissADME website (38) (http://www.swissadme.ch), respectively.

2.6. Molecular dynamic simulation analysis

After docking, the coral compounds with the highest scores for each protein were selected for molecular dynamics (MD) simulation analysis. The structures obtained from the docking simulations were refined using MD simulations. To refine the protein-ligand complex, we performed molecular dynamics (MD) simulations using GROMACS 2018. The topology file was created according to the AMBER03 force field. The structure was placed in the center of a dodecahedral box and filled with water using the TIP3 water model. To neutralize the system, some water molecules were randomly replaced with Cl⁻ or Na⁺. After neutralization, energy minimization was performed using the Steepest descent algorithm. Equilibration of the system was performed with a 100 ps NVT at the temperature of 298 K, followed by a 100 ps NPT ensemble at the pressure of 1 bar. Electrostatic interactions were calculated by PME, and the LINCS method was applied to constrain all bonds between hydrogen atoms. The final MD simulation was run for 100 ns without any restrictions. Using GROMACS software, the compound with the best docking score for each protein was selected for molecular dynamics simulations(39).

2.7. Binding energy throughout the MM-PBSA method

The binding free energy of each protein binding complex during MD simulations was calculated by applying ΔGMMPBSA. The residual contribution was calculated using the energy decomposition function of gmx_MMPBSA using the MMPBSA method. ΔGMMPBSA is the sum of the free energy of the gas phase (ΔG Gas), and the free energy of the dissolved phase (ΔG Solv). ΔG gas is the energy resulting from the sum of bond and nonbond energies. Bond energy consists of bond energy, angular energy, and dihedral angular energy, while nonbond energy is contributed by van der Waals energy and electron energy. In the ΔGMMPBSA calculation, ΔG Solv is contributed by Poisson-Boltzmann energy, nonpolar solvation energy, and dispersion energy. Poisson-Boltzmann energy is polar energy, others are non-polar energy (40).

3.1. Docking analysis

The best algae, hard, and soft corals compounds for GP63, FPPS, and NMT proteins were identified based on their binding energy. The molecular docking results of the proteins medicinal compounds of algae are presented in Table 2. Alga-utd-01 was the best compound with the highest binding affinity for GP63 (Table 2 and Fig. 1). Alga-utd-05 was the best compound for interaction with FPPS (Table 2 and Fig. 2). Alga-utd-14 was the compound with the highest affinity for NMT (Table 2 and Fig. 3).

Molecular docking results for proteins and coral compounds are shown in Table 3. Coral-utd-01 was the best compound with the highest binding affinity for GP63 (Table 3 and Fig. 4). Coral-utd-02 was the best compound to interact with FPPS (Table 3 and Fig. 5). Coral-utd-03 was the compound with the highest affinity for NMT (Table 3 and Fig. 6). Molecular docking results for blockers and proteins are shown in Table 3.

3.2. The common compounds with a high affinity for each protein

Alga-utd-05, Alga-utd-22, and Alga-utd-16 were the common compound of algae between all three proteins. The docking score of each compound for each protein is shown in Table 4. Coral-utd-01, Coral-utd-02, Coral-utd-03, Coral-utd-06, and Coral-utd-12 were the common compound of corals between all three proteins. The docking score of each compound for each protein is shown in Table 4.

Table 4

Affinity analysis of Common Compounds of algae with the high affinity for GP63, FPPS, and NMT proteins.
protein Name	Common Compounds	Docking score kCal/mol
GP63	Alga-utd-05 Alga-utd-22 Alga-utd-16	-8.6 -8 -8
FPPS	Alga-utd-05 Alga-utd-22 Alga-utd-16	-8.9 -7.5 -7.7
NMT	Alga-utd-05 Alga-utd-22 Alga-utd-16	-10.3 -10.2 -9.5

Table 5

Affinity analysis of Common Compounds of corals with the high affinity for GP63, FPPS, and NMT proteins.
protein Name	Common Compounds	Docking score kCal/mol
GP63	Coral-utd-01 Coral-utd-02 Coral-utd-03 Coral-utd-06 Coral-utd-12	-10.3 -10.1 -9.8 -9.3 -9
FPPS	Coral-utd-01 Coral-utd-02 Coral-utd-03 Coral-utd-06 Coral-utd-12	-8.4 -9.6 -8 -8.8 -8.5
NMT	Coral-utd-01 Coral-utd-02 Coral-utd-03 Coral-utd-06 Coral-utd-12	-10.6 -9.3 -11.4 -9 -10.3

3.3. The results of the interactions of proposed compounds with amino acids of 3 important proteins of Leishmania major and blocker

The interactions of bisphosphonate compounds inhibiting GP63 protein in its active site are as follows. Five hydrogen bonds with His235, Gly230, Ala126, Leu125 and Ala250 amino acids with distances of 3.485, 3.323, 2.657, 2.285 and 2.444 angstroms, respectively. It also has electrostatic interactions with Glu166 and His165, with a docking score of -5.7 kCal/mol. The compound Coral-utd-01 has the following interactions with the active site of GP63 protein: hydrophobic interaction with amino acids Leu158, Leu125, Ala250. Also, this compound has two hydrogen bonds with amino acids Ser243 and Ala128 with distances of 3.0675 and 2.833 angstroms, respectively, and a hydrophobic bond with Leu262 and a docking score of -10.3 kCal/mol.

The interactions of bisphosphonate compounds that inhibit the active site of FPPS proteins are as follows: It has four hydrogen bonds with amino acids Thr43, Tyr49, Asn51, and Gln91, with distances of 3.180, 2.545, 2.338, and 2.263. It also has two electrostatic interactions with Lys48 and Arg51, with a docking score of -6.6 kCal/mol. The combination of Coral-utd-02 and the active site of the FPPS protein has five hydrogen bonds with amino acids Arg108, Gly109, Lys264, and Gly263 at distances of 2.231, 1.565, 2.242, 2.449, and 3.564, respectively, and a hydrophobic bond with Leu262 and a docking score of -9.6 kCal/mol.

The interactions of bisphosphonate compound inhibiting NMT protein in its active site are as follows. It has four hydrogen bonds with amino acids Glu72, Val71, Tyr335, His209 with distances of 3.040, 3.698, 2.204, 3.336. It also has hydrophobic interaction with Phe80 and electrostatic interaction with Asp386 and Tyr207, with a docking score of -6.4 kCal/mol. The combination of Coral-utd-03 with the active site of NMT protein has three hydrophobic interactions with Phe80, Val71 and Tyr207 amino acids respectively, and a hydrophobic bond with Leu262 and a docking score of -11.4 kCal/mol.

As mentioned earlier, the proposed compounds form strong bonds including covalent, hydrogen and ionic bonds with amino acids in the active site of 3 important Leishmania proteins. Also, the mentioned compounds completely cover the active site of the protein and thus limit its activity by preventing it from forming bonds with other ligands (Fig. 1–6). Molecular docking scores of blockers are presented in Table 6.

Table 6

Affinity analysis of blocker with GP63, FPPS, and NMT proteins.
protein Name	Blocker	Docking score kCal/mol
GP63	Bisphosphonate	-5.7
FPPS	Bisphosphonate	-6.6
NMT	Bisphosphonate	-6.4

3.4. Results from the Lazar and WaytoDrug Databases

The results obtained from Lazar and WaytoDrug are presented in Table 7.

Table 7

Different characteristics of compounds of algae and corals with high affinity to GP63, FPPS, and NMT proteins.
Name of compound			activity ●	Carcinogenicity (Mouse) *
Alga-utd-01			Retinoic acid receptor agonist, Antiacne, Dermatologic, Antipsoriatic, Retinol dehydrogenase inhibitor, TP53 expression enhancer, Anticarcinogenic, Antioxidant, Chemopreventive, Antineoplastic, MMP9 expression inhibitor, HMOX1 expression enhancer, Prostate cancer treatment, Proliferative diseases treatment, EIF4E expression inhibitor, Antieczematic, TNF expression inhibitor, Antineoplastic (non-Hodgkin's lymphoma), Antipruritic, allergic, Antipruritic, Antimetastatic	non-carcinogenic				non-carcinogenic
Alga-utd-05			Methylenetetrahydrofolate reductase (NADPH) inhibitor, Methionyl-tRNA formyltransferase inhibitor, Cytostatic, Folate transporter 1 inhibitor, Gamma-glutamyl hydrolase inhibitor, Pteridine reductase inhibitor, AICAR transformylase inhibitor, Hematopoietic, Pancreatic ribonuclease inhibitor, Antineoplastic (lymphocytic leukemia), Antiviral (Herpes), Antiviral (Poxvirus)		non-carcinogenic				non-carcinogenic
Alga-utd-14			Photosensitizer, Gluconate 2-dehydrogenase (acceptor) inhibitor, Chemosensitizer, Antieczematic, Hepatoprotectant, Antipsoriatic, HMOX1 expression enhancer, Antiviral (Hepatitis B, Herpes), Antineoplastic (solid tumors), Antineoplastic, Antibacterial		N/D			N/D
Coral-utd-01			Antineoplastic (multiple myeloma), Antineurotic, Antineoplastic alkaloid, Antineoplastic (lymphoma), Antineoplastic (solid tumors), Antiprotozoal, Antitussive, Antibacterial, Antiinflammatory, Antiparasitic, Antimetastatic, Antiprotozoal (Leishmania), Antifungal, Antimitotic, Antibiotic, Antipsoriatic, Antieczematic, TP53 expression enhancer,			N/D			N/D
Coral-utd-02		Apoptosis agonist, Antineoplastic, Antieczematic, Antibacterial, Antifungal, Antiinflammatory, Antiviral (Rhinovirus), Antineoplastic (lung cancer), Antineoplastic (breast cancer), Antimetastatic, Antiprotozoal (Leishmania),					N/D			N/D
Coral-utd-03	Antineoplastic, Antiinflammatory, Antiprotozoal, Antipruritic, allergic, Prostate disorders treatment, Antiprotozoal (Leishmania), Antiseborrheic						non-carcinogenic		N/D

FPPS, and NMT proteins.

● The activity of each active substance is gained from the pass online database (http://www.pharmaexpert.ru/passonline/index.php). * The Blood-Brain Barrier, Carcinogenicity, and Maximum Recommended Daily Dose prediction were obtained from the Lazar database (https://lazar.in-silico.ch). * not defined

3.5. Results from the SwissADME database

The physicochemical properties of the pharmaceutical compounds retrieved from the database are shown in Table 8. Add properties of the compounds including lipophilicity, water solubility, and pharmacokinetics are shown in Tables 9, 10, and 11, respectively. According to Table 7, compound Alga-utd-01 has more lipophilicity than others and Coral-utd-01 is more lipophilic among coral compounds and as a result, they have better absorption. The LIPO (lipophilicity), SIZE, POLAR (polar), INSOLU (insoluble), INSATU (unsaturation), and FLEX (flexibility) of each compound are shown in Fig. 7.

Table 8

Different physicochemical properties of compounds of algae and corals with high affinity to GP63, FPPS, and NMT proteins
compound name	Alga-utd-01	Alga-utd-05	Alga-utd-14	Coral-utd-01	Coral-utd-02	Coral-utd-03
Formula	C40H56O	C19H19N7O6	C35H32N4O5	C40H62O8S	C38H46O12	C28H36O5
Molecular weight	552.87 g/mol	441.40 g/mol	588.65 g/mol	702.98 g/mol	694.76 g/mol	452.58 g/mol
Num. heavy atoms	41	32	44	49	50	33
Num. arom. heavy atoms	0	16	5	0	0	0
Fraction Csp3	0.50	0.21	0.23	0.85	0.63	0.75
Num. rotatable bonds	10	10	6	4	3	1
Num. H-bond acceptors	1	9	8	8	12	5
Num. H-bond donors	0	6	3	2	2	0
Molar Refractivity	183.95	111.92	182.90	195.57	178.22	125.33
TPSA*	12.53 Å²	213.28 Å²	136.70Å²	143.42Å²	179 Å²	69.67 Å²
*Topological Polar Surface Area

Table 9

Different lipophilicity characteristics of compounds of algae and corals with high affinity to GP63, FPPS, and NMT proteins
Lipophilicity	Alga-utd-01			Alga-utd-14	Coral-utd-01	Coral-utd-02	Coral-utd-03
Log P_o/w (iLOGP)	7.72		0.04	3.27	5.25	3.51	3.72
Log P_o/w (XLOGP3)	12.66	-1.08		2.39	5.52	1.27	5.47
Log P_o/w (WLOGP)	11.82	-0.38		3.36	7.23	3.06	4.65
Log P_o/w (MLOGP)	7.96	-0.62		1.52	4.17	0.28	4.29
Log P_o/w (SILICOS-IT)	12.50	0.24		7.68	6.35	4.32	3.94
Consensus Log P_o/w	10.54	-0.36		3.65	5.70	2.49	4.41

Table 10

Different water solubility characteristics of compounds of corals and algae with high affinity to GP63, FPPS, and NMT proteins
compound Name	Alga-utd-01	Alga-utd-05	Alga-utd-14	Coral-utd-01				Coral-utd-03
LogS (ESOL)	-10.58	-1.61	-4.68	-7.41	-4.75			-6.3
Solubility	1.44e-08 mg/ml ; 2.61e-11 mol/l	1.09e + 01 mg/ml ; 2.48e-02 mol/l	1.22e-02 mg/ml ; 2.07e-05 mol/l	2.72e-05 mg/ml ; 3.87e-08 mol/l	1.24e-02 mg/ml ; 1.78e-05 mol/l				4.26e-04 mg/ml ; 9.42e-07 mol/l
Class	Insoluble	Very soluble	Moderately soluble	Poorly soluble	Moderately soluble			Poorly soluble
Log S (Ali)	-12.95	-2.91	-4.90	− 8.29			-4.64	-6.69
Solubility	6.18e-11 mg/ml ; 1.12e-13 mol/l	5.44e-01 mg/ml ; 1.23e-03 mol/l	7.38e-03 mg/ml ; 1.25e-05 mol/l	3.60e-06 mg/ml ; 5.12e-09 mol/l			1.57e-02 mg/ml ; 2.27e-05 mol/l	9.23e-05 mg/ml ; 2.04e-07 mol/l
Class	Insoluble	soluble	Moderately soluble	Poorly soluble	Moderately soluble			Poorly soluble
Log S (SILICOS-IT)	-7.31	-4.60	-7.08	− 6.76			-5.43	− 4.29
Solubility	2.70e-05 mg/ml ; 4.88e-08 mol/l	1.12e-02 mg/ml ; 2.54e-05 mol/l	5.08e-05 mg/ml ; 8.63e-08 mol/l	1.21e-04 mg/ml ; 1.72e-07 mol/l		2.57e-03 mg/ml ; 3.70e-06 mol/l		2.33e-02 mg/ml ; 5.15e-05 mol/l
Class	Poorly soluble	Moderately soluble	Poorly soluble	Poorly soluble	Moderately soluble			Moderately soluble

Table 11

Different pharmacokinetics characteristics of compounds of corals and algae with high affinity to GP63, FPPS, and NMT proteins
compound Name	Alga-utd-01			Alga-utd-14	Coral-utd-01	Coral-utd-02	Coral-utd-03
GI absorption	Low	Low		Low	Low	Low	High
BBB permeant	No	No		No	No	No	Yes
P-gp substrate	Yes	No		Yes	Yes	Yes	Yes
CYP1A2 inhibitor	No	No		No	No	No	No
CYP2C19 inhibitor	No	No		No	No	No	No
CYP2C9 inhibitor	No	No		Yes	No	No	Yes
CYP2D6 inhibitor	No	No		No	No	No	No
CYP3A4 inhibitor	No	No		No	No	No	No
Log K_p (skin permeation)	-0.68 cm/s		− 9.76 cm/s	-8.19 cm/s	-6.67 cm/s	-9.64 cm/s	-5.18 cm/s

3.6. Results of molecular dynamic simulation analysis

The GP63/ Coral-utd-01, FPPS/ Coral-utd-02 and NMT/ Coral-utd-03 results from the docking analysis were refined using MD simulation. Finally, 100 ns scale MD simulations were performed to understand the stability of the interactions. Plots of RMSD and RMSF, Radius of gyration (Rg), MM/PBSA binding energy, and solvent accessible surface area (SASA) were examined (Fig. 8–14).

3.6.1. RMSD

The Root Mean Square Deviation (RMSD) is an important metric to assess the structural stability of biomolecular systems during molecular dynamics simulations. RMSD quantifies the average deviation of the position of a particular atom in the simulated structure from the initial structure during the simulation. The RMSD values for GP63/Coral-utd-01 ranged from 0.1 to 0.4 nm (Fig. 8, A). According to the results of FPPS/Coral-utd-02 (Fig. 1) The RMSD values of Fig. 8, B) ranged from 0.1 to 0.5 nm, and the RMSD values of NMT/Coral-utd-03 protein ranged from 0.10 to 0.22 nm (Fig. 8, C). Figure 8 shows the smallest fluctuations in the range of 20–50 ns, indicating the stability of the interaction.

3.6.2. RMSF

The Root Mean Square Fluctuation (RMSF) is a valuable metric for understanding the flexibility and dynamics of individual atoms in molecular systems. Quantify how much the positions of atoms change over time during molecular dynamics simulations. The RMSF value indicates how much each atom deviates from its average position. High RMSF values indicate flexibility or mobility, and low values indicate stiffness. The three proteins of L major showed no unintended fluctuations in the current simulations, demonstrating the stability of the interaction (Fig. 9).

3.6.3. The Rg analysis

The radius of gyration (often referred to as Rg) is a measure of how compact a structure is. This provides insight into the overall spatial distribution of atoms within the molecule. Rg is particularly useful for understanding the compactness of polymer chains and protein structures. As the Rg value decreases, the molecule becomes more compact, suggesting that it is tightly folded. An increasing Rg value indicates that the molecule is expanding and unfolding. The results shown in Fig. 10 demonstrates the stability of major protein-compound interactions and eventually the fluctuations will stabilize.

Figure 10. The radius gyration diagram of A: GP63/ Coral-utd-01. B: FPPS/ Coral-utd-02. C: NMT/ Coral-utd-03.

3.6.4. H-bond

A hydrogen bond diagram shows the number of hydrogen bonds formed between a protein and a ligand. These bonds play important roles in molecular interactions and influence protein-ligand binding and stability. In general, approximately 2, 2 and 1 h-bonds were observed for GP63/ Coral-utd-01, FPPS/ Coral-utd-02, NMT/ Coral-utd-03, respectively (Fig. 11).

3.6.5. The SASA analysis:

Solvent accessible surface area (SASA) is a measure of the surface area of a biomolecule (such as a protein) or other molecular structure that is accessible to solvent molecules. SASA calculations provide insight into the exposure of different parts of the molecule to the surrounding solvent. This analysis was calculated SASA per time, SASA per protein residue, and SASA per protein atom. This provides information about how the solvent-accessible surface of the protein changes during the simulation. Figure 12 shows the solvent accessible surface area of the three studied proteins GP63 (A), FPPS (B), and NMT (C). The SASA value does not change significantly between 20,000 ps and 50,000 ps.

3.6.6. Mmpbsa

The binding energy of the protein-ligand complex was calculated using the gmx-MMPBSA method. The results are shown in Fig. 13. In all figures, the last right column shown the final binding energies. Data and graphs showed that GP63/Coral-utd-01 interaction has a total binding energy of -33.94 (Fig. 13, A), and FPPS/Coral-utd-02 interaction has a total binding energy of -39.85 (Fig. 13, B). Total binding energy of NMT/ Coral-utd-03 interaction is -34.53(Fig. 13, C).

3.6. Systematic review flow diagram

In this study, we docked 389 coral compounds and 624 algae compounds against three important Leishmania target proteins to find the best drugs to inhibit these proteins. Among them, six active compounds showing the highest affinity for the protein were obtained, while three algae compounds, and five coral compounds showed high binding affinity for all three proteins. The results of molecular binding analysis showed that the binding tendencies of compounds examined in this study to bind to GP63, FPPS and NMT proteins were higher than existing blockers such as bisphosphonates. Our proposed algae and coral compounds are capable of strong interactions (ionic, hydrogen covalent) with important amino acids in the active site of proteins, which inactivates them because it has a better binding score and stability than conventional inhibitors, which improves the strength and the stability of interactions. Furthermore, good results were obtained from MD (RMSD, RMSF, Rg, PCA, MMPBSA, and SASA), indicating that the equil.

and high stability of interactions. Considering all these cases, these compounds can effectively inhibit the active proteins of Leishmania, and most likely, by inhibiting these proteins, they have a significant effect in inhibiting the Leishmania parasite.

Leishmaniasis is an important disease in subtropical and tropical regions. Several species of Leishmania parasites cause parasitic infections. Leishmaniasis is transmitted by the bite of a female sand fly that carries the parasite (41). 350 million individuals are at risk of contracting leishmaniasis, which is endemic in 98 nations (42). Affected patients face many problems, including toxicity, prolonged treatment duration, high treatment costs, and lack of oral preparations. Therefore, the development of new drugs to treat leishmaniasis is a global priority (43).

The in vitro anti-leishmanial activity of medicinal plants was studied by Mothana et al. against the promastigotes of three different species of Leishmania, L. tropica, L. infantum, and L. major, and several members of the Lamiaceae family. It was claimed that the species had anti-leishmania properties. (44). Algae and corals are found to be rich sources of several metabolites such as alkaloids, terpenoids, polysaccharides, pigments, cyclic peptides, phenols, lipid, and vitamins. The fractioned extracts of algae have shown interesting and exciting biological activities including antibacterial, antifungal, antiinflammatory, antioxidants, anticoagulant, anticancer, and antiprotozoal and antiviral. The recent trends in the drug discovery from marine microalgae with these activities are of attracted attention(45).

In 2020, Mordi et al, with a review study on the biological activity of carotenoids, concluded that these compounds, including Alga-utd-01, play a significant role in cancer treatment due to their antioxidant activity(46). Kim et al. (2021) observed the antiparasitic effect of Phaeodactylum tricornutum against the unicellular guppy fish parasite Gyrodactylus turnbulli and the antibacterial effect against Streptococcus in vivo and under laboratory conditions(15). In 2020, Veas et al. reported the anti-trypanosomal activity of ocular algal extracts of Chlamydomonas reinhardtii, Arthrospira platensis, and Nannochromopsis(47). Vaitkovicius et al. in 2022, with treated humans infected with L. infantum type (VL) with bioactive extracts of Dunaliella tertiolecta and Arthorspira platensis and comped their therapeutic effect with the reference drugs miltefosine (MTF) and N-methylglucamine, antimoniate (Sb^V) their stronger effect observed on the parasite(48). In one study, researchers investigated the anti-leishmanial and anti-cancer effects of three microalgae species, Nannochloris spp., Picochlorum spp., and Desmochloris spp. infected mice(49). In 2013, Kumar et al. reported antifungal and anti-Ieishmania activity in Candida albicans and Leishmania donovain as targets of Aphanothece(50).

In 2016, during a re-examination of Sinularia spp, Ofwegan et al. reported that Sinularia polydactyla is synonymous with Sinularia candidula(51). Soft coral Sinularia candidula extract has potent antiviral activity against avian influenza H5N1 strain(52). In 2020, the cytotoxic, anti-inflammatory, anti-angiogenic, and neuroprotective acivity of sinularia polydactyla compounds, as well as their effect on androgen receptor-regulated transcription was evaluated in vitro in human tumor and non-cancerous cells which inhibitory effects of endothelial cell migration and tumor cytotoxicity were reported(53). A study on four soft corals, including Sinularia polydactyla, discovered their antiviral effects against SARS-COV2 and HPV(54).

In one study, researchers reported significant anti-inflammatory and neuroprotective activities of Coral-utd-01(55). Li Song et al. in 2018 showed significant cytotoxicity from soft coral Sinularia spp. against tumor cells(56). In another study, researchers obtained anti-Leishmania donovani, anti-fungal effects on Candida albican and anti-bacterial Sinularia brassica effects against Staphylococcus aureus and Escherichia coli due to the presence of terpenoid compounds and sinolactol A and B in coral extract(57). In 2020, Yang et al. reported the anti-inflammatory and TNF-α inhibitory effects of Sinularia depressa extract(58). Another study on Sarcophyton glaucum found that the coral extract had leishmancidal activity and was highly toxic to HepG2 and MCF7 tumor cells(59). Aceret et al. in 1998, reported the antibiotic effects of Sinularia flexibilis(23). Another study demonstrated the antibacterial effects of Sinularia spp. and among others Sinularia polydactyla against Escherichia coli and Staphylococcus aureus(60). In 2016, Pham et al. discovered the antibacterial activity of Alkionium digitatum against the bacteria Bacillus subtilis, Staphylococcus epidermidis, Escherichia coli and the yeast Candida albicans(61). In confirmation of the obtained results, Tasdemir et al. study on the anti-trypanosomal and an-tileishmanial activity of flavonoids and their analogues such as flavones, anthroquinones, withanolides, and isoquinolines, the flavone-luteolin has an IC₅₀ value of 0.8 μg/ml, has been shown to be one of the most effective compounds in inhibiting L. donovani. His IC₅₀ value was almost the same as that of the clinically used anti-leishmania drug miltefosine (IC₅₀, 0.34 g/ml). Although a single OH group did not significantly change the benzochromone component of the flavone structure, the addition of two OH functional groups significantly increased the Leishman killing ability. C-5, C-7, and C-8 were very important positions. Hydroxylation of ring B had a significant effect on the activity, but no clear SAR could be observed. For example, luteolin, which has a catechol moiety (3,4-dihydroxyphenyl) and a 5,7-dihydroxybenzochromone structure, has an IC₅₀ value of 0.8 g/ml, which is twice as potent as apigenin (IC₅₀, 1.9 g/ml)). It has a p-hydroxyphenyl side chain. Luteolin showed the best combination as it had four OH groups at C-5, C-7, C-3', and C4' positions(62). Fouad et al. in 2021, and 2022 by in-vitro studies, showed the anti-L. major and anti-cancer effect of methanol extract of the soft corals Sarcophyton spongiosum and Sarcophyton trocheliophorum on three cancer cell lines, A549, HepG2, and MCF-7(24, 63).

According to drug databases like Pass Online, the Alga-utd-01, Alga-utd-05, Alga-utd-14, Coral-utd-01, Coral-utd-02 and Coral-utd-03 compounds have been predicted to have low side effects, antioxidant, anticarcinogenic, anti-inflammatory, antiviral, antineoplastic, antifungal, antibacterial, antiprotozoal, strengthening the immune system, and wound healing properties. In addition, the MD results indicate relatively stable interactions between these ligands and the mentioned proteins. Investigations of previous articles revealed that no bioinformatic studies, especially related to L. major have been performed on these compounds, and only Alga-utd-01 and Coral-utd-01 have been clinically investigated. Therefore, most of these compounds have been proposed for the first time in this study. Furthermore, most of the algae and corals that contain these compounds have unfortunately not been studied for leishmaniasis especially L. major, and the compounds and their mechanisms of action on parasites and disease have not been investigated in detail. The current research determines which vital protein of L. major can be inhibited through which effective algae and coral substance and to what extent, by preventing the occurrence of which stages of the disease, leishmaniasis is inhibited and treated. However, more experimental research and clinical trial tests on these proposed compounds, algae, and coral are needed to make sure of their antileishmanial effects.

In this study, we docked 389 coral compounds and 624 algae compounds against three important Leishmania target proteins to find the best drugs to inhibit these proteins. Among them, six active compounds showing the highest affinity for the protein were obtained, while three algae compounds, and five coral compounds showed high binding affinity for all three proteins. The results of molecular binding analysis showed that the binding tendencies of compounds examined in this study to bind to GP63, FPPS and NMT proteins were higher than existing blockers such as bisphosphonates. Our proposed algae and coral compounds are capable of strong interactions (ionic, hydrogen covalent) with important amino acids in the active site of proteins, which inactivates them because it has a better binding score and stability than conventional inhibitors, which improves the strength and the stability of interactions. Furthermore, good results were obtained from MD (RMSD, RMSF, Rg, PCA, MMPBSA, and SASA), indicating that the equil.

and high stability of interactions. Considering all these cases, these compounds can effectively inhibit the active proteins of Leishmania, and most likely, by inhibiting these proteins, they have a significant effect in inhibiting the Leishmania parasite.

Leishmaniasis is an important disease in subtropical and tropical regions. Several species of Leishmania parasites cause parasitic infections. Leishmaniasis is transmitted by the bite of a female sand fly that carries the parasite (41). 350 million individuals are at risk of contracting leishmaniasis, which is endemic in 98 nations (42). Affected patients face many problems, including toxicity, prolonged treatment duration, high treatment costs, and lack of oral preparations. Therefore, the development of new drugs to treat leishmaniasis is a global priority (43).

The in vitro anti-leishmanial activity of medicinal plants was studied by Mothana et al. against the promastigotes of three different species of Leishmania, L. tropica, L. infantum, and L. major, and several members of the Lamiaceae family. It was claimed that the species had anti-leishmania properties. (44). Algae and corals are found to be rich sources of several metabolites such as alkaloids, terpenoids, polysaccharides, pigments, cyclic peptides, phenols, lipid, and vitamins. The fractioned extracts of algae have shown interesting and exciting biological activities including antibacterial, antifungal, antiinflammatory, antioxidants, anticoagulant, anticancer, and antiprotozoal and antiviral. The recent trends in the drug discovery from marine microalgae with these activities are of attracted attention(45).

In 2020, Mordi et al, with a review study on the biological activity of carotenoids, concluded that these compounds, including Alga-utd-01, play a significant role in cancer treatment due to their antioxidant activity(46). Kim et al. (2021) observed the antiparasitic effect of Phaeodactylum tricornutum against the unicellular guppy fish parasite Gyrodactylus turnbulli and the antibacterial effect against Streptococcus in vivo and under laboratory conditions(15). In 2020, Veas et al. reported the anti-trypanosomal activity of ocular algal extracts of Chlamydomonas reinhardtii, Arthrospira platensis, and Nannochromopsis(47). Vaitkovicius et al. in 2022, with treated humans infected with L. infantum type (VL) with bioactive extracts of Dunaliella tertiolecta and Arthorspira platensis and comped their therapeutic effect with the reference drugs miltefosine (MTF) and N-methylglucamine, antimoniate (Sb^V) their stronger effect observed on the parasite(48). In one study, researchers investigated the anti-leishmanial and anti-cancer effects of three microalgae species, Nannochloris spp., Picochlorum spp., and Desmochloris spp. infected mice(49). In 2013, Kumar et al. reported antifungal and anti-Ieishmania activity in Candida albicans and Leishmania donovain as targets of Aphanothece(50).

In 2016, during a re-examination of Sinularia spp, Ofwegan et al. reported that Sinularia polydactyla is synonymous with Sinularia candidula(51). Soft coral Sinularia candidula extract has potent antiviral activity against avian influenza H5N1 strain(52). In 2020, the cytotoxic, anti-inflammatory, anti-angiogenic, and neuroprotective acivity of sinularia polydactyla compounds, as well as their effect on androgen receptor-regulated transcription was evaluated in vitro in human tumor and non-cancerous cells which inhibitory effects of endothelial cell migration and tumor cytotoxicity were reported(53). A study on four soft corals, including Sinularia polydactyla, discovered their antiviral effects against SARS-COV2 and HPV(54).

In one study, researchers reported significant anti-inflammatory and neuroprotective activities of Coral-utd-01(55). Li Song et al. in 2018 showed significant cytotoxicity from soft coral Sinularia spp. against tumor cells(56). In another study, researchers obtained anti-Leishmania donovani, anti-fungal effects on Candida albican and anti-bacterial Sinularia brassica effects against Staphylococcus aureus and Escherichia coli due to the presence of terpenoid compounds and sinolactol A and B in coral extract(57). In 2020, Yang et al. reported the anti-inflammatory and TNF-α inhibitory effects of Sinularia depressa extract(58). Another study on Sarcophyton glaucum found that the coral extract had leishmancidal activity and was highly toxic to HepG2 and MCF7 tumor cells(59). Aceret et al. in 1998, reported the antibiotic effects of Sinularia flexibilis(23). Another study demonstrated the antibacterial effects of Sinularia spp. and among others Sinularia polydactyla against Escherichia coli and Staphylococcus aureus(60). In 2016, Pham et al. discovered the antibacterial activity of Alkionium digitatum against the bacteria Bacillus subtilis, Staphylococcus epidermidis, Escherichia coli and the yeast Candida albicans(61). In confirmation of the obtained results, Tasdemir et al. study on the anti-trypanosomal and an-tileishmanial activity of flavonoids and their analogues such as flavones, anthroquinones, withanolides, and isoquinolines, the flavone-luteolin has an IC₅₀ value of 0.8 µg/ml, has been shown to be one of the most effective compounds in inhibiting L. donovani. His IC₅₀ value was almost the same as that of the clinically used anti-leishmania drug miltefosine (IC₅₀, 0.34 g/ml). Although a single OH group did not significantly change the benzochromone component of the flavone structure, the addition of two OH functional groups significantly increased the Leishman killing ability. C-5, C-7, and C-8 were very important positions. Hydroxylation of ring B had a significant effect on the activity, but no clear SAR could be observed. For example, luteolin, which has a catechol moiety (3,4-dihydroxyphenyl) and a 5,7-dihydroxybenzochromone structure, has an IC₅₀ value of 0.8 g/ml, which is twice as potent as apigenin (IC₅₀, 1.9 g/ml)). It has a p-hydroxyphenyl side chain. Luteolin showed the best combination as it had four OH groups at C-5, C-7, C-3', and C4' positions(62). Fouad et al. in 2021, and 2022 by in-vitro studies, showed the anti-L. major and anti-cancer effect of methanol extract of the soft corals Sarcophyton spongiosum and Sarcophyton trocheliophorum on three cancer cell lines, A549, HepG2, and MCF-7(24, 63).

According to drug databases like Pass Online, the Alga-utd-01, Alga-utd-05, Alga-utd-14, Coral-utd-01, Coral-utd-02 and Coral-utd-03 compounds have been predicted to have low side effects, antioxidant, anticarcinogenic, anti-inflammatory, antiviral, antineoplastic, antifungal, antibacterial, antiprotozoal, strengthening the immune system, and wound healing properties. In addition, the MD results indicate relatively stable interactions between these ligands and the mentioned proteins. Investigations of previous articles revealed that no bioinformatic studies, especially related to L. major have been performed on these compounds, and only Alga-utd-01 and Coral-utd-01 have been clinically investigated. Therefore, most of these compounds have been proposed for the first time in this study. Furthermore, most of the algae and corals that contain these compounds have unfortunately not been studied for leishmaniasis especially L. major, and the compounds and their mechanisms of action on parasites and disease have not been investigated in detail. The current research determines which vital protein of L. major can be inhibited through which effective algae and coral substance and to what extent, by preventing the occurrence of which stages of the disease, leishmaniasis is inhibited and treated. However, more experimental research and clinical trial tests on these proposed compounds, algae, and coral are needed to make sure of their antileishmanial effects.

According to the study, effective substances Alga-utd-01, Alga-utd-05, Alga-utd-14, Coral-utd-01, Coral-utd-02 and Coral-utd-03 had the greatest binding affinity for vital proteins of Leishmania GP63, FPPS, and NMT. In addition, the review of the mentioned drug databases showed that none of them have side effects, and they may have antioxidant, anticarcinogenic, anti-inflammatory, antiviral, antineoplastic, antifungal, antibacterial, antiprotozoal, strengthening the immune system, and wound healing properties. As a result, algae and corals containing these substances, especially those containing one or more of the desired compounds, are suggested for effective treatment of L. major. So, these compounds could be an effective treatment for leishmaniasis. Although this research has in silico approach, more clinical studies are needed to make sure of their antileishmanial effects on the L. major parasite.

Author Contribution

All authors wrote the main manuscript text and Hajar Shabandoust , Negar Balmeh, Seyed Mahmoud Mousavi , Zahra Alimardan prepared figures. All authors reviewed the manuscript."

Data availability statement

Data is provided within the manuscript file.

leishmaniasis 2023 [updated 12 January 202312 January 2023]. Available from: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis.
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Table 2 and 3 are available in the Supplementary Files section.

No competing interests reported.

Table2and3.docx

In silico investigation of the anti-leishmanial role of algae and corals active substances using molecular dynamic simulation and Molecular docking methods

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Version 1

Abstract

Materials and Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and methods