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Item In silico study of physicochemical, pharmacokinetic, toxicity, metabolism and molecular docking of aristolactam E, a constituent of Aristolochia elegans Mast(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Seneviratne, K. N.; Jayathilaka, N.Aristolochia elegans Mast, also known as Calico flower, is an ornamental plant found around the world including Asia. In traditional medicine, the plant has been used to treat a variety of disorders by employing its broad spectrum of pharmacological properties such as antibacterial, antitumoral, antidiarrheal, anti-snake venom, and anti-scorpion venom. It is also rich in aristolactam alkaloids, which have a variety of biological effects including anti-inflammatory, antibacterial, and anticancer activities. Aristolactam E (AE) is one of the less studied aristolactam alkaloids found in Aristolochia elegans Mast. Therefore, this computational study was designed to investigate (a) physicochemical properties and drug likeness, (b) pharmacokinetic and toxicity, (c) mode of antibacterial, anticancer and antiinflammatory action, (d) sites of metabolism mediated by cytochrome P450 (CYP) 3A4 isoform and (e) probable metabolites of AE compound. The physicochemical attributes and drug likeness were assessed using molinspiration server. SwissADME, LASAR, and Pred-hERG 4.2 servers were used to estimate pharmacokinetic and toxicological characteristics. Molecular docking was performed using AutoDock 4.2 software to determine the binding affinity and molecular interactions of AE with protein targets namely, dihydropteroate synthase (DHPS), dihydroneopterin aldolase (DHNA), phospholipase A2 (PLA2) and tankyrase 2 (TNK2). SOMP, SMARTcyp, and RS-WebPredictor webservers were used to predict the sites of AE metabolism mediated by the CYP 3A4 isoform. Furthermore, the BioTransformer and GLORYx online tools were used to forecast the potential AE metabolites. AE demonstrated favorable physicochemical qualities and met the requirements for oral bioavailability and druggability by following both Lipinski's rule of five and the Veber rule. It also showed a high rate of human intestinal absorption and no blood-brain barrier permeability. Furthermore, the toxicity predictions revealed that AE was a mutagenic chemical, but it was not carcinogenic or cardiotoxic in the mouse model. The AE showed binding affinity of -5.45 kcal/mol, -8.79 kcal/mol, -7.32 kcal/mol, -8.30 kcal/mol and -8.41 kcal/mol with DHPS, DHNA, PLA2, TNK2 (nicotinamide binding site) and TNK2 (adenosine binding site) respectively. The AE exhibited a stronger binding affinity than the control compounds of DHNA and PLA2 while showing close binding affinity to the control of TNK2 (nicotinamide binding site). The overall evaluation identified two sites of metabolism of AE based on a consensus of different metabolism site predictions by CYP3A4 using SOMP, SMARTcyp and RS-WebPredictor webservers. Two probable metabolites of AE were proposed based on the consensus of the results of BioTransformer and GLORYx web tools. This computational analysis can aid in the development of AE derivatives with improved pharmacokinetic and toxicological profiles, hence accelerating drug discovery against microbial infections and inflammation.Item Identification of carbazole alkaloids from Murraya koenigii as potential main protease inhibitors of SARS-CoV-2 Omicron variant(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Jayathilaka, N.; Seneviratne, K. N.Despite of COVID-19 vaccination, immune escape of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has created an urgent priority to identify additional antiviral drugs. In the short span of two years, SARS-CoV-2 has evolved to raise five variants of concern out of which Omicron has currently become the dominant variant all over the world. Targeting main protease (Mpro) expressed by SARS-CoV-2 Omicron variant, is a therapeutic strategy for drug development due to its prominent role in viral replication cycle. Leaves of Murraya koenigii are used in various traditional medicinal applications and this plant is known as a rich source of carbazole alkaloids. Previous research reports have shown that the leaves, roots and bark of this plant are high in carbazole alkaloids. Many drug compounds containing a carbazolic core have been discovered, and some have demonstrated antiviral action. Thus, this computational study was designed to investigate the inhibitory potential of carbazole alkaloids from Murraya koenigii against Mpro. Molecular docking was performed using AutoDock Vina software to determine the binding affinity and molecular interactions of carbazole alkaloids and the reference inhibitor (3WL) in the active site of SARS-CoV-2 Omicron variant Mpro (PDB ID: 7TLL). The top scoring compounds were further assessed for physicochemical properties and drug likeness, pharmacokinetic and toxicity (ADME/T) properties, antiviral activity, pharmacophore modeling and molecular dynamics (MD). Two carbazole alkaloids namely, koenine (-7.8 kcal/mol) and girinimbine (-7.6 kcal/mol) displayed a unique binding mechanism that shielded the catalytic dyad (His41 and Cys145) of Mpro with stronger binding affinities and molecular interactions than 3WL (-7.2 kcal/mol). Furthermore, the two compounds with high affinity displayed favorable physicochemical and ADME/T properties that satisfied the criteria for oral bioavailability and druggability. The pharmacophore modeling study showed shared pharmacophoric features (aromatic ring, hydrophobic area, hydrogen bond donor/acceptor and positively ionizable region) of those compounds for their biological interaction with Mpro. During the molecular dynamics simulation of 100 ns, the MD simulation trajectories of root mean square deviation (RMSD), root mean square fluctuation (RMSF) and radius of gyration (Rg) of top two complexes exhibited high stability. Therefore, koenine and girinimbine from Murraya koenigii, may have the potential to restrict SARS-CoV-2 replication by inactivating the Mpro catalytic activity thus offering potential hits that may be further structurally modified and evaluated in vitro and in vivo for the discovery of novel SARS-COV-2 Mpro inhibitors.Item Identification of phytochemical inhibitors against papain-like protease of SARS-CoV-2: molecular docking, molecular dynamics and absorption, distribution, metabolism, excretion and toxicity (ADMET) study(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Mannapperuma, U.; Jayathilaka, N.The COVID-19 outbreak has created a huge social and economic disruption worldwide due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro) of SARS-CoV-2 plays a crucial role in viral replication and host innate immunity suppression. Therefore, it is an ideal therapeutic target to develop inhibitors. Thus, the goal of this study was to use virtual screening methods to identify potential phytochemical inhibitors of this dual therapeutic target. Virtual docking was performed for 31 phytochemicals with documented anti-SARS-CoV-1 PLpro activity and two positive controls using AutoDock 4.2 software to determine the binding affinity, inhibition constant and ligand efficiency of each compound within the S3/S4 binding pocket of SARS-CoV-2 PLpro (PDB ID: 6WX4). Based on the docking results, top twelve compounds were subjected to protein-ligand interaction analysis utilizing the Discovery Studio Visualizer software. Physicochemical properties were analyzed using molinspiration web server. Moreover, pharmacokinetics and toxicity descriptors were assessed using pkCSM and StopTox web servers, respectively. Molecular dynamics simulations (MD) were carried out for 100 ns for each top docking complex and PLpro of SARS-CoV-2 inhibitors. Hirsutenone (from Alnus japonica), broussoflavan A (from Broussonetia papyrifera) and broussochalcone A (from Broussonetia papyrifera) displayed the strongest binding affinities (-8.23 kcal/mol, -8.13 kcal/mol and -7.78 kcal/mol), the lowest inhibition constants (920.39 nM, 1.1 μM and 1.97 μM ) and the highest ligand efficiencies (0.34, 0.26 and 0.31) among all phytochemicals towards the S3/S4 binding pocket of SARS-CoV-2 PLpro, demonstrating superiority to positive control, GRL0617 while hirsutenone and broussoflavan A exhibited superiority to both positive controls, 3k and GRL0617. In addition, hirsutenone, broussoflavan A and broussochalcone A possessed favorable physicochemical properties satisfying Lipinski’s and Veber’s rules. Furthermore, in silico pharmacokinetics and toxicity predictions revealed that the three phytochemicals are water soluble, non-mutagenic, non-hepatotoxic. These compounds were not toxic for acute inhalation and acute dermal exposure. They also showed no eye irritation, skin irritation or corrosive properties. MD confirmed the stability of broussoflavan A and broussochalcone A. However, hirsutenone showed less stability due to fluctuations during the simulation period. Hence, broussoflavan A and broussochalcone A might be exploited to expedite the drug discovery process against the ongoing COVID-19 infection.Item Identification of potential inhibitors against prostate cancer metastasis drug target, human fatty acid binding protein-12: An in silico study(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Seneviratne, K. N.; Jayathilaka, N.Fatty acid-binding proteins (FABPs) play a vital role in fatty acid metabolism, cell growth and proliferation and cancer development in humans. Recent studies have revealed that FABP-12 can promote prostate cancer through activation of peroxisome proliferator-activated receptor gamma (PPAR-) which has previously been reported as a driver of metastasis in prostate cancer. Hence targeting the human FABP-12 might be a therapeutic strategy to control prostate cancer metastasis. Due to the absence of crystal structure of this protein, a 3D homology model of FABP-12 was generated using crystal structure of human myelin protein P2 (PDB ID: 2WUT, Resolution: 1.85 Å) as the template by Modeller 9.23 software. The hypothetical model showed the backbone root mean square deviation (RMSD) value of 0.128 Å after superimposition with the template. Further, the structural quality of the model was validated through QMEAN, VERIFY3D, ERRAT, PROCHECK and PROSA tools. Structure based drug discovery of the FABP-12 protein was performed using AutoDock4.2 software with a library of ligands consisting experimentally known FABP family inhibitors (BMS309403 and SBFI-26) and anti-prostate cancer phytochemicals that have been reported earlier. Molecular interactions were explored to understand the nature of intermolecular bonds between ligand and the protein binding site residues using BIOVIA Discovery Studio Visualizer. The in silico hepatoxicity, mutagenicity and cytotoxicity end points for experimentally known FABP family inhibitors and top docked phytochemicals were examined using ProTox-II web server. The BMS309403 showed the highest binding affinity of -10.02 kcal/mol closely followed by celastrol, SBFI-26 and glycyrrhetinic acid with binding affinities of -9.39 kcal/mol, - 9.24 kcal/mol and -9.39 kcal/mol respectively. The inhibition constant (Ki) of BMS309403, celastrol, SBFI-26 and glycyrrhetinic acid were 44.94 nM, 131.65 nM, 167.52 nM and 200.62 nM respectively. Moreover, the in-silico toxicity results revealed that BMS309403 has a weak hepatotoxic potential. Notably, other three compounds obtained negative results for all toxicity descriptors, implying no severe human side effects. These computational findings indicate that celastrol, SBFI-26, and glycyrrhetinic acid have the ability to suppress FABP-12 activity and hence could be utilized as a starting point for future drug development to treat prostate cancer metastasis.Item In silico evaluation of coconut milk phenolic antioxidants and their metabolites by human gut bacteria(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Chamikara, V; Perera, S. M.; Seneviratne, K. N.; Jayathilaka, N.Coconut milk (CM) or the aqueous extract of grated coconut meat is used in culinary applications when preparing both vegetarian and non-vegetarian foods in many Asian countries. Despite CM is traditionally known to be beneficial for gut health, scientific evidence supporting such claims is extremely limited. The beneficial properties of phenolic antioxidants on gut microbiota suggest that phenolic-rich CM may also benefit gut health. Human intestinal gut microbiota plays a vital role in metabolizing dietary components that enter the intestinal tract. These metabolites possess molecular characteristics as well as biological activities, pharmacokinetic and toxicity properties that are different from their parent compounds. Due to a lack of experimental data to examine the gut microbial metabolism of phenolic compounds of CM, a computational study was designed to predict its phenolic metabolites. Our previous high-performance liquid chromatography (HPLC) study reported seven phenolic acids in the CM extract. BioTransformer 3.0 web-based tool was used to predict metabolic transformations of the seven phenolic acids by human gut bacteria. In silico studies were conducted to predict the antioxidant, intestinal anti-inflammatory, antibacterial activities and toxicity of the phenolic compounds and their metabolites using PASS and ProTox-II web servers respectively. The safety of the phenolic compounds of CM and their metabolites were also evaluated based on the endocrine-disrupting effect and the probability of interaction with multiple human receptors using Endocrine Disruptome web server. The in silico analysis of human gut microbial biotransformation predicted the formation of 41 metabolites from 7 parent phenolic compounds present in CM. Most of the parent phenolic acids and the predicted metabolites of CM were shown to have moderate to high antioxidant, intestinal anti-inflammatory activity and antibacterial activity with Probable activity (Pa) > Probable inactivity (Pi) values. Most of the metabolites had a low probability of binding to human nuclear receptors, causing small risks to the endocrine system and posing minimal risk to human health. Moreover, the results revealed that only a few compounds have a weak mutagenic and hepatotoxic potential, while all compounds were devoid of cytotoxicity. However, further in vitro and in vivo testing is required to confirm the impact of these parent phenolic acids and their metabolites on diverse gut microbes and human health and in promoting CM as a functional food as well as a vegan replacement for cow’s milk.