Molecular docking and dynamics simulation of Orthosiphon stamineus against SGLT1 and SGLT2.

Orthosiphon stamineus Benth a traditional medicine used in the treatment of diabetes and kidney diseases. Sodium-glucose co-transporter (SGLT1 and SGLT2) inhibitors are the novel group of drugs used to treat patients with type 2 diabetes mellitus. In this study 20 phytochemical compounds from Orthosiphon stamineus Benth were obtained from 3 databases viz Dr.Duke's phytochemical, Ethno botanical database and IMPPAT. They were subjected to physiochemical, drug likeliness, and ADMET and toxicity predictions. Homology modeling and molecular docking against SGLT1 and SGLT2 were performed and the stability of the selected drug molecule was validated by molecular dynamic (MD) simulation for 200 ns. Among the 20 compounds, 14-Dexo-14-O-acetylorthosiphol Y alone showed higher binding affinity with SGLT1 and SGLT2 protein with the binding energy of -9.6 and -11.4 Kcal/mol respectively and had highest affinity towards SGLT2 inhibitor. This compound also satisfied Lipinski rule of 5 and had a good ADMET profile. The compound is non-toxic to marine organisms and to normal cell lines and non-mutagenic. The RMSD value attained equilibrium at 150 ns with the stability around 4.8 Å and no significant deviation was reported from 160 to 200 ns for SGLT2. Our study suggests that 14-Dexo-14-O-acetylorthosiphol Y showed promising results against the SGLT2 and could be considered as a potent anti-diabetic drug.Communicated by Ramaswamy H. Sarma.

Genetic Polymorphism and Gene Expression of ß-Defensin-1 in Periodontitis Associated With Type 2 Diabetes Mellitus.

Introduction Periodontitis is a multifactorial disease caused by periodontopathic bacteria and influenced by both genetic and environmental factors. Genetic predispositions are found to play a crucial part in the onset and progression of periodontal disease. There is a two-way relationship between diabetes and periodontitis with severe periodontal tissue destruction seen in diabetic patients. Antimicrobial peptide, ß-defensin-1 (DEFB1 gene), plays an important role in the innate immune responses and forms the first line of host defense against periodontal pathogens. Single nucleotide polymorphisms (SNPs) in the specific genetic loci of the DEFB1 gene and its expression level could confer a degree of risk or protection from periodontitis associated with diabetes. The present study determined the association between SNPs at the 5' untranslated region (UTR) in the DEFB1 gene and susceptibility to periodontitis associated with type 2 diabetes mellitus (T2DM) and analyzed the effect of 5' UTR polymorphisms on DEFB1 gene expression. Methods SNPs in the 5' UTR of the DEFB1 gene (-20G>A (rs11362), -44C>G (rs1800972), and -52G>A (rs1799946)) were genotyped by polymerase chain reaction (PCR) followed by Sanger sequencing. The study group included periodontitis (n = 40), periodontitis with T2DM (n = 20), and periodontally and systemically healthy as controls (n = 40). DEFB1 gene expression was determined by real-time PCR in the study group comprising periodontitis (n = 20), periodontitis with T2DM (n = 15), and healthy controls (n = 20). The effect of 5' UTR polymorphisms on the expression was analyzed by statistical tools. Results Statistically significant higher prevalence of the variant AA genotype of rs11362 was observed in periodontitis (odds ratio (OR) = 3.64, 95% confidence Interval (CI) = 1.16-11.43, p = 0.04) and periodontitis with T2DM (OR = 5.14, 95% CI = 1.29-20.5, p = 0.03) in comparison with healthy controls. Moreover, there was a significant increase of the variant AA genotype of rs1799946 in periodontitis (OR = 3.88, 95% CI = 1.19-12.68, p = 0.04) compared to healthy controls. DEFB1 gene expression was downregulated in periodontitis and upregulated in periodontitis with T2DM patients when compared to healthy controls but was not statistically significant. No significant association was found for the effect of SNPs of the DEFB1 gene on its expression. Conclusion From the SNP analysis, it can be inferred that the presence of SNPs at the 5' UTR (rs11362 and rs1799946) in the DEFB1 gene may be an important predictive factor for periodontitis.

The Role of ß-Defensin 1 Against Porphyromonas gingivalis Lipopolysaccharide-Mediated Inflammation in the THP-1 Cell Line.

Introduction Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) is one of the crucial virulence factors of periodontitis. Antimicrobial peptides (AMPs) are emerging as alternatives or adjuncts to antibiotics in the treatment of microbial infections. In this study, cytotoxicity, anti-inflammatory activity, anti-oxidative stress, cell cycle analysis, and apoptosis properties of AMP, ß-defensin 1, were studied in Pg-LPS-stimulated THP-1 (Tohoku Hospital Pediatrics - 1) cell line. Methods The cytotoxic nature of Pg-LPS and ß-defensin 1 was studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The cytotoxic effect of ß-defensin 1 on Pg-LPS-stimulated THP-1 cells was also studied by the same method. The anti-inflammatory role of ß-defensin 1 against cyclooxygenase (COX), lipoxygenase (LOX), myeloperoxidase (MPO), and inducible nitric oxide synthase activities were studied. The anti-oxidative nature of ß-defensin 1 was analyzed by measuring reactive oxygen species (ROS) generation by dichlorodihydrofluorescein diacetate (DCFDA) assay. Cell cycle distribution and apoptosis were studied by flow cytometry. The hemolytic nature of ß-defensin 1 was predicted using the HemoPred web tool. Results The results of the study demonstrated that Pg-LPS showed dose-dependent cytotoxicity to THP-1 cells. ß-Defensin 1 had dose-dependent cytotoxicity to THP-1 cells and showed a protective effect on THP-cells up to 1 µg/mL of Pg-LPS, beyond which cell viability decreased. ß-Defensin 1 inhibited COX, LOX, MPO, and inducible nitric oxide synthase activities in a concentration-dependent manner. ß-Defensin 1 showed anti-oxidative activity by suppressing the generation of ROS measured through fluorescence intensity. From the cell cycle analysis, it was found that ß-defensin 1 was able to reduce the Pg-LPS-induced cell cycle arrest at the G0/G1 phase. From the apoptosis profile, ß-defensin 1 was found to increase the live cells when compared to THP-1 cells stimulated only with Pg-LPS, indicating that ß-defensin 1 provided a protective role to THP-1 cells. ß-Defensin 1 was found to be hemolytic in nature by the HemoPred web tool. Conclusion ß-Defensin 1 exerted multifunctional activities and can be considered a promising agent for controlling periodontitis.

Determination of deleterious single-nucleotide polymorphisms of human LYZ C gene: an in silico study.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) have a crucial function in affecting the susceptibility of individuals to diseases and also determine how an individual responds to different treatment options. The present study aimed to predict and characterize deleterious missense nonsynonymous SNPs (nsSNPs) of lysozyme C (LYZ C) gene using different computational methods. Lyz C is an important antimicrobial peptide capable of damaging the peptidoglycan layer of bacteria leading to osmotic shock and cell death. The nsSNPs were first analyzed by SIFT and PolyPhen v2 tools. The nsSNPs predicted as deleterious were then assessed by other in silico tools - SNAP, PROVEAN, PhD-SNP, and SNPs & GO. These SNPs were further examined by I-Mutant 3.0 and ConSurf. GeneMANIA and STRING tools were used to study the interaction network of the LYZ C gene. NetSurfP 2.0 was used to predict the secondary structure of Lyz C protein. The impact of variations on the structural characteristics of the protein was studied by HOPE analysis. The structures of wild type and variants were predicted by SWISS-MODEL web server, and energy minimization was carried out using XenoPlot software. TM-align tool was used to predict root-mean-square deviation (RMSD) and template modeling (TM) scores. RESULTS: Eight missense nsSNPs (T88N, I74T, F75I, D67H, W82R, D85H, R80C, and R116S) were found to be potentially deleterious. I-Mutant 3.0 determined that the variants decreased the stability of the protein. ConSurf predicted rs121913547, rs121913549, and rs387906536 nsSNPs to be conserved. Interaction network tools showed that LYZ C protein interacted with lactoferrin (LTF). HOPE tool analyzed differences in physicochemical properties between wild type and variants. TM-align tool predicted the alignment score, and the protein folding was found to be identical. PyMOL was used to visualize the superimposition of variants over wild type. CONCLUSION: This study ascertained the deleterious missense nsSNPs of the LYZ C gene and could be used in further experimental analysis. These high-risk nsSNPs could be used as molecular targets for diagnostic and therapeutic interventions.

In silico targeting of red complex bacteria virulence factors of periodontitis with ß-defensin 1.

BACKGROUND: Periodontitis is a multi-factorial infection with red complex bacteria playing a crucial role in the pathogenesis. As bacteria are tending to develop resistance against conventional antibiotics, new treatment modalities need to be developed. Antimicrobial peptides (AMPs) are potential tools for drug development and are gaining widespread interest. ß-defensin 1 is an important AMP and forms the first-line host defense mechanism. The present study analyzed the structure and molecular docking of ß-defensin 1 with the virulence factors of red complex bacteria of periodontitis. The physico-chemical properties of ß-defensin 1 were determined by various online tools such as ProtParam, ProteinPredict, ToxinPred, and BioPep web servers. The structure of ß-defensin 1was predicted by the SWISS-MODEL web server and the structure was evaluated by different web tools. The structure of lipopolysaccharide of Porphyromonas gingivalis was drawn using Chem3D ultra 11.0 software. The structure of important protein virulence factors of red complex bacteria of periodontitis was determined by the SWISS-MODEL web server. The interaction study between ß-defensin 1 and virulence factors was carried out by molecular docking using Auto dock version 4.0 software and pyDock WEB server. RESULTS: Using online tools, ß-defensin 1 was predicted to be stable and non-toxic. SWISS-MODEL web server predicted Ramachandran score as 94.12% and clash score 0.0 for ß-defensin 1. Auto dock version 4.0 software and pyDock WEB server analyzed the interaction to have low binding energies and hydrogen bonds were formed between the peptide and virulence factors. CONCLUSION: ß-defensin 1 was found to have good binding interaction with the disease-causing factors of red complex bacteria of periodontitis and in turn could play a role in reducing the severity of infection. ß-defensin 1 could be a potential candidate for drug development for periodontitis.