Nitric Oxide Inhibition Assay and the Respective Target Identification of an Aptamer Designed to Control Atherosclerosis.

INTRODUCTION: Aptamers are emerging newer therapeutics and diagnostics whichcan be designed to bind any kind of target proteins. Vascular endothelial damage by the excess amount of nitric oxide production in systemic circulation leads to the secretion of inflammatory chemoattractants and cell adhesions are the prime pro-atherogenic events in the formation of plagues at atrial intimal layers due to oxidation - sensitive mechanisms. Nitric oxide inhibition assay is one of the valuable qualitative anti-atherosclerosis matrices. METHODS: In this research, Nitric oxide inhibition efficiency of an ssDNA aptamer on cell lines was studied, and the respective targets of that aptamer were identified by network analysis. The aptamer used here was originally designed for Selectin P Ligand Protein to control the atherogenic process. 20 nM of aptamer solution in LipofectamineTM 2000 sshowshighest level 70.5% inhibition of nitric oxide liberation on 24 hours cultured medium of lipopolysaccharide stimulated murine macrophage RAW 264.7 cell lines. RESULTS: Protein interaction network analysis of the nitric oxide synthesis pathway interactors and the molecular docking analysis with network resulted in proteins such as AKT serine/threonine kinase 1, calmodulin, estrogen receptor 1, and nitric oxide synthase-3, which confirmed that the G - quadruplex Model of 18-mer sequence effectively bound to the active sites of estrogen receptor 1, and nitric oxide synthase-3. CONCLUSION: The aptamer designed for atherosclerotic target has also exertedsignificant nitric oxide inhibition to control the atherogenic events through the proteins such as, AKT1, NOS3 and ESR1.

In-silico strategies for identification of potent inhibitor for MMP-1 to prevent metastasis of breast cancer.

Matrix Metalloproteinase-1 (MMP-1) has been often upregulated in advanced breast cancers, known to participate in ECM degradation, migration, invasion, thus leading to metastasis. Due to these effects, the condition is often reported to inversely correlate with survival in advanced breast cancers. In the present study, in-silico method was adopted based on selective non zinc binding inhibitors of MMP-1. ADME properties were predicted for PASS filtered compounds and docking calculations were performed using Glide XP and IFD protocols of Schrodinger program. We identified six ligands as potent inhibitors and validated by observing structures and the interactions of MMP-1. The identified hits were validated using molecular dynamics simulation studies. Electronic structure analysis was performed for two top hit compounds myricetin and quercetin using density function theory (DFT) at B3LYP/6-31**G level to understand their molecular reactivity. Finally, one compound myricetin has emerged as the structurally stable compound with -7.801 kcal/mol and reasonable pose inside the binding site. Molecular dynamics results indicated that myricetin forms a stable interaction with the key amino acid residues such as Glu209, Glu219, Tyr240 and Pro238. In addition, it did not form any binding with the catalytic zinc at its active site. The interaction pattern of myricetin at its substrate binding site exhibited to be potent MMP-1 inhibitor. DFT study also showed that it has more potent inhibitory effect and solubility. These factors altogether show that myricetin could be considered as the best among the compounds evaluated in inhibiting MMP-1 thereby preventing metastasis of breast cancer. Communicated by Ramaswamy H. Sarma.

Structural insights into the binding mode of flavonols with the active site of matrix metalloproteinase-9 through molecular docking and molecular dynamic simulations studies.

Cartilage degradation in rheumatoid arthritis is mediated principally by the collagenases and gelatinases. Gelatinase B (also called matrix metalloproteinase 9 - MMP-9), is a valid target molecule which is known to participate in cartilage degradation as well as angiogenesis associated with the disease and inhibition of its activity shall prevent cartilage damage and angiogenesis. The focus of this study is to investigate the possibilities of MMP-9 inhibition by flavonol class of bioflavonoids by studying their crucial binding interactions at the active site of MMP 9 using molecular docking (Glide XP and QPLD) and further improvisation by post-docking MM-GBSA and molecular dynamic (MD) simulations. The results show that flavonols can convincingly bind to active site of MMP-9 as demonstrated by their stable interactions at the S1' specificity pocket and favourable binding energies. Gossypin has emerged as a promising candidate with a docking score of -14.618 kcal/mol, binding energy of -79.97 kcal/mol and a stable MD pattern over 15 ns. In addition, interaction mechanisms with respect to catalytic site zinc are also discussed. Further, the drug-like characters of the ligands were also analysed using ADME analysis.

A pilot study on the infiltrating cells and cytokine levels in the tear of fungal keratitis patients.

AIM: To determine the cellular profile and cytokine levels in the tear fluid of fungal keratitis patients. MATERIALS AND METHODS: Tear samples were collected from six fungal keratitis patients (Group I) from active stages of the disease up to resolution. Tears collected from the following served as controls: uninfected fellow eye (Group II A) of Group I, patients undergoing cataract surgery (Group II B) and acute conjunctivitis (Group II C). The cellular profile was evaluated. Interleukines (IL-6, IL-8 and IL-1beta) were estimated using sandwich enzyme immunoassay. Statistical analysis was carried out using nonparametric two-sample median test. RESULTS: Polymorphonuclear leukocytes (PMN) were the predominant infiltrating cells in Group I. During the initial stages of fungal infection, levels of IL-6 and IL-8 in the tear samples were found to be significantly increased when compared with Group II A (P=0.019 for IL-6, P<0.001 for IL-8). This was also true for IL -8 (P=0.008) levels in Group I and Group II B). While IL-6 levels decreased significantly towards healing, IL-8 remained slightly elevated even after healing. These cytokines were at the base level in Group II A. Lymphocytes and PMN were present in equal proportions in Group II C, which showed elevated levels of cytokines but not to the extent of Group I. CONCLUSION: This horizontal study indicates that understanding the nature of the inflammatory response in the tears of fungal keratitis patients is of considerable interest and warrants further investigations.