In-silico approach to investigate death domains associated with nano-particle-mediated cellular responses.

The current research is based on computational study of the Death Domain (DD) superfamily of proteins involved in the cytoplasmic inflammasome complexes associated with apoptosis and inflammation in response to the nanoparticle treatment. The PYD domains of the DD superfamily proteins, NALP3 and ASC, were chosen for investigation as they are found to be primarily involved in the regulation of innate immunity and are associated with apoptosis and inflammation. The in-vitro studies of these proteins have proven to be a challenge as the proteins have a tendency to aggregate under laboratory conditions. The interactions between PYD-PYD domains of NALP3 & ASC proteins as well as PYD-PYD domains of NALP3 and ASC2 proteins were studied using the computational tools. In our study, the protein structures were taken from Protein Data Bank, and molecular dynamics simulation was performed using NAMD software followed by molecular docking studies using HADDOCK. The generated protein models were validated using PROCHECK and then the protein-protein interactions were analyzed using the molecular visualization tool CHIMERA. We noticed that the affinity between PYD of NALP3-ASC complex is better when compared to the NALP3-ASC2 complex based on their binding energies and docking scores. In the case of NALP3-ASC complex, seven key amino acids of ASC-PYD protein interface interact with four key amino acids of NALP3-PYD protein interface. However, in the case of NALP3-ASC2 complex, six key amino acids of ASC-PYD protein interact with four key amino acids of NALP3-PYD protein. Although, there is not much difference in the number of interacting amino acid residues between the two protein complexes, we understand that the NALP3-ASC exhibits better binding interaction and stability than the NALP3-ASC2 protein complex because the number of hydrogen bonding between them is more when compared to the latter. The hydrogen bonds between the interacting amino acids of the NALP3-ASC protein interface are 12, whereas it is 6 in the case of NALP3-ASC2 protein interaction. The protein-protein interaction seems to be dominated by the energetically significant hydrogen bonding followed by the electrostatic interaction in the NALP3-ASC protein interface, whereas both hydrogen bonding and the interaction between charged residues appears to play a significant role at the NALP3-ASC2 protein interfaces.

Gold and Silver Nanoparticles Biomimetically Synthesized Using Date Palm Pollen Extract-Induce Apoptosis and Regulate p53 and Bcl-2 Expression in Human Breast Adenocarcinoma Cells.

Recently, several attempts have been made to use the phytopharmaceuticals from plant extracts as reducing, capping and stabilizing agents for the biomimetic synthesis of various metal nanoparticles conjugated to the phytopharmaceuticals. These biogenic metal nanoparticles are non-toxic and can be used as contrast agents, drug delivery vehicles and photothermal agents for cancer therapy. Herein, we report the synthesis of both silver and gold nanoparticles using the pollen extract of Phoenix dactylifera (Date Palm), characterization using UV-visible spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy, quantitation of phytochemicals capping the nanoparticles using Folin - Ciocalteu's method, cytotoxicity studies on MCF-7 breast cancer cells, cancer cell death analysis using fluorescent microscopy, and modulation of expression of the pro-apoptotic p53 and anti-apoptotic Bcl-2 proteins. The biosynthesis resulted in stable and poly-dispersed silver nanoparticles and gold nanoparticles, exhibiting strong and broad surface plasmon absorption peaks. The elemental analysis confirmed the presence of gold and silver of high purity and also the organic moieties from the plant extract acting as capping and stabilizing agents. The biogenic nanoparticles also exhibited dose-dependent cytotoxicity on MCF-7 cells and showed signs of apoptotic cell death. Immunoassays revealed the upregulation of the pro-apoptotic protein p53 and down-regulation of the anti-apoptotic protein Bcl-2 after the nanoparticle treatment.

Doxorubicin loaded polymeric gold nanoparticles targeted to human folate receptor upon laser photothermal therapy potentiates chemotherapy in breast cancer cell lines.

The current research focuses on the application of folate conjugated and doxorubicin loaded polymeric gold nanoparticles (GNPs) for the targeted treatment of folate receptor overexpressing breast cancers, augmented by adjunctive laser photothermal therapy. Herein, GNPs surface modified with folate, drug doxorubicin and polyethylene glycol were engineered and were used as vehicles for folate receptor targeted delivery of doxorubicin into cancer cells. Subsequently, the GNPs were photo-excited using laser light for mediating hyperthermia in the cancer cells. In vitro studies were performed to validate the efficacy of the combined modality of folate conjugated and doxorubicin loaded polymeric GNP mediated chemotherapy followed by photothermal therapy in comparison to treatment with free drug; and the combination modality showed better therapeutic efficacy than that of plain doxorubicin treatment in MDA-MB-231 breast cancer cells that express increased levels of surface folate receptors when compared to MCF-7 breast cancer cells that express low levels of folate receptor. The mechanism of cell death was investigated using fluorescent microscopy. Immunoassays showed the up-regulation of the pro-apoptotic protein p53 and down-regulation of the anti-apoptotic protein Bcl-2. Collectively, these results suggest that the folate tagged doxorubicin loaded GNPs are an attractive platform for targeted delivery of doxorubicin and are agents suitable for photothermal cancer therapy.

Reduced catalytic activity of human CYP2C9 natural alleles for gliclazide: molecular dynamics simulation and docking studies.

Amongst sulfonylureas, gliclazide is one of the mostly prescribed drugs to diabetic patients and is metabolized extensively by P450 CYP2C9. Among 24-CYP2C9 alleles, the *2/*2 and *3/*3 genotypes showed significantly lower gliclazide clearances with reductions of 25 and 57%, respectively. However, the reason for the change in drug-metabolizing activity induced by these natural alleles is unknown. In the present study, we used molecular dynamics simulation and autodocking studies to provide models for gliclazide-bound complexes of CYP2C9*2, *3 and *2/*3 mutants, which give insight into CYP2C9-gliclazide interactions and explain the reduced enzymatic activity seen in these variants. Our data shows that the size of the substrate-access entry site is significantly reduced in mutants, which limits the access of gliclazide to heme and the active site. The distance from the substrate oxidation site and heme is >5Å in *3 and *2/*3. Therefore, the addition of an active oxygen molecule by heme-Fe is hindered. The absence of F100, F114 and F476 in the interacting amino acid pocket in *3 reduces catalytic efficiency toward gliclazide. In *1, gliclazide is stabilized by the formation of two hydrogen bonds with R108 while it is absent in mutants. Further in *3 and *2/*3, the key heme-stabilizing residue, R97 stabilization is greatly reduced. Therefore, the decreased catalytic activity of these variants can be explained from the reduced access of the gliclazide to heme, and the interaction between heme and substrate is affected due to their instability in the active site.