Computational protocol: Evidences for Piperine inhibiting cancer by targeting human G-quadruplex DNA sequences

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Protocol publication

[…] Fluorescence titration experiment was performed on Synergy H1 multi-mode microplate reader using 96-well microplates at 25 °C. The excitation and emission wavelengths for Piperine were obtained by performing its absorption and fluorescence scan diluted in potassium phosphate buffer. The readings were taken for Piperine: G-quadruplex DNA titration at emission wavelength of 486 nm when excited at the wavelength of 341 nm. Each sample was tested in duplicates in 75 μL reaction volume. 5–10 μM of G-quadruplex DNA and a final concentration of 100 μM for CT-DNA were serially diluted; with the last well serve as blank (no DNA). Data were analyzed using SigmaPlot 12.0 software (Systat Software, Chicago, USA) according to the following equation that accounts for two receptor binding sites with two different affinities kd1 and kd2: Bmax = maximum number of binding sites.Kd = equilibrium binding constant. [...] The structure of G-quadruplex Pu24T (PDB code: 2MGN) was taken as the starting model and the required replacements, addition of residues, optimization of G-quadruplex structure and Piperine structure using charmM forcefield were performed on Discovery studio 3.5 The molecular docking studies were carried out on Autodock 4.0 in which G-quadruplex DNA was treated as rigid body. All the other parameters used were set to their default values. Pu24T and Piperine structures were converted to AD4 format files and Gesteiger charges were assigned to the atoms. The grid was set in such a manner that it covers complete DNA structure so that ligand can explore the whole conformational space. The Lamarckian genetic algorithm was used for the search and the results were analyzed based on binding energy. For molecular dynamic (MD) simulation studies, the best conformation of Pu24T- Piperine complex obtained from docking studies was used as input. Second molecule of Piperine was placed manually in a way as obtained from docking studies. The complex was typed in charmM forcefield and solvated with periodic TIP3P orthorhombic water box containing 2091 water molecules. This complex was first minimized then subjected to simulated annealing molecular dynamics by employing standard dynamic cascade. In this cascade, the system was heated to 700 K and equilibrated for 10 ps under constant pressure. The production was done at 300 K for 100 ns in an NPT ensemble and long range electrostatics were treated with the Particle Mesh Ewald (PME) method with a 14 Å cut-off radius counted the non-bonded distances. The SHAKE algorithm was applied during the whole simulation runs in order to constrain the motion of Hydrogen bonds. […]

Pipeline specifications

Software tools SigmaPlot, CHARMM, AutoDock
Applications Miscellaneous, Protein interaction analysis
Organisms Homo sapiens
Diseases Neoplasms