In silico and in vitro screening for potential anticancer candidates targeting GPR120

The G-protein coupled receptor - GPR120 has recently been implicated as a novel target for colorectal cancer (CRC) and other cancer managements. In this study, a homology model of GPR120S (short isoform) was generated to identify potential anti-cancer compounds targeting the GPR120 receptor using a combined in silico docking-based virtual screening (DBVS), structure-activity relationships (SAR) and in vitro screening approach. SPECS database of synthetic chemical compounds (~350,000) was screened using the developed GPR120S model to identify molecules binding to the orthosteric binding pocket followed by an AutoDock SMINA rigid-flexible docking protocol. The best 13 hit molecules were then tested in vitro to evaluate their cytotoxic activity against SW480 - human CRC cell line expressing GPR120. The test compound 1 (3-(4-methylphenyl)-2-[(2-oxo-2-phenylethyl)sulfanyl]-5,6-dihydrospiro(benzo[h]quinazoline-5,1\u27-cyclopentane)-4(3H)-one) showed ~ 90% inhibitory effects on cell growth with micromolar affinities (IC50 = 23.21-26.69 µM). Finally, SAR analysis of compound 1 led to the identification of a more active compound from the SPECS database showing better efficacy during cell-based cytotoxicity assay -5 (IC50 = 5.89-6.715 µM), while a significant reduction in cytotoxic effects of 5 was observed in GPR120-siRNA pre-treated SW480 cells. The GPR120S homology model generated, and SAR analysis conducted by this work discovered a potential chemical scaffold, dihydrospiro(benzo[h]quinazoline-5,1\u27-cyclopentane)-4(3H)-one, which will aid future research on anti-cancer drug development for CRC management

3D mammalian cell culture models in toxicology testing

3D cell culture can be successfully used as an alternative to laboratory animals, and as a cost effective and time-saving tissue culture technique, which also reduces the trial period for drug testing

Structure based prediction of a novel GPR120 antagonist based on pharmacophore screening and molecular dynamics simulations

The G-protein coupled receptor, GPR120, has ubiquitous expression and multifaceted roles in modulating metabolic and anti-inflammatory processes. Recent implications of its role in cancer progression have presented GPR120 as an attractive oncogenic drug target. GPR120 gene knockdown in breast cancer studies revealed a role of GPR120-induced chemoresistance in epirubicin and cisplatin-induced DNA damage in tumour cells. Higher expression and activation levels of GPR120 is also reported to promote tumour angiogenesis and cell migration in colorectal cancer. Some agonists targeting GPR120 have been reported, such as TUG891 and Compound39, but to date development of small-molecule inhibitors of GPR120 is limited. Herein, following homology modelling of the receptor a pharmacophore hypothesis was derived from 300 ns all-atomic molecular dynamics (MD) simulations on apo, TUG891-bound and Compound39-bound GPR120S (short isoform) receptor models embedded in a water solvated lipid bilayer system. We performed comparative MD analysis on protein–ligand interactions between the two agonist and apo simulations on the stability of the “ionic lock” – a Class A GPCRs characteristic of receptor activation and inactivation. The detailed analysis predicted that ligand interactions with W277 and N313 are critical to conserve the “ionic-lock” conformation (R136 of Helix 3) and prevent GPR120S receptor activation. The results led to generation of a W277 and N313 focused pharmacophore hypothesis and the screening of the ZINC15 database using ZINCPharmer through the structure-based pharmacophore. 100 ns all-atomic molecular dynamics (MD) simulations were performed on 9 small molecules identified and Cpd 9, (2-hydroxy-N-{4-[(6-hydroxy-2-methylpyrimidin-4-yl) amino] phenyl} benzamide) was predicted to be a small-molecule GPR120S antagonist. The conformational results from the collective all-atomic MD analysis provided structural information for further identification and optimisation of novel druggable inhibitors of GPR120S using this rational design approach, which could have future potential for anti-cancer drug development studies

Estimation of daily soil CO2 emission using instantaneous measurements

Non-Peer Reviewe

B835: Landfills and Municpal Solid Waste in Maine

Municipal leaders need current information about alternative disposal methods to make rational decisions on handling their town\u27s waste. To provide an overview of landfilling and other waste-handling methods used in the upper New England states, a group of university researchers from New Hampshire, Maine, and Vermont initiated a study of landfills and solid waste management practices. The study involved a comprehensive mail survey of municipalities in Maine, New Hampshire, and Vermont. This report focuses upon and discusses the results of the landfill and solid waste management survey for Maine.https://digitalcommons.library.umaine.edu/aes_bulletin/1019/thumbnail.jp

U-251MG Spheroid Generation Using Hanging Drop Method Protocol

The use of 3D cell culture has been a major step in developing cellular models that can mimic physiological tissues. Traditional 2D cell cultures are often unable to accurately represent the cellular functions and responses that are present in tissues, as a result, research findings based on 2D cultures tend to be skewed with limited predictive capability. 3D cell cultures can be grown from cells obtained from cancer organoids in patients. These models are useful for understanding disease mechanisms and exploring drug therapeutics in areas such as toxicity and efficacy. In order to gather more physiologically relevant data, a variety of 3D cell culture techniques have been developed to mimic the in vivo characteristics of physiological tissues. This protocol describes in vitro generation of U-251MG spheroids using the hanging drop method. Advantages of using hanging drop plate method are, able to produce uniform size spheroids, low cost, comfortable to handling and suitable for short term culture. The main downside of this method is medium change, different drug treatment at different time points are impossible and labor intensive. This method uses the Perfecta3D hanging drop plate, a novel cell culture device that simplifies the process of spheroid formation, testing and analysis. Rather than having to invert the plates which often results in spillage or detachment, these plates are designed to create hanging drops using a plateau structure at the bottom of the plate

U-251MG Spheroid generation using a scaffold based method protocol

3D cell culture is a technique that is used to grow cells in vitro that will mimic an in vivo environment. 3D cell models are a helpful learning tool for researchers to better understand disease mechanisms and to explore different therapeutic properties of drugs. 3D cell cultures can be developed using patient derived cancer cells. Once they have been grown, these 3D cells can be used to screen for small molecule drugs or for genetic modification in for analysis of disease pathways or to predict drug treatments toxicity or efficacy. 3D cell cultures are a big step towards the more ethical testing of drug toxicity and efficacy as they decrease the need to use animals in research as well as providing more reliable results as the cells used are of human physiology. Cellusponge are 3D porous hydroxipropylcellulose scaffolds that are designed for use with cells that do not require specific ligands. As well as the standard non-coated cellusponge, there are two more of the same type of scaffold available for use that are made with two different coatings to allow for improved adaptation of different cell types, these are called Cellusponge-Gal and Cellusponge-Col. Cellusponge is a no-coating approach that is intended for use in the development of general soft tissue 3D culture. It has been used as soft matrix for 3D cell culture and 3D tumour model

U-251MG Spheroid generation using low attachment plate method protocol

3D cell culture is a process used to grow cells in vitro to mimic an in vivo environment. 3D cell models are very useful for understanding disease mechanisms and exploring drug therapeutics. 3D cultures can be grown from cells taken from cancer organoids in patients. Once grown, they can be used to screen for small molecule drugs or they can be genetically modified in order to analyse disease pathways or predict the toxicity or efficacy of a drug treatment. These cultures decrease the need to use animals in research and provides more reliable results as it uses human physiology. This protocol describes the in vitro generation of spheroids using the low attachment plate method. This method uses low-adhesion plates that are coated with hydrophilic polymer to allow cells to cluster together, forming their own extracellular matrix, rather than sticking to the plate surface. The scaffold-free 3D cell culture models produced can more accurately reflect an in vivo microenvironment making them useful in the study of oncology, hepatotoxicity, neurology, nephrology and stem cell biology