Thermal Proteome Profiling of drug treated c. elegans
Ontology highlight
ABSTRACT: Thermal Proteome Profiling raw mass spectrometer files from control and drug treated C. elegans used for identification of proteins which interacted with the ligand
Project description:Early identification of a compound’s mode of action can greatly benefit the discovery process. Thermal proteomics profiling (TPP) is a powerful, unbiased tool that can be used to identify potential ligands of protein targets. TPP takes advantage of the fact that a ligand binding to its target protein can significantly stabilise that protein, increasing its melting temperature (Tm). We previously optimised this technique for use in drug target deconvolution in the kinetoplastid parasite, Leishmania donovani1 and here report the optimisation and validation of TPP in the malaria-causing parasite P. falciparum.
Project description:The target deconvolution of MMV897615 was evaluated using thermal proteome profiling (TPP), a chemical proteomics approach based on the stabilisation of protein targets upon ligand binding. In these TPP experiments we used a whole-cell strategy, exposing cells rather than lysates to the drug
Project description:Global challenges with anthelmintic failure and resistance development lends impetus to the development of new nematocides (anthelmintics) with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is as an important model organism used for drug discovery and a powerful tool for anthelmintic screening, evaluation and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the ‘Pandemic Response Box’ (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans. Here, we explored this nematocidal pharmacophore on C. elegans, and then tested a series of 46 analogues for human hepatoma (HepG2) toxicity, revealing five compounds whose potency was similar or greater than that of ABX464. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico docking algorithm to identify prime ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing of ABX464 and associated analogues against a panel of parasitic nematodes, towards the development of a new anthelmintic with a novel mechanism of action.
Project description:To realistically evaluate the effects of the environment in space, it is necessary to understand the effects of external factors during sample transport from Earth to space. The present study focused on temperature, profiling the altered gene expression that develops under low cultivation temperatures in C. elegans, used as a space life science model. The 7903 genes were selected as differentially expressed genes, and divided into five sets with similar expression patterns using k-means clustering. Results from Gene Ontology analysis are significantly indicated that the cell cycle related genes, and the TGFβ/insulin-like signal pathway related genes changed. The TGFβ/insulin-like signal pathway is expected to be activated due to low temperatures, as well as by other stressors. To determine the genes whose expression changed four thermal conditions (10, 15, 20, and 25 °C), DNA microarray analysis was performed. The data consisted of 12 samples, consisting of three biological replicates at each temperature.
Project description:In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to characterize compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified 1.498 million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.
Project description:Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the complexity of pathogenesis and molecular mechanisms and pathways perturbed by such compounds requires target deconvolution. Thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), recently emerged as a method to identify small molecule–target interactions in living cells and cell extracts in a variety of organisms, including unicellular eukaryotic pathogens. Ligand binding induces a thermal stability shift—stabilizing or destabilizing proteins that change conformationally in response to the ligand—that can be measured by mass spectrometry (MS). Cells are incubated with different concentrations of ligand and heated, causing thermal denaturation of proteins. The soluble protein is extracted and quantified with multiplexed, quantitative MS, giving rise to thousands of thermal denaturation profiles. Proteins engaging the ligand can be identified by their concentration-dependent thermal shift. The protocol provided here can be used to identify compound-target interactions and assess the impact of environmental or genetic perturbation on the thermal stability of the proteom in T. gondii and other eukaryotic pathogens. Here, we generate a reference dataset of the thermal profiles of extracellular T. gondii tachyzoites.
Project description:In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to characterize compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified 1.498 million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.