Project description:The membrane proteins are essential targets to understand cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system- level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy was aiming to simultaneous identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants most frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering the proteome solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed globular, and membrane proteome. In the presence of surfactant micelles, the changes in protein solubility analyzed after the thermal shift assay are affected by the thermal dependent modification of the micelles size, and their interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to explore thermal-independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanism of actions from pharma, biomedicine, analytical chemistry, or toxicology and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.
Project description:Chemical biology and the application of small molecules has proven to be a potent perturbation strategy especially for the functional elucidation of proteins, their networks and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins as well as off-targets in living cells. Here, we asked the question if isothermal dose-response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein-protein interactions (PPI). Applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose-responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose-response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named "small molecule arranged thermal proximity coaggregation" (smarTPCA).
Project description:Palbociclib (Ibrance, Pfizer) is a recent drug approved by the FDA for phase 3 clinical trials in treating estrogen-receptor-positive and HER2-negative breast cancer. In vitro, palbociclib, a selective inhibitor of CDK4 and CDK6, was shown to reduce cellular proliferation of breast cancer cell lines by blocking progression of cells from G1 into S phase of the cell cycle. While the primary targets of palbociclib have been deciphered, the molecular mechanisms leading to drug resistance as well as off-targets effects of palbociclib are not known. To identify new palbociclib targets we propose a quantitative mass spectrometry and a Cellular Thermal Shift Assay (CETSA) that works under the assumption that protein-drug interaction stabilises the protein thus, leading to an increase in thermostability
Project description:This project describe a novel lysine reactivity profiling method for proteome-wide identification of drug targets and binding sites
Project description:Exploring the therapeutic effects of bioactive compounds has been traditionally approaching by phenotypic screenings followed by functional validation by in vitro assays. The thermal proteome profiling (TPP) has been successfully applied to study drug targets and off-target. We applied a modified protocol based on TPP to elucidate the mechanism of actions (MOA)s of novel bioactives compounds from marine biodiscovery. We have modified the method to gain the specificity for its application to compounds with limited chemical or structural characterization. Method implementation includes increasing the centrifugation force to precipitate the microsomal fraction previous to the thermal shift assay. The range of temperatures has been reduced to optimize the analysis without compromising resolution. Finally, the mass spectrometry analysis was based on label-free quantitative proteomics. Comparison of the targets among methodologies confirmed that the precipitation of the microsomal membranes before TPP is an essential step to discriminate between true targets from proteins precipitates after subcellular fractionation by centrifugation force. As a probe of concept, a novel marine bioactive compound has been analyzed on the hepatic cell line HepG2. We found that 2 targets proteins, aldehyde dehydrogenase and isocitrate dehydoregenase, can be related with beneficial properties towards obesity and obesity-related comorbidities. Identification of targets is key to decipher the MOAs of bioactive compound, predict the mode of action as well as possible harmful effects.
Project description:Determining protein targets directly bound by drug molecules remains
challenging. Here we present the isothermal shift assay, iTSA, for rapid unbiased
identification of drug targets. Compared with thermal proteome profiling, the
prevailing method for target engagement, iTSA offers a simplified workflow, 4-
fold higher throughput, and a multiplexed experimental design with higher
replication. We demonstrate its application to determination of target engagement
for several kinase inhibitors in lysates and living cells.
Project description:We introduce tissue Thermal Proteome Profiling (tissue-TPP) which enables the discovery of drug targets and off-targets as well as measurements of target occupancy in tissue samples derived from dosed animals. Here we report the first proteome-wide thermal stability map of tissues and organ-specific target profiles of the non-covalent histone deacetylase inhibitor, panobinostat. In addition, we devised blood-CETSA/TPP to monitor target and off-target engagement directly in whole blood.
Project description:We introduce tissue Thermal Proteome Profiling (tissue-TPP) which enables the discovery of drug targets and off-targets as well as measurements of target occupancy in tissue samples derived from dosed animals. Here we report the first proteome-wide thermal stability map of tissues and organ-specific target profiles of the non-covalent histone deacetylase inhibitor, panobinostat. In addition, we devised blood-CETSA/TPP to monitor target and off-target engagement directly in whole blood.
Project description:We introduce tissue Thermal Proteome Profiling (tissue-TPP) which enables the discovery of drug targets and off-targets as well as measurements of target occupancy in tissue samples derived from dosed animals. Here we report the first proteome-wide thermal stability map of tissues and organ-specific target profiles of the non-covalent histone deacetylase inhibitor, panobinostat. In addition, we devised blood-CETSA/TPP to monitor target and off-target engagement directly in whole blood.
Project description:We introduce tissue Thermal Proteome Profiling (tissue-TPP) which enables the discovery of drug targets and off-targets as well as measurements of target occupancy in tissue samples derived from dosed animals. Here we report the first proteome-wide thermal stability map of tissues and organ-specific target profiles of the non-covalent histone deacetylase inhibitor, panobinostat. In addition, we devised blood-CETSA/TPP to monitor target and off-target engagement directly in whole blood.