Celecoxib or Celebrex, an NSAID (non-steroidal anti-inflammatory drug), is one of the most common medicines for treating inflammatory diseases. Recently, it has been shown that celecoxib is associated with implications in complex diseases such as Alzheimer's disease and cancer, as well as with cardiovascular risk assessment and toxicity, suggesting that celecoxib may affect multiple unknown targets. In this project, we detected targets of celecoxib within the nervous system using a label-free TPP (Thermal Proteome Profiling) method. First, proteins of the rat hippocampus were treated with multiple drug concentrations and temperatures. Next, we separated the soluble proteins from the denatured and sedimented total protein load by ultracentrifugation. Subsequently, the soluble proteins were analyzed by nano-liquid chromatography-mass spectrometry to determine the identity of the celecoxib targeted proteins based on structural changes by thermal stability variation of targeted proteins towards higher solubility in the higher temperatures. In the analysis of the soluble protein extract at 67 centigrade, 44 proteins were uniquely detected in drug-treated samples out of all 478 identified proteins at this temperature. Rab4a, one out of these 44 proteins, has previously been reported as one of the celecoxib off-targets in the rat CNS. Furthermore, we provide more molecular details through biomedical enrichment analysis to explore the potential role of all detected proteins in the biological systems. We show that the determined proteins play a role in the signaling pathways related to neurodegenerative disease-and cancer pathways. Finally, we fill out molecular supporting evidence for using celecoxib towards the drug repurposing approach by exploring drug targets.
Celecoxib, or Celebrex, a nonsteroidal anti-inflammatory drug, is one of the most common medicines for treating inflammatory diseases. Recently, it has been shown that celecoxib is associated with implications in complex diseases, such as Alzheimer disease and cancer as well as with cardiovascular risk assessment and toxicity, suggesting that celecoxib may affect multiple unknown targets. In this project, we detected targets of celecoxib within the nervous system using a label-free thermal proteome profiling method. First, proteins of the rat hippocampus were treated with multiple drug concentrations and temperatures. Next, we separated the soluble proteins from the denatured and sedimented total protein load by ultracentrifugation. Subsequently, the soluble proteins were analyzed by nano-liquid chromatography tandem mass spectrometry to determine the identity of the celecoxib-targeted proteins based on structural changes by thermal stability variation of targeted proteins toward higher solubility in the higher temperatures. In the analysis of the soluble protein extract at 67°C, 44 proteins were uniquely detected in drug-treated samples out of all 478 identified proteins at this temperature. Ras-associated binding protein 4a, 1 out of these 44 proteins, has previously been reported as one of the celecoxib off targets in the rat central nervous system. Furthermore, we provide more molecular details through biomedical enrichment analysis to explore the potential role of all detected proteins in the biologic systems. We show that the determined proteins play a role in the signaling pathways related to neurodegenerative disease-and cancer pathways. Finally, we fill out molecular supporting evidence for using celecoxib toward the drug-repurposing approach by exploring drug targets. SIGNIFICANCE STATEMENTThis study determined 44 off-target proteins of celecoxib, a nonsteroidal anti-inflammatory and one of the most common medicines for treating inflammatory diseases. It shows that these proteins play a role in the signaling pathways related to neurodegenerative disease and cancer pathways. Finally, the study provides molecular supporting evidence for using celecoxib toward the drug-repurposing approach by exploring drug targets.
: The search for disease-related targets and studying drug-protein and protein-protein interactions are central issues that would accelerate the clinical approval of a drug. Also, by developing an accurate method in this regard, time and resource consumption will significantly decrease. The low efficiency of some drugs in humans is a grave issue leading to a low rate of FDA approval after spending billions of dollars and decades of research. Several strategies and methods have been expanded to fill this gap, such as drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), and finally, thermal proteome profiling (TPP). The TPP is based on the combination of CETSA and quantitative mass spectrometry. Among recently introduced proteomics technologies, TPP demonstrates the ability to offer detailed proteomic profiles for the large-scale analysis of protein-ligand interactions, including endogenous ligands and proteins like cofactors and metabolites. TPP facilitates the identification of the markers governing drug efficacy and toxicity and provides an unbiased measure for estimating the rate of drug-target engagement. At a glance at TPP steps, after protein extraction, the molecule is exposed to different temperatures and drug concentrations. After discarding solubilized and stabilized proteins, the protein’s identity is investigated by mass spectrometry analysis. As a result of the protein’s structural stabilization after binding to its substrate, TTP helps to accurately identify target proteins with high throughput. In this study, we aimed to introduce the basics of this method and review most recent studies on this technique.
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