Identification of Celecoxib targeted proteins using label-free thermal proteome profiling on rat hippocampus.

Celecoxib 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 Alzheimer9s 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. Significance Statement In this study, we determined forty-four off-target proteins of celecoxib, a non-steroidal anti-inflammatory, and one of the most common medicines for treating inflammatory diseases. We showed that these proteins play a role in the signaling pathways related to neurodegenerative disease and cancer pathways. Finally, we provided molecular supporting evidence for using celecoxib towards the drug repurposing approach by exploring drug targets.