Figure 1. Schematic representation of TPP-based drug target discovery using samples of rat hippocampus. Then, we extract proteins from the hippocampus following tissue homogenization and cell lysis. Next, the samples were treated with a range of compound concentrations. Each concentration was treated with four serial temperatures, i.e., 37C, 47C, 57C, 67C. Then, soluble proteins were separated and tryptic digested before Mass spectrometry. Protein identification was made using nano LC-ESI-Thermo Q Exactive Plus Orbi-Trap MS followed by Proteome Discoverer software. Finally, data processing and computational analysis were performed to compare with previously identified celecoxib targets in different databases, and to explore the possible enriched biological annotations in the identified protein targets.

Result

The amount of soluble proteins significantly decreased with increasing temperature (Supplementary Figure 1). The declining pattern was observed for all the five drug concentrations; 20µM, 10 µM, 5 µM, 1 µM, 0.1 µM, and two negative controls, i.e., water and DMSO. Finally, the protein sample treated in 20 µM drug concentration and 67℃ was chosen for further analysis. In fact, proteins start unfolding at high temperature unless the binding energy of any binding partner such as a drug is high enough [48, 49]. We used the highest temperature to avoid detecting the weak and transient interactions among Celecoxib and the proteins. Also, we selected the highest dosage of Celecoxib to detect all potent drug-target interactions.