Elham Gholizadeh1, Reza Karbalaei2, Ali Khaleghian1, Mona Salimi3, Kambiz Gilany4, Rabah Soliymani5, Ziaurrehman Tanoli6, Hassan Rezadoost7, Marc Baumann5, Mohieddin Jafari6*, Jing Tang6*
1Department of Biochemistry, Faculty of Medicine, Semnan University of Medical Science, Semnan, Iran. Gholizadeh.bio@gmail.com, khaleghian.ali@gmail.com
2Department of Psychology, College of Science and Technology, Temple University. reza.karbalaei@temple.edu
3Physiology and Pharmacology Department, Pasteur Institute of Iran, P.O. Box 13164, Tehran, Iran. salimi_mona@yahoo.com
4Avicenna Research Institute, Shahid Beheshti University, Darakeh, Tehran, Iran. k.gilany@ari.ir
5Medicum, Biochemistry/Developmental Biology and HiLIFE, Meilahti Clinical Proteomics Core Facility, University of Helsinki, Helsinki, Finland; rabah.soliymani@helsinki.fi , marc.baumann@helsinki.fi
6 Research Program in Systems Oncology, Faculty of Medicine, University of Helsinki, 00270 Helsinki, Finland; mohieddin.jafari@helsinki.fi, jing.tang@helsinki.fi
7Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran. rezadoosthassan@gmail.com
* Correspondence:
Mohieddin Jafari,
mohieddin.jafari@helsinki.fi
Jing Tang,
jing.tang@helsinki.fi

Data Availability Statement

Data available on request from the authors

Abstract

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.
Keywords : Celecoxib, thermal proteome profiling, rat hippocampus, proteomics, signaling network

Introduction

Celecoxib is a non-steroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic and antipyretic properties. Celecoxib prevents the synthesis of lipid compounds called prostaglandins, by selectively inhibiting cyclooxygenases-2 (COX-2) [1, 2]. COX has an essential role in the synthesis of prostaglandins (PGs) derived from arachidonic acid [3]. There are two isoforms of COX: COX-1, and COX-2. COX-1, as a gastric cytoprotectant, is physiologically constitutive and responsible for renal and platelet homeostasis. COX-2, which is considered to be inductive, is arising only in situations of tissue trauma and infections [4, 5]. All types of classic NSAIDs can inhibit both COX-1 and COX-2 isoforms with a predominant effect on COX-1 [6]. Most NSAIDs have broad side effects such as bleeding, ulceration, and perforation on gastrointestinal tract, while celecoxib selectively inhibits COX-2, and does not have side effects on the digestive system [7, 8]. Since celecoxib suppresses pain and inflammation, it is one of the most commonly prescribed drugs and accounts for 5-10% of prescriptions per year [9-11]. Celecoxib can easily access the central nervous system (CNS), while the mechanism of action (MoA) through its protein targets in CNS has not yet been fully elucidated [12].
Determining the affinity of a drug for all its potential targets is the main challenge for understanding the MoA in pharmaceutical sciences. Target-based drug discovery (TDD) starts by identifying molecular targets, which are supposed to have an essential role in the disease of interest [13-15], opposed to phenotypic-based drug discovery (PDD). The mechanism of drug performance, that is essential for designing a drug, is not often considered in PDD investigations [16]. However, also TDD research has its limitations, i.e. proving the presence of a protein target in a particular biological pathway, or its involvement in disease, is a time- and cost-consuming process. Therefore, the development of alternative strategies for target deconvolution is on-demand. Some successful options are Drug Affinity Responsive Target Stability (DARTS) [17], Stability of Proteins from Rates of OXidation (SPROX) [18], CEllular Thermal Shift Assay (CETSA) [19] and Thermal Proteome Profiling (TPP) [20]. TPP, a recently suggested method, can be done in high-throughput to identify drug targets [21]. It can also be applied in living cells in addition toin vitro studies without requiring compound labeling. It is an approach combining CETSA and quantitative mass spectrometry, enabling monitoring of changes in protein thermal stability across heat scaling up. Identifying drug targets in TPP is based on changes in the thermal stability of proteins after their binding to the substrates, i.e. drugs [22, 23]. This stability is mostly related to the protein melting temperature (TM), a temperature in which the process of unfolding will happen [24].
Thermal stress usually causes some irreversible changes in the structure of a protein leading to unfolding. This process leads to the exposure of the hidden hydrophobic core of a protein, and finally, to its aggregation [25, 26]. For proteins connected to a ligand (e.g. a drug), more energy is needed for unfolding because the dissociation of a ligand from the protein requires some energy itself [22]. In other words, binding of a ligand to a protein causes the formation of a complex with increased stability compared to the free protein. Therefore, these proteins are more resistant to the process of unfolding induced by heat, a fact that is the basis of TPP [20, 27-29]. TPP can be used to investigate any change in the structure of the protein [27]. TPP is unique in having the following advantages: While not requiring any labeling, it can be applied to living cells, and it permits an objective search of drug targets [30].
In the present study we have investigated targets of celecoxib, a high prevalence drug, using a label-free TPP method in rat hippocampus. We also provide supporting computational evidence related to biological annotations of the targets to explain the potential repurposing implications of this NSAID [31, 32]. We further show that several proteins related to cancer and inflammation pathways are the targets of Celecoxib. The results of these experiments are also compared with the available knowledge across all drug-target interaction databases. In addition to reinforcing previous findings, we especially explore more potential off-targets of Celecoxib within the nervous system. Based on these results we suggest a conceivable repurposing strategy of this drug for neuronal inflammation as well as cancer.

Materials and Methods

Preparation rat brain for protein extraction

Five rats were used as biological replicates, considering not affecting the present study by two crucial variables (i.e., gender and weight). Therefore, five male rats of Rattus norvegicus were prepared by the weight of 200+_10 gr. After dissecting the hippocampus under complete anesthesia, tissue was washed two times with cold PBS. Experiments were approved by the local Animal Ethics Committee (National Institute for Medical Research Development Ethics Board, NIMAD, No.964580). Immediately after washing, the hippocampus was homogenized and lysed in RIPA buffer. Then, the homogenates were centrifuged at 20,000 g for 20 min at 4°C in order to separate the protein extracts from precipitates [33]. Bradford assay was used to measure protein concentrations.

Drug treatment and heating procedure

A solution of celecoxib in dimethyl sulfoxide (DMSO) was added to the protein extracts to have a 0.1% final DMSO concentration. In this study, five concentrations of celecoxib including 20 µM, 10 µM, 5 µM, 1 µM and 0.1µM, were used, based on the pharmaceutical implications as described previously [34-37]. Two negative controls, i.e., control with DMSO and control with pure DD water were also used. The starting protein amounts in each tube were 1600 µg in total of 400 µl solution. The extracts were incubated for 10 min at 23°C, and then divided into four aliquots of 100 ml.
These 4 aliquots were heated at the following temperatures: 37°C, 47 °C, 57 °C, and 67 °C for 3 min. This was followed by cooling down at room temperature for 3 minutes. Subsequently, the extracts were centrifuged at 60,000 g for 30 min at 4°C and finally, the supernatant which contained soluble targeted proteins was collected and stored at -20°C for further investigations as previously described [20, 38].

Sample preparation, proteolytic digestion, and nano LC-ESI-MS/MS

Next, the extracted proteins treated with the highest drug concentration, i.e., 20 µM at the highest temperature, i.e., 67°C was selected for the protein identification step. The highest dosage of Celecoxib and the highest temperature was used not to detect the weak or transient interactions of Celecoxib and the proteins. The same temperature was used to analyze and identify proteins in the control negative samples.
The protein samples were digested in Amicon Ultra-0.5 centrifugal filters using a modified FASP method [39, 40]. In brief, reduction and alkylation of samples were performed by the addition of tris (2-carboxyethyl) phosphine (TCEP) and iodoacetamide to a final concentration of 2 mM and 50 mM respectively and incubation in the dark for 30 min. The trypsin solution was added in a ratio of 1:50 w/w in 50 mM ammonium bicarbonate and incubate overnight at room temperature. The peptide samples were cleaned using C18-reverse-phase ZipTipTM (Millipore). Dried peptide digest was re-suspended in 1% TFA, and sonicated in a water bath for 1 min before injection. Fractionated protein digests were analyzed in nano-LC-Thermo Q Exactive Plus Orbi-Trap MS. Each sample run was followed by two empty runs to wash out any remaining peptides from previous runs. The peptides were separated by Easy-nLC system (Thermo Scientific) equipped with a reverse-phase trapping column Acclaim PepMapTM 100 (C18, 75 μm × 20 mm, 3 μm particles, 100 Å; Thermo Scientific), followed by an analytical Acclaim PepMapTM 100 RSLC reversed-phase column (C18, 75 µm × 250 mm, 2 µm particles, 100 Å; Thermo Scientific). The injected sample analytes were trapped at a flow rate of 2 µl min-1 in 100% of solution A (0.1 % formic acid). After trapping, the peptides were separated with a linear gradient of 120 min comprising 96 min from 3% to 30% of solution B (0.1% formic acid/80% acetonitrile), 7 min from 30% to 40% of solution B, and 4 min from 40% to 95% of solution B.
LCMS data acquisition was done with the mass spectrometer settings as follows: The resolution was set to 140,000 for MS scans, and 17,500 for the MS/MS scans. Full MS was acquired from 350 to 1400 m/z, and the 10 most abundant precursor ions were selected for fragmentation with 30 s dynamic exclusion time. Ions with 2+, 3+, and 4+ charge were selected for MS/MS analysis. Secondary ions were isolated with a window of 1.2 m/z. The MS AGC target was set to 3 x 106 counts, whereas the MS/MS AGC target was set to 1 x 105. Dynamic exclusion was set with a duration of 20 s. The NCE collision energy stepped was set to 28 kJ mol–1.

Proteomic data and bioinformatic analysis

Following LC-MS/MS acquisition, the raw files were qualitatively analyzed by Proteome Discoverer (PD), version 2.4.0.305 (Thermo Scientific, USA). The identification of proteins by PD was performed against the UniProt Rat protein database (release 11-2019 with 8086 entries) using the built-in SEQUEST HT engine. The following parameters were used: 10 ppm and 0.25 Da were tolerance values set for MS and MS/MS, respectively. Trypsin was used as the digesting enzyme, and two missed cleavages were allowed. The carbamidomethylation of cysteines was set as a fixed modification, while the oxidation of methionine and deamidation of asparagine and glutamine were set as variable modifications. The false discovery rate was set to less than 0.01 and a minimum length of six amino acids (one peptide per protein) was required for each peptide hit.
Following the identification of proteins, for better understanding of the role and importance of proteins, enrichment analysis was used to determine the corresponding biological processes by EnrichR [41]. Eight different libraries were selected to explore biomedical annotations of drug targets, including gene ontology (GO), molecular function (MF), GO Cellular Component (CC), GO Biological Process (BP), DisGeNet [42], HumanPhen [43], Mouse Genome Informatics (MGI) [44], PheWeb [45] and WikiPathways [46]. We used Enrichr’s combined scores and adjusted p-values to sort annotations descendingly. Also, PEIMAN software was used to determine possible enriched post-translational modifications (PTM) in the list of protein targets [47].