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].