Takinib Inhibits Inflammation in Human Rheumatoid Arthritis Symptoms Synovial Fibroblasts by Individuals Janus Kinase-Signal
Transducer and Activator of Transcription 3
(JAK/STAT3) Path
Paul M. Panipinto 1,† , Anil K. Singh 1,† , Farheen S. Shaikh 1
, Ruby J. Siegel 1
, Mukesh Chourasia 2
and Salahuddin Ahmed 1,3,*
Citation: Panipinto, P.M. Singh,
A.K. Shaikh, F.S. Siegel, R.J.
Chourasia, M. Ahmed, S. Takinib
Inhibits Inflammation in Human
Rheumatoid Arthritis Symptoms Synovial
Fibroblasts by Individuals Janus
Kinase-Signal Transducer and
Activator of Transcription 3
(JAK/STAT3) Path. Int. J. Mol. Sci.
2021, 22, 12580. https://doi.org/
10.3390/ijms222212580
Academic Editors: Sander W. Tas and
Jan Piet van Hamburg
Received: 30 September 2021
Recognized: 19 November 2021
Printed: 22 November 2021
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4./).
1 Department of Pharmaceutical Sciences, Washington Condition College College of Pharmacy and
Pharmaceutical Sciences, Spokane, WA 99202, USA [email protected] (P.M.P.)
[email protected] (A.K.S.) [email protected] (F.S.S.) [email protected] (R.J.S.)
2 Center for Computational Biology and Bioinformatics, Amity Institute of Biotechnology, Amity College
Uttar Pradesh, Noida 201301, India [email protected]
3 Division of Rheumatology, College of Washington Med school, San antonio, WA 98109, USA
* Correspondence: [email protected] Tel.: 1-509-368-6566
† Equal contribution.
Abstract: TGF ß-activated kinase 1 (TAK1) is a vital participant in inflammatory pathogenesis
for illnesses for example rheumatoid arthritis symptoms (RA) and gouty joint disease. The central position it occupies
between your mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NF-?B) pathways
causes it to be a beautiful therapeutic target. Because this field is promoting recently, several novel
inhibitors happen to be presented as getting specific activity that cuts down on the TAK1 function either
covalently as with the situation of 5Z-7-oxozeanol (5Z7O) or reversibly (NG-25). However, the mechanism
by which takinib elicits its anti-inflammatory activity remains elusive. Although this inhibitor
shows great promise, an intensive analysis of their inhibitor function and it is potential off-target effects is
necessary before addressing its clinical potential or its use within inflammatory conditions. An analysis
through Western blot demonstrated an unpredicted rise in IL-1ß-caused TAK1 phosphorylation-a pre requisite for and indicator of their functional potential-by takinib while concurrently demonstrating
the inhibition from the JAK/STAT path in human rheumatoid arthritis symptoms synovial fibroblasts (RASFs)
in vitro. In THP-1 monocyte-derived macrophages, takinib again brought towards the lipopolysaccharide caused phosphorylation of TAK1 with no marked inhibition from the TAK1 downstream effectors,
namely, of c-Jun N-terminal kinase (JNK), phospho-c-Jun, NF-?B phospho-p65 or phospho-I?Ba.
Taken together, these bits of information indicate that takinib inhibits inflammation during these cells by targeting
multiple signaling pathways, most particularly the JAK/STAT path in human RASFs.
Keywords: takinib TAK1 rheumatoid arthritis symptoms RASFs THP-1 MAPK NF-kB JAK/STAT
1. Introduction
Rheumatoid arthritis symptoms is definitely an autoimmune disease with complex etiology leading
to progressive discomfort, inflammation, and eventual joint failure [1]. Major contributions
to RA pathogenesis are created by activated synovial fibroblasts (RASFs), which produce
inflammatory mediators and recruit inflammatory immune cells whilst destroying
cartilage via invasion and matrix metalloproteinase (MMP) production [2,3]. Invasive
RASFs further propagate RA pathogenesis via producing interleukin-6 (IL-6), which
facilitates bone resorption and prevents bone production [4-6]. Our knowledge of
the etiology and pathology of the inflammatory condition is promoting dramatically,
however, effective and safe therapy remains elusive.
Int. J. Mol. Sci. 2021, 22, 12580. https://doi.org/10.3390/ijms222212580 https://world wide web.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2021, 22, 12580 2 of 13
While traditional therapeutics for RA include disease modifying anti-rheumatic
drugs (DMARDs), the development of cytokine-targeted biologics as treatments introduced
the opportunity of novel, more effective therapy. The prosperity of tocilizumab (an anti IL-6R antibody) and tofacitinib being an inhibitor of JAK/STAT brought towards the effective use
of JAK/STAT targeted therapy in clinics for RA [7,8]. Its clinical usage was short-resided,
however, as publish-market surveillance revealed elevated vulnerability to opportunistic
infections, cardiovascular and thromboembolic occasions [9-11]. The ongoing look for a
therapeutic target by having an acceptable safety profile has now use novel targets. TAK1 has
become a leading therapeutic target and it is proximal towards the IL-1ß, Tumor Necrosis
Factor-alpha (TNFa) and Toll-like (TLR) receptors and also the upstream of both cytokine producing nuclear factor kappa-B (NF-?B) and mitogen activated protein kinase (MAPK)
pathways [11-15]. The expectation would be that the inhibition of both NF-?B and MAPK will
result in reduced inflammation via inhibited cytokine production. Mutational research has
uncovered substantial mechanistic insights into TAK1’s function, such as the importance
of phosphorylation of Thr184/187 within the kinase activation loop [16-18].
Several TAK1 inhibitors have reached preclinical use, such as the covalent type-1
inhibitor 5Z-7-oxozeanol (5Z7O) and also the DFG-out conformation reversable type-2 inhibitor
NG-25, that mechanisms of action are very well established [19,20]. Another inhibitor,
takinib, continues to be proven to lessen proinflammatory mediators and it is suggested to do something like a
specific inhibitor of TAK1 [21,22] although its mechanism have yet to be completely
resolved. To higher comprehend the signaling mechanisms by which takinib exhibits its
anti-inflammatory activity, we tested the result of takinib on IL-1ß-activated human RASFs
and also on IL-6-activated RASFs in vitro. Furthermore, we used lipopolysaccharide
(LPS)-activated THP-1 monocyte-derived macrophages to look for the aftereffect of takinib on
TLR4-caused inflammatory signaling. Within this study, we aimed to decipher the actual
mechanism of action of takinib and identified the JAK/STAT path like a potential target
of takinib in IL-1ß-activated human RASFs in vitro. These bits of information claim that takinib
functions inside a non-specific manner that varies by cell and stimulation.
2. Results
2.1. Inhibition of TAK1 Reduces Proinflammatory Mediators Secreted by RASFs and THP-1
Monocyte-Derived Macrophages
We started this research having a comparison of known TAK1 inhibitors takinib, 5Z7O,
and NG-25 to research their relative inhibition of professional-inflammatory cytokines and
chemokines. Human RASFs were serum-starved overnight, pre-treated for 2 hrs
with TAK1 inhibitors adopted with a 24 h activation period with recombinant human IL-
1ß (10 ng/mL). The conditioned media was examined for secreted proteins by ELISA.
Takinib (.1-20 µM) displayed significant dose-dependent decreases in IL-1ß-activated
ENA-78/CXCL5, IL-6, and MCP-1/CCL2 production (Figure 1a). IL-8 shown a
downward trend and arrived at record significance in a 10 µM takinib dosing inside a one-way
ANOVA comparison towards the IL-1ß stimulated samples (Figure 1a). Compared to takinib,
1 µM 5Z7O displayed statistically significant reductions when compared with both IL-1ß stimulated
samples. Treatment with 1 µM NG-25 considerably decreased all pro-inflammatory
mediators in every case. In THP-1 monocyte-derived macrophages (Figure 1b), 1 µM Takinib
reduced IL-1ß production by typically 26% over the replicates when compared with LPS
treated (10 µg/mL) samples, while 1 µM of 5Z7O or NG-25 reduced LPS-caused IL-1ß
supernatant concentrations by 63% and 36%, correspondingly (Figure 1b). Takinib demon strated a powerful inhibition of TNFa at even .1 µM concentrations (-54%), though 5Z7O
and NG-25 treatments reduced LPS-caused TNFa production with a considerably greater
degree (-86% and -75%, correspondingly). These results show takinib truly does
have significant anti-inflammatory ability, though inhibition is not as effective as for that
known inhibitors of TAK1. Furthermore, our MTT-based cell viability results revealed
takinib and 5Z7O to become cytotoxic at 24 h in the 10 and 1 µM concentrations, correspondingly
(Extra Figure S1) suggesting the decrease in takinib at greater concentrations
might be partly related to losing cell viability.
Int. J. Mol. Sci. 2021, 22, 12580 3 of 13 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 13
takinib truly does have significant anti-inflammatory ability, though inhibition is less
effective compared to the known inhibitors of TAK1. Furthermore, our MTT-based cell viabil ity results revealed takinib and 5Z7O to become cytotoxic at 24 h in the 10 and 1 µM concentra tions, correspondingly (Extra Figure S1) suggesting the decrease in takinib at
greater concentrations might be partly related to losing cell viability.
(a) (b)
Figure 1. Inhibition of TAK1 reduces proinflammatory mediators. (a) RASFs were pre-given selected inhibitors
adopted by 24 h stimulation with IL-1ß (10 ng/mL). Supernatants were collected and examined by ELISA. Takinib demon strates inhibition of CXCL5, IL-6, IL-8, and MCP-1 though less completely as 5Z7O or NG-25. (b) THP-1 monocyte derived macrophages pre-given selected inhibitors adopted by 24 h stimulation with LPS (10 µg/mL). Supernatants
collected and examined by ELISA demonstrate significant decrease in both IL-1ß and TNFa. 5Z7O decreased both cytokines
considerably greater than any management of takinib. Significance bars represent p < 0.05 in one-way ANOVA with Dunnett’s
post hoc test for multiple comparisons to either IL-1ß or LPS.
2.2. Takinib Inhibits STAT3 and JNK Phosphorylation in IL-1ß, But Not IL-6 Stimulated
Human RASFs
Next, we investigated the effect of takinib on the signaling capability of major inflam matory cytokine pathways in human RASFs. Overnight-starved human RASFs were pre treated as described above with a dose range of takinib (0.1-20 µM) followed with 30 min
IL-1ß stimulation. Whole-cell extracts were analyzed by Western Blotting. Surprisingly,
takinib induced dose-dependent phosphorylation of TAK1Thr184/187 in IL-1ß-treated sam ples while also demonstrating a significant inhibition of STAT3Tyr705 and STAT3Ser727 phos Figure 1. Inhibition of TAK1 reduces proinflammatory mediators. (a) RASFs were pre-treated with selected inhibitors
followed by 24 h stimulation with IL-1ß (10 ng/mL). Supernatants were collected and analyzed by ELISA. Takinib
demonstrates inhibition of CXCL5, IL-6, IL-8, and MCP-1 though not as completely as 5Z7O or NG-25. (b) THP-1 monocyte derived macrophages pre-treated with selected inhibitors followed by 24 h stimulation with LPS (10 µg/mL). Supernatants
collected and analyzed by ELISA demonstrate significant reduction in both IL-1ß and TNFa. 5Z7O lowered both cytokines
significantly more than any treatment of takinib. Significance bars represent p < 0.05 in one-way ANOVA with Dunnett’s
post hoc test for multiple comparisons to either IL-1ß or LPS.
2.2. Takinib Inhibits STAT3 and JNK Phosphorylation in IL-1ß, but Not IL-6 Stimulated
Human RASFs
Next, we investigated the effect of takinib on the signaling capability of major in-
flammatory cytokine pathways in human RASFs. Overnight-starved human RASFs were
pre-treated as described above with a dose range of takinib (0.1-20 µM) followed with
30 min IL-1ß stimulation. Whole-cell extracts were analyzed by Western Blotting. Surpris ingly, takinib induced dose-dependent phosphorylation of TAK1Thr184/187 in IL-1ß-treated
samples while also demonstrating a significant inhibition of STAT3Tyr705 and STAT3Ser727
phosphorylation (Figure 2a,b). As expected, JNK phosphorylation decreased with an in creasing takinib concentration and the total STAT3 levels were unaffected. Treatment with
takinib did not lead to a significant inhibition of p38 signaling and was insufficient to pre vent the degradation of IRAK1 and I-?Ba triggered by IL-1ß. We then examined whether
STAT3 inhibition functions via canonical or non-canonical STAT3 activation by stimulating
human RASFs with an IL-6 and IL-6 receptor. In RASFs, IL-6 trans-signaling induced
Int. J. Mol. Sci. 2021, 22, 12580 4 of 13
JAK/STAT3 activation was not inhibited by takinib pretreatment, whereas tofacitinib (a
JAK inhibitor) demonstrated a complete inhibition of JAK/STAT signaling (Figure 2c).
Additionally, takinib was unable to inhibit the JNK signaling pathway as would normally
be expected from upstream TAK1 kinase inhibition.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 13
phorylation (Figure 2a,b). As expected, JNK phosphorylation decreased with an increas ing takinib concentration and the total STAT3 levels were unaffected. Treatment with
takinib did not lead to a significant inhibition of p38 signaling and was insufficient to
prevent the degradation of IRAK1 and I-?Ba triggered by IL-1ß. We then examined
whether STAT3 inhibition functions via canonical or non-canonical STAT3 activation by
stimulating human RASFs with an IL-6 and IL-6 receptor. In RASFs, IL-6 trans-signaling
induced JAK/STAT3 activation was not inhibited by takinib pretreatment, whereas tofa citinib (a JAK inhibitor) demonstrated a complete inhibition of JAK/STAT signaling (Fig ure 2c). Additionally, takinib was unable to inhibit the JNK signaling pathway as would
normally be expected from upstream TAK1 kinase inhibition.
(a)
(b) (c)
Figure 2. Treatment of RASFs with takinib and either IL-1ß or IL-6 trans-signaling demonstrates differential activation of
signaling pathways. (a) RASFs were pre-treated for two hours with takinib followed by 30 min stimulation with IL-1ß (10
ng/mL). Whole cell extracts were collected and analyzed by Western Blot. Takinib induces phosphorylation of TAK1 while
reducing STAT3 phosphorylation at the Tyr705 and Ser727 residues in n = 4 RASF cell lines. (b) Takinib demonstrates
increasing phosphorylation of the Thr184/187 residues in the kinase loop of TAK1. (c) RASFs were pre-treated with takinib
followed by 30 min stimulation with IL-6 and IL-6R (100 ng/mL). Whole cell extracts analyzed by Western Blot show no
effect on STAT3 phosphorylation by Takinib in n = 3 RASF lines. * = p < 0.05, ** = p < 0.001, *** = p < 0.0001, **** = p < 0.00001.
Figure 2. Treatment of RASFs with takinib and either IL-1ß or IL-6 trans-signaling demonstrates differential activation of
signaling pathways. (a) RASFs were pre-treated for two hours with takinib followed by 30 min stimulation with IL-1ß
(10 ng/mL). Whole cell extracts were collected and analyzed by Western Blot. Takinib induces phosphorylation of TAK1
while reducing STAT3 phosphorylation at the Tyr705 and Ser727 residues in n = 4 RASF cell lines. (b) Takinib demonstrates
increasing phosphorylation of the Thr184/187 residues in the kinase loop of TAK1. (c) RASFs were pre-treated with takinib
followed by 30 min stimulation with IL-6 and IL-6R (100 ng/mL). Whole cell extracts analyzed by Western Blot show no
effect on STAT3 phosphorylation by Takinib in n = 3 RASF lines. * = p < 0.05, ** = p < 0.001, *** = p < 0.0001, **** = p < 0.00001.
2.3. Takinib Inhibits STAT3 Nuclear Translocation and DNA-Binding Activity in Human RASFs
To further examine the impact of STAT3 phosphorylation inhibition by takinib, we
isolated the nuclear extract from IL-1ß stimulated human RASFs, with or without takinib
pre-treatment. A Western blot analysis of the purified nuclear extract from RASFs re vealed markedly reduced levels of nuclear pSTAT3Tyr705, but not of pSTAT3Ser727 in IL-
1ß-stimulated samples treated with either 10 µM takinib or 5 µM tofacitinib (Figure 3a).
Int. J. Mol. Sci. 2021, 22, 12580 5 of 13
Surprisingly, takinib (10 µM) was not potent enough to inhibit IL-1ß-induced nuclear
translocation of NF-?Bp65, suggesting that STAT3 and JNK pathways are the primary
target of takinib in IL-1ß-activated signaling in human RASFs (Figure 3a).
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 13
2.3. Takinib Inhibits STAT3 Nuclear Translocation and DNA-Binding Activity in
Human RASFs
To further examine the impact of STAT3 phosphorylation inhibition by takinib, we
isolated the nuclear extract from IL-1ß stimulated human RASFs, with or without takinib
pre-treatment. A Western blot analysis of the purified nuclear extract from RASFs re vealed markedly reduced levels of nuclear pSTAT3Tyr705, but not of pSTAT3Ser727 in IL-1ß-
stimulated samples treated with either 10 µM takinib or 5 µM tofacitinib (Figure 3a). Sur prisingly, takinib (10 µM) was not potent enough to inhibit IL-1ß-induced nuclear trans location of NF-?Bp65, suggesting that STAT3 and JNK pathways are the primary target
of takinib in IL-1ß-activated signaling in human RASFs (Figure 3a).
We hypothesized that this reduction in Tyr705 phosphorylation may interfere with
the ability of STAT3 to act as a transcription factor by reducing its ability to bind to DNA.
To test this hypothesis, we used a purified nuclear extract from human RASFs from the
above-mentioned treatment for determining the DNA-binding activity. Results from the
DNA binding ELISA of nuclear extracts showed that takinib significantly inhibited IL-1ß-
induced STAT3 DNA binding activity, which was comparable to the inhibitory potential
of the JAK inhibitor tofacitinib in IL-1ß treated samples
Figure 3. Management of RASFs with takinib or Tofacitinib and IL-1ß reduces nuclear phospho-STAT3Tyr705 and STAT3’s
DNA-binding ability. (a) RASFs were pre-given takinib or tofacitinib adopted by 30 min stimulation with IL-1ß (10
ng/mL). Purified nuclear extracts from n = 3 cell lines were collected and examined by Western Blot. In n = 3 RASF cell
lines. (b) RASFs were pre-given takinib adopted by 30 min stimulation with IL-1ß (10 ng/mL). Nuclear extracts
examined by ELISA show reduced DNA binding activity in n = 3 RASF cell lines given takinib and tofacitinib. * = p
< 0.05, ** = p < 0.001.
2.4. Takinib Phosphorylates TAK1 and Fails to Inhibit NF-?B or MAPK Signaling in LPS Stimulated THP-1 Macrophages
Next, we examined the effect of takinib treatment on TLR4-induced TAK1 signaling.
Using phorbol 12-myristate 13-acetate (PMA) treated THP-1 monocytes, pre-treated with
Figure 3. Treatment of RASFs with takinib or Tofacitinib and IL-1ß reduces nuclear phospho-STAT3Tyr705 and STAT3’s
DNA-binding ability. (a) RASFs were pre-treated with takinib or tofacitinib followed by 30 min stimulation with IL-1ß
(10 ng/mL). Purified nuclear extracts from n = 3 cell lines were collected and analyzed by Western Blot. In n = 3 RASF cell
lines. (b) RASFs were pre-treated with takinib followed by 30 min stimulation with IL-1ß (10 ng/mL). Nuclear extracts
analyzed by ELISA show reduced DNA binding activity in n = 3 RASF cell lines treated with takinib and tofacitinib.
* = p < 0.05, ** = p < 0.001.
We hypothesized that this reduction in Tyr705 phosphorylation may interfere with
the ability of STAT3 to act as a transcription factor by reducing its ability to bind to DNA.
To test this hypothesis, we used a purified nuclear extract from human RASFs from the
above-mentioned treatment for determining the DNA-binding activity. Results from the
DNA binding ELISA of nuclear extracts showed that takinib significantly inhibited IL-1ß-
induced STAT3 DNA binding activity, which was comparable to the inhibitory potential of
the JAK inhibitor tofacitinib in IL-1ß treated samples (Figure 3b).
2.4. Takinib Phosphorylates TAK1 and Fails to Inhibit NF-?B or MAPK Signaling in
LPS-Stimulated THP-1 Macrophages
Next, we examined the effect of takinib treatment on TLR4-induced TAK1 signaling.
Using phorbol 12-myristate 13-acetate (PMA) treated THP-1 monocytes, pre-treated with
takinib and stimulated for 30 min with 10 µg/mL LPS, we isolated the whole-cell extract
and performed Western Blotting. Pretreatment with takinib further increased LPS-induced
TAK1 phosphorylation at Thr184/187, with a modest inhibitory effect on Ser727 phosphory lation site (Figure 4). However, takinib did not lead to an inhibitory effect on LPS-induced
pNF-?Bp65 and led to a potential increase in pJNKp46/p54 (Figure 4). Additionally, a
densitometric analysis of Western blots showed no significant inhibition of pJNKp46/p54
Int. J. Mol. Sci. 2021, 22, 12580 6 of 13
or its downstream substrate p-c-Jun. Finally, STAT3 signaling in LPS-stimulated cells was
unaffected by treatment with takinib.
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 13
takinib and stimulated for 30 min with 10 µg/mL LPS, we isolated the whole-cell extract
and performed Western Blotting. Pretreatment with takinib further increased LPS-in duced TAK1 phosphorylation at Thr184/187, with a modest inhibitory effect on Ser727 phos phorylation site (Figure 4). However, takinib did not lead to an inhibitory effect on LPS induced pNF-?Bp65 and led to a potential increase in pJNKp46/p54 (Figure 4). Addition ally, a densitometric analysis of Western blots showed no significant inhibition of
pJNKp46/p54 or its downstream substrate p-c-Jun. Finally, STAT3 signaling in LPS-stim ulated cells was unaffected by treatment with takinib.
Figure 4. Takinib phosphorylates TAK1 and fails to inhibit the MAPK/NF-kB pathways in THP-1 macrophages. THP-1
monocytes were treated with 100 ng/mL PMA for 3 h, left to rest in fresh RPMI with 10% FBS overnight, followed by 2 h
treatment with takinib and an additional 30 min LPS (10 µg/mL) stimulation. NF-kB p65, IkBa, JNK, and c-Jun phosphor ylation were all uninhibited, while TAK1 phosphorylation increased under LPS and takinib treatment. * = p < 0.05, ** = p <
0.001.
2.5. Docking Simulations Using Takinib and STAT3 Demonstrate a Potential Site of Interaction
The electrostatic potential surface of STAT3 shows that the ligand binding cavity of
STAT3 is mainly lined by hydrophobic residues (Figure 5a). The binding site of STAT3 is
shallow and wide. The docked takinib aligned well with the orientation of the core of the
co-crystallized ligand (SD-36) in the binding cavity and presented similar active site bind ing partners [22]. Both STAT3 and takinib demonstrated a binding energy of -6.610
Kcal/mol. The H-boding and hydrophobic interactions contribute to the binding. The
E638, Q644 and Y657 residues form the network of the H-bond with the ligand, while
W623, Y640, Y657 and I659 residues presented hydrophobic interaction with the ligands.
Their similarities are further observed in the p-p interaction between W623 and the ben zimidazole ring of takinib (Figure 5b). A list of potential interacting residues modeled
using Schrodinger suit 2020.3 [23] and their impact on STAT3 functions can be found in
Table 1.
Figure 4. Takinib phosphorylates TAK1 and fails to inhibit the MAPK/NF-kB pathways in THP-1 macrophages. THP-1
monocytes were treated with 100 ng/mL PMA for 3 h, left to rest in fresh RPMI with 10% FBS overnight, followed by
2 h treatment with takinib and an additional 30 min LPS (10 µg/mL) stimulation. NF-kB p65, IkBa, JNK, and c-Jun
phosphorylation were all uninhibited, while TAK1 phosphorylation increased under LPS and takinib treatment. * = p < 0.05,
** = p < 0.001.
2.5. Docking Simulations Using Takinib and STAT3 Demonstrate a Potential Site of Interaction
The electrostatic potential surface of STAT3 shows that the ligand binding cavity of
STAT3 is mainly lined by hydrophobic residues (Figure 5a). The binding site of STAT3 is
shallow and wide. The docked takinib aligned well with the orientation of the core of the
co-crystallized ligand (SD-36) in the binding cavity and presented similar active site binding
partners [22]. Both STAT3 and takinib demonstrated a binding energy of -6.610 Kcal/mol.
The H-boding and hydrophobic interactions contribute to the binding. The E638, Q644 and
Y657 residues form the network of the H-bond with the ligand, while W623, Y640, Y657
and I659 residues presented hydrophobic interaction with the ligands. Their similarities
are further observed in the p-p interaction between W623 and the benzimidazole ring of
takinib (Figure 5b). A list of potential interacting residues modeled using Schrodinger suit
2020.3 [23] and their impact on STAT3 functions can be found in Table 1.
Table 1. Predicted takinib/STAT3 Binding Interactions and Known STAT3 Residue Function.
Residue Takinib Interaction Function
E638 H-Bond Amide H-Bond works with Q644 to stabilize ligand [24]
Q644 H-Bond Stabilizes side chain H-bonds [25]
Y640 Hydrophobic Modulates Tyr705 phosphorylation, pocket
hydrophobicity [26]
Y657 H-bond, Hydrophobic Works with E638 & Y640 to modulate pocket
hydrophobicity [22,24,26]
I659 Hydrophobic Ligand stabilization, pocket hydrophobicity [24,26]
W623 Hydrophobic, p-p
Induces ligand conformational fit, stabilizes ligand in
pocket [24,27]
Int. J. Mol. Sci. 2021, 22, 12580 7 of 13 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 7 of 13
(a) (b)
Figure 5. Docked pose of takinib in the binding site of STAT3 (PDB ID: 6NJS): (a) Showing electrostatic potential surface
of the STAT3 with co-crystallized ligand (SD-36) and docked takinib. The red and blue color depict electronegative and
electropositive surfaces while white color denotes hydrophobic surface. (b) The interaction of takinib with the binding site
residues of STAT3. The light grey labelling represents the residues that leads to hydrophobic interaction with the ligand.
The yellow and blue dotted line represent H-bonding and p-p interaction, respectively. The ligand in the active site is
represented by thick stick model while interacting residues are represented in thin stick model.
Table 1. Predicted takinib/STAT3 Binding Interactions and Known STAT3 Residue Function.
Residue Takinib Interaction Function
E638 H-Bond Amide H-Bond works with Q644 to stabilize ligand [24]
Q644 H-Bond Stabilizes side chain H-bonds [25]
Y640 Hydrophobic Modulates Tyr705 phosphorylation, pocket hydropho bicity [26]
Y657 H-bond, Hydrophobic Works with E638 & Y640 to modulate pocket hydropho bicity [22,24,26]
I659 Hydrophobic Ligand stabilization, pocket hydrophobicity [24,26]
W623 Hydrophobic, p-p Induces ligand conformational fit, stabilizes ligand in
pocket [24,27]
3. Discussion
The positioning of TAK1 in the nexus from the MAPK and NF-kB pathways causes it to be an
appealing target for anti-inflammatory therapy. This research shows that although
takinib is an efficient inhibitor of inflammation, its mode of action differs from the
formerly described target, TAK1. Our findings show takinib preferentially
inhibited IL-1ß-caused STAT3 phosphorylation, nuclear translocation, and DNA-bind ing in human RASFs in vitro. In addition, while takinib displayed a modest inhibition
of JNK path in IL-1ß stimulated RASFs, it unsuccessful to duplicate this inhibition of inflam mation brought on by canonical IL-6 trans-signaling in human RASFs, or perhaps in LPS stimulated
THP-1 macrophages. Importantly, takinib caused the phosphorylation of TAK1 at
Thr184/187 residues, that are important kinase domain and ATP-binding sites of TAK1, in
both human RASFs and THP-1 macrophages. Molecular docking studies claim that
takinib interacts with a few of the key residues that other STAT3 inhibitors are recognized to
require. Within our study, we discovered that the participation of Y640, Y675, I659 supplies a hy drophobic pocket by which E638, Q644 and W623 orient and stabilize takinib, in accordance with
STAT3, that is in line with formerly printed reports of other STAT3 inhibitors.
Together, these bits of information prove takinib may target signaling proteins that
don’t crosstalk using the TAK1/MAPK path and may hinder inflammatory mediators
through the STAT3 path.
Figure 5. Docked pose of takinib within the binding site of STAT3 (PDB ID: 6NJS): (a) Showing electrostatic potential surface
from the STAT3 with co-crystallized ligand (SD-36) and docked takinib. The blue and red color illustrate electronegative and
electropositive surfaces while white-colored color denotes hydrophobic surface. (b) The interaction of takinib using the binding site
residues of STAT3. The sunshine gray labelling represents the residues leading to hydrophobic interaction using the ligand.
The yellow and blue dotted line represent H-connecting and p-p interaction, correspondingly. The ligand within the active website is
symbolized by thick stick model while interacting residues are symbolized in thin stick model.
The positioning of TAK1 in the nexus from the MAPK and NF-kB pathways causes it to be an
appealing target for anti-inflammatory therapy. This research shows that although
takinib is an efficient inhibitor of inflammation, its mode of action differs from the
formerly described target, TAK1. Our findings show takinib preferentially
inhibited IL-1ß-caused STAT3 phosphorylation, nuclear translocation, and DNA-binding
in human RASFs in vitro. In addition, while takinib displayed a modest inhibition of JNK
path in IL-1ß stimulated RASFs, it unsuccessful to duplicate this inhibition of inflammation
brought on by canonical IL-6 trans-signaling in human RASFs, or perhaps in LPS stimulated THP-1
macrophages. Importantly, takinib caused the phosphorylation of TAK1 at Thr184/187
residues, that are important kinase domain and ATP-binding sites of TAK1, both in
human RASFs and THP-1 macrophages. Molecular docking studies claim that takinib
interacts with a few of the key residues that other STAT3 inhibitors are recognized to require.
Within our study, we discovered that the participation of Y640, Y675, I659 supplies a hydrophobic
pocket by which E638, Q644 and W623 orient and stabilize takinib, in accordance with STAT3,
that is in line with formerly printed reports of other STAT3 inhibitors. Together,
these bits of information prove takinib may target signaling proteins that don’t
crosstalk using the TAK1/MAPK path and may hinder inflammatory mediators through the
STAT3 path.
The inflammatory milieu and heterogenous nature of RA pathogenesis applies
to complex overlapping cell signaling mechanisms along with a difficulty in targeting a particular
kinase or protein for therapeutic purposes. As the roles of numerous inflammatory mediators
are very well known, questions remain with regards to the inflammatory hierarchy and also the best
method for safe, effective therapy [28]. This complexity makes the aim of one-drug,
one-target therapy more and more difficult and needs pharmacologists within the field to become
aware of the significance of inflammatory cytokines in disease pathogenesis. The
limitations of presently used biologics that concentrate on proinflammatory cytokines, for example
TNFa and IL-6R, have brought to the quest for small molecule inhibitors that can
target common signaling kinases, that are central to mediating inflammatory signals,
with TAK1 proving itself to be one promising therapeutic target for RA [12,15]. Two specific
classes of validated TAK1 inhibitors (a covalent-binding irreversible type I (5Z7O) and
a DGF-out conformation based reversible type II (NG-25)) have given possibilities
Int. J. Mol. Sci. 2021, 22, 12580 8 of 13
for preclinical testing. Takinib was proven to competitively hinder TAK1 ATP-binding in
DFG-in conformation to boost TNFa-caused apoptosis in RASFs [21] and also to improve
bovine collagen-caused joint disease (CIA) in rodents [29,30], however, its impact on TAK1 kinase activity
during these cells has not yet been validated. Importantly, although TAK1 is activated by TNFa [31],
it doesn’t solely depend on connection to TRAF2 to create an activation complex [32],
which enables for that bifurcation of cell signaling to MAPK and NF-?B pathways in the
TAK1/TAB1 complex, and therefore TAK1 becomes dispensable in TNFa-driven signaling.
IL-1ß plays a huge role in synovial inflammation and bone/cartilage destruction
in RA [33,34]. Even just in TNFa-driven arthritic human TNF-transgenic (hTNF-tg) rodents, IL-1
inhibition completely abolished cartilage and bone destruction, suggesting that it is a crucial
mediator of RA pathogenesis [35-37]. Our findings prove, in human RASFs,
takinib inhibits inflammatory markers largely by disturbing IL-1ß-caused STAT3 and
JNK, which implies that takinib doesn’t directly effect TAK1, but rather effects multiple
pathways in RASFs to induce its anti-inflammatory activity. In addition, we evidence,
using in silico docking studies, that sites where takinib communicate with STAT3 residues
are essential for confirmational changes, consequential phosphorylation or activation,
as well as in nuclear translocation for DNA binding activity. It was attested by our findings
in human RASFs that indeed takinib inhibits IL-1ß-caused nuclear translocation as well as in
DNA binding activity of STAT3. These finding claim that the anti-inflammatory activity
of takinib is mediated by multiple pathways in RASFs and THP-1 macrophages, which are
separate from TAK1 within the signaling hierarchy of those cells. Furthermore, the present
findings, that takinib maintained TAK1 phosphorylation at Thr184/187 in RASFs
and THP-1 macrophages via IL-1ß or LPS stimulation, correspondingly, and inhibited p-JNK
expression, claim that takinib could have a more profound impact on JNK kinase rather
than you are on TAK1 in the mechanisms of suppressing inflammation.
Within the study by Totzke et al. [21] treating human RASFs with takinib caused
apoptosis and reduced IL-6 production. Inside a follow-up study by Scarneo et al., the authors
provided evidence that takinib, in a similar dose range, doesn’t have adverse impact on RASF via bility, but considerably inhibited chemokine production by TNFa-activated human RASFs.
However, the mechanisms by which takinib pertains to the inhibition of TAK1 kinase
during these cells remains elusive. In addition, the activation of TNFa-caused apoptosis
signaling kinase 1 (ASK1) soon after TRAF2 activation, branches signaling towards the
JNK path that includes to programmed cell dying in order to apoptosis, as observed
in situation of takinib. Our findings, which elucidate the function of STAT3, bridges the space in
knowledge of the medicinal action of takinib by identifying a principal target
of takinib in human RASFs. While these answers are available to different interpretations, the
available literature detailing the function of STAT3 in LPS-caused TNFa along with other forms
of chemokine production in macrophage cells [38,39] further verify our argument that
takinib elicits its anti-inflammatory effects mainly through suppression of JAK/STAT
path, not TAK1 driven signaling, in human RASFs and perhaps macrophages.
To conclude, this is actually the first study to check the result of takinib in IL-1ß stimulated
inflammation primary human RASFs and also to provide molecular insights in to the possi ble mechanism of their anti-inflammatory activity in vitro. Our findings says the
medicinal action of takinib in lessening cytokine production might not be because of its
inhibition of TAK1, but of other signaling proteins including STAT3 during these cells. Addition ally, the elevated phosphorylation of TAK1 and also the don’t have any inhibition from the MAPK and
NF-kB pathways in macrophages raise further questions regarding the selectivity of takinib
in cases of IL-1ß-driven illnesses. These bits of information warrant further analysis before
takinib is validated like a selective TAK1 inhibitor, by testing its therapeutic potential.
4. Materials and techniques
4.1. Antibodies and Reagents
Recombinant IL-1ß were purchased in R&D Systems (Minneapolis, MN). Antibodies
for p-TAK1(Thr184/187), TAK1(Ser412), p-IRAK4(Thr345/Ser346), p-IRAK1(Thr209)
, p-TAB2(Ser372)
Int. J. Mol. Sci. 2021, 22, 12580 9 of 13
IL-1ß, p-STAT3(Tyr705), p-STAT3(Ser727), p-SAPK/JNK(Thr183/Tyr185), and that i?Ba were pur chased from Cell Signaling Technologies (Danvers, MA Cat# 90C7, 9339S, D6D7, T209,
8155, D3U3E, 9145S, 9136S, 4671S and 4812S). ß-Actin and IRAK1 were purchased in
Santa Cruz Biotechnology (Santa Cruz, CA, USA sc-47778 H273). Lipopolysaccharide
(LPS), takinib and Tofacitinib were purchased in Sigma (St. Louis, MO, USA). The
STAT3 Transcription Factor Assay Package (Cat: 601950), 5Z-7-oxozeaenol, and NF-?B (p65)
Transcription Factor Assay Package (Cat: 10007889) were purchased in Cayman Chemicals
(Ann Arbor, MI, USA) and NG 25 trihydrochloride was purchased in Axon Medchem
(Reston, Veterans administration, USA).
4.2. Culturing of Human RASFs and THP1
De-identified human RA synovium tissues were acquired from Cooperative Human
Tissue Network (CTHN Columbus, OH, USA) and National Disease Research Interchange
(NDRI Philadelphia, PA USA). Normal and RA tissues were acquired from total joint
substitute surgical procedures or synovectomy under an Institutional Review Board (WSU-IRB)
approved protocol, in compliance using the Helsinki Declaration. The donor population
incorporated both men and women identified as having RA whereby the typical chronilogical age of the
contributors was 57 ± 27 years. RA tissues were digested in collagenase, prior to being seeded
in 72 cm2 flasks. Cells were grown within an RPMI 1640 medium supplemented with 15%
fetal bovine serum (FBS), 5000 U/mL penicillin, 5 mg/mL streptomycin, and 10 µg/mL
gentamicin. Upon confluency (>85%), cells were passaged with brief trypsinization. The
experiments were performed using cells which were passed a minimum of four to five occasions to make sure pure
fibroblast population. For experimental purposes, RASFs between passages 5-10 were utilised.
All treatments were performed in serum free media. All experiments were performed
on a minimum of 3-4 cell lines established from various RA contributors with this study. Human
monocytic leukemia (THP-1) cells were purchased in ATCC (88081201-1VL, Manassas,
Veterans administration, USA) and maintained within an RPMI 1640 culture media with 10% fetal bovine serum
(FBS) and antibiotics. For that experiments, THP-1 cells were differentiated to some macrophage
population with phorbol 12-myristate 13-acetate (100 ng/mL) for several h, adopted by media
substitute with fresh Opti-MEM media overnight.
4.3. Management of RASFs and THP-1
RASFs were seeded in 6-well plates or 100 mm dishes, grown to >85% confluency.
Cells were put into serum-free media overnight just before treatments. To review inhibitors,
cells were pre-incubated with takinib (.1-20 µM), 5Z-7-oxozeaenol (1 µM), NG 25 tri hydrochloride (1 µM) for just two h just before IL-1ß (10 ng/mL) stimulation for 30 min to review
alterations in signaling, or 24 h, to judge producing IL-6, IL-8, CXCL5 and
MCP-1. Likewise, overnight-differentiated THP-1 cells were pre-incubated with takinib
(.1-20 µM), 5Z-7-oxozeaenol (1 µM), NG 25 trihydrochloride (1 µM) for just two h just before
stimulation from LPS (10 ng/mL) for 30 min or 24 h and exposed to Western blotting
or ELISA.
4.4. Assay for Cytokine Production
The conditioned media from RASFs or THP-1 was collected from 24 h IL-1ß- or LPS stimulated samples that have been spun lower at 10,000 revoltions per minute for 10 min at 4 ?C to get rid of
particulate matter where then collected in fresh Eppendorf tubes. The collected super natants were examined for human IL-1ß, TNFa, ENA-78/CXCL5, MCP-1/CCL2, IL-6 and
IL-8 levels using colorimetric sandwich ELISA kits (R&D Systems, Minneapolis, MN, USA)
as reported by the manufacturer’s instructions.
4.5. Western Immunoblotting
The entire-cell extract was prepared using RIPA buffer (50 mM Tris pH 7.6, 150 mM
NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM DTT, .5% sodium deoxycholate, and .1% SDS)
that contains protease and phosphatase inhibitors (Roche, Basel, Europe). Protein disadvantage-
Int. J. Mol. Sci. 2021, 22, 12580 10 of 13
tent was measured using Bio-Rad Electricity method (Bio Rad, Hercules, CA, USA). The same
sum of proteins (25 µg) was loaded for every sample and separated on acrylamide gel
prior to being used in a PVDF membrane (EMD Millipore, Billerica, MA, USA). Blots
were then blocked in TBST that contains 5% nonfat dry milk for 2 hrs just before overnight
incubation using the particular primary antibody (see section on antibodies and reagents)
with dilution based on the manufacturer’s instructions. Protein bands were visualized
using chemiluminescence and examined using Image Lab software (Bio-Rad) for band
intensity. Blots were probed with ß-actin to make sure equal loading. Relative STAT3 phos phorylation was resolute by normalizing pSTAT3 and total STAT3 bands with ß-actin,
utilizing a ratio of pSTAT3/Total STAT3. We performed pTAK1 phosphorylation utilizing a ratio
of pTAK1/ß-actin.
4.6. Cell Fractionation
The fractionation approach to Zhu et al. was modified accordingly [12,14]. RASFs were
grown in 100 mm plates having a confluency as high as 90%, pretreated with takinib, tofacitinib
or untreated, adopted by IL-1ß (10 ng/mL) stimulation for twenty-four h. Later on, cells were
washed once in cold 1X PBS and lysed with 1 mL of Cytoplasmic lysis buffer (10 mM
Tris-HCL, pH 7.9, .34 M Sucrose, 3 mM CaCl2, 2 mM magnesium acetate, .1 mM EDTA,
1 mM DTT, .5% CA630 and protease inhibitors). The cell pellet that created was lightly
resuspended utilizing a wide mouth tip, adopted by incubation on ice for 30 min. Nuclei
were pelleted by centrifugation at 3500× g for 15 min at 4 ?C. After this, centrifuge
cytoplasmic extract was kept in a pre-chilled 1.5 mL Eppendorf tube.
The rest of the nuclei pellet was washed with 1 mL of Cytoplasmic lysis wash buffer
as before, adopted by lysis in .2 mL of RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl,
1% Triton X-100, 1 mM EDTA, 1 mM DTT, .5% sodium deoxycholate, and .1% SDS) and
stored on ice for 30 min. The nuclear extract was collected via centrifugation at 15,000× g for
30 min at 4 ?C inside a fresh Eppendorf tube.
4.7. DNA Binding Assay for STAT3
Five µg of nuclear extract was utilized for DNA binding activity, to create unstimulated
and IL-1ß- (10 ng/mL) stimulated samples without or with takinib and Tofacitinib treat ments for just two h at 70 degrees for binding as reported by the manufacturer’s instructions.
4.8. Molecular Dynamics (MD) Simulation Studies
To recognize potential interactions of takinib with STAT3 in silico, the 3D structures of
human STAT3 (PDB ID: 6NJS) [22] were prepared using protein preparation wizard. All
docking calculations were performed using Schrodinger suit 2020.3 [23]. The protonation
states of all of the titratable residues were assigned in a physiological pH using PROPKA.
Retrained minimization was performed using .30 Å root-mean-square deviation (RMSD)
via enhanced potentials for that liquid simulation’s extended (OPLS3e) pressure field. The
30 Å grid was generated round the co-crystallized small molecule (SD-36). The takinib
structure, employed for docking, was prepared at pH 7. ± 2. using LigPrep module. The
docking calculations were performed using GLIDE module. The takinib molecule was initially
posted for normal Precision (SP) docking to create 10 docking poses. These poses
were then posted to Extra Precision (XP) docking. Selecting the greater appropriate
pose is made based on the power and interaction of takinib using the active site
residues. To higher comprehend the interactions between your active site and takinib, these
poses were then posted for inducedFit docking. In caused fit docking, we maintained
the versatility of active site residues and takinib so they could adjust themselves
to some better binding pose and affinity.
Int. J. Mol. Sci. 2021, 22, 12580 11 of 13
Extra Materials: Listed here are available on the web at https://world wide web.mdpi.com/article/
10.3390/ijms222212580/s1.
Author Contributions: Conceptualization, S.A., A.K.S., P.M.P.; methodology, S.A., A.K.S., P.M.P.;
software, M.C.; validation, A.K.S., P.M.P., F.S.S., R.J.S. and M.C.; formal analysis, P.M.P., F.S.S., R.J.S.
and M.C.; investigation, A.K.S., P.M.P., F.S.S., R.J.S. and M.C.; resources, P.M.P., F.S.S., R.J.S. and
M.C.; data curation, S.A., A.K.S., P.M.P., F.S.S., R.J.S. and M.C.; writing—original draft preparation,
P.M.P.; writing—review and editing, P.M.P., F.S.S.; visualization, P.M.P.; supervision, S.A.; project
administration, S.A. and P.M.P.; funding acquisition, S.A. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by NIH R01, grant number AR072615.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors would like to thank the Cooperative Human Tissue Network
(CHTN; Columbus, OH, USA) and the National Disease Research Interchange (NDRI; Philadelphia,
PA, USA) for providing RA synovial tissues as well as David A. Fox (University of Michigan, Ann
Arbor, MI, USA) for providing some RASF cell lines generated in his laboratory.
Conflicts of Interest: The authors declare no conflict of interest.
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