Discovery of an Orally Active Small-Molecule Tumor Necrosis Factor-α Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.0c00377

Tumor necrosis factor α (TNF-α) is an important therapeutic target for rheumatoid arthritis, inflammatory bowel disease, and septic hepatitis. In this study, structure-based virtual ligand screening combined with in vitro and in vivo assays were applied.

Full text of DOI:10.1021/acs.jmedchem.0c00377

Subscriber access provided by the University of Exeter
Article
Discovery of an Orally Active Small Molecule TNF-# Inhibitor
Weiguang Sun, Yanli Wu, Mengzhu Zheng, Yueying Yang, Yang Liu,
Canrong Wu, Yirong Zhou, Yonghui Zhang, Lixia Chen, and Hua Li
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00377 • Publication Date (Web): 15 Jul 2020
Downloaded from pubs.acs.org on July 15, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
Discovery of an Orally Active Small Molecule TNF-α Inhibitor
6
7
8
9
Weiguang Sun†, #, Yanli Wu‡, #, Mengzhu Zheng†, #, Yueying Yang‡, Yang Liu‡, Canrong Wu†,
Yirong Zhou†, *, Yonghui Zhang†, *, Lixia Chen‡, *, Hua Li†, ‡,*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
† Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of
Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan
430030, China
‡ Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery,
Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 47
1
2
3
4
5
6
7
8
9
ABSTRACT: Tumor necrosis factor α (TNF-α) is an important therapeutic target for rheumatoid
arthritis, inflammatory bowel disease and septic hepatitis. In this study, structure-based virtual
ligand screening combined with in vitro and in vivo assays was applied. A lead compound,
benpyrine, could directly bind to TNF-α and block TNF-α-trigged signaling activation.
Furthermore, endotoxemia murine model showed that benpyrine could attenuate TNF-α-induced
inflammation, thereby reducing liver and lung injury. Meanwhile, administration of benpyrine by
gavage significantly relieved the symptoms of collagen-induced arthritis and imiquimod-induced
psoriasiform inflammation in mice. Thus, our study discovered a novel, highly specific and
orally active small molecule TNF-α inhibitor that is potentially useful for treating TNF-α
mediated inflammatory and autoimmune disease.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
ACS Paragon Plus Environment

Page 3 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
■ INTRODUCTION
6
7
8
9
Tumor necrosis factor α (TNF-α), which is mainly secreted by active macrophages
and monocytes, is a pleiotropic cytokine that plays crucial roles in immune functions,
including inflammation, antitumor responses, and infections.1 An elevated serum level
of TNF-α is associated with tumorigenesis, diabetes, and particularly autoimmune
disorders, such as rheumatoid arthritis (RA), psoriatic arthritis, multiple sclerosis, and
Crohn’s disease.2 Therefore, direct inhibition of TNF-α has become a major
therapeutic approach for the treatment of these diseases. The well-known commercial
monoclonal antibodies adalimumab and infliximab, and the fusion protein etanercept
have been proven to directly bind to TNF-α, further preventing its interaction with the
tumor necrosis factor receptor (TNFR).3, 4 To date, these biomacromolecular agents
have shown excellent therapeutic effects and high specificity for the treatment of
autoimmune diseases.5 However, several severe limitations, such as poor stability,
cost-ineffective commercial-scale production, and serious side effects, have also
emerged, prompting the demand for novel small molecule TNF-α inhibitors.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To date, several selective small molecule antagonists of TNF-α have been
identified, most of which engender the inhibitory effects by targeting key molecules
of the intracellular TNF-α pathway, inhibiting TNF-α converting enzyme (TACE) or
down-regulating the expression of TNF-α.7 Although progress has been made in
developing small molecules that are capable of directly disrupting TNF-α and TNFR
interactions, this area of research remains a huge challenge. To our knowledge, some
small molecules that directly antagonize TNF-α, such as the polysulfonated
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 47
1
2
3
4
5
6
7
8
9
naphthylurea suramin analogues and the indole-linked chromone derivative SPD304,
often show low potency, poor selectivity, and tend to cause adverse side effects,
making them unsuitable for anti-TNF-α therapies.8-10 Furthermore, SPD304, which
contains a toxic 3-alkylindole moiety, is metabolized by cytochrome P450 enzymes
through a dehydrogenation pathway similar to that of the potent pneumotoxin
3-methylindole, producing reactive electrophilic iminium materials that could react
with protein and DNA targets.11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Thus, additional efforts to identify effective and highly specific TNF-α inhibitors
with relatively lower toxicity are urgently demanded.12-14 In the present study, a novel
small molecule inhibitor targeting TNF-α was identified using a structure-based
virtual screening method in combination with in vitro and in vivo activity assays.
■ RESULTS AND DISCUSSION
Structure-based virtual ligand screen for TNF-α inhibitors. In this study, the
X-ray co-crystal structure of a TNF-α dimer with SPD304 (PDB code: 2AZ5) was
used as the model to screen 6 million drug-like compounds from the ZINC database in
silico. The top seven highest scoring compounds (1-7) were selected from the initial
high-throughput virtual screening campaign. Subsequently, we applied these
compounds at concentrations of 1 and 10 μM in the initial tests to examine their
ability to inhibit TNF-α-induced cytotoxicity in L929 cell line. Compound 7,
(S)-4-((9H-purin-6-yl)amino)-1-benzylpyrrolidin-2-one, which was designated
benpyrine, exhibited dose-dependent inhibitory effects that were consistent with the
4
ACS Paragon Plus Environment

Page 5 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
screening result. Therefore, this compound was selected for further characterization
6
7
and functional validation in vitro and in vivo.
8
9
Benpyrine directly binds to TNF-α. The microscale thermophoresis (MST)
method was utilized to assess the ability of these compounds binding to TNF-α and to
further validate the results of the molecular docking simulations.15, 16 Encouragingly,
the KD value of benpyrine (82.1 ± 5.0 μM) was even smaller than that of SPD304
(91.7 ± 6.3 μM). In comparison, no statistically significant binding was detected when
TNF-α was treated with compounds 1-6 (Table S1 and Figure S1 in SI ). Based on
these data, benpyrine and SPD304 displayed relatively stronger binding affinity to
TNF-α. Another MST assay was performed to further determine whether the direct
binding between benpyrine and TNF-α was related to the interaction between TNF-α
and TNFR1 or not. Briefly, after incubation with 10 μM benpyrine for 30 min, the KD
value of TNF-α-TNFR1 binding was increased approximately 18-fold (from 0.23 μM
to 4.26 μM) comparing with the interaction in the absence of benpyrine. In addition,
the KD value was further increased to 19.63 μM after the addition of 100 μM
benpyrine (Figure 2A-2D). All these results confirmed the specific binding of
benpyrine to TNF-α, and partially confirmed that benpyrine suppressed the binding of
TNF-α to TNFR1. Meanwhile, the ELISA assay further revealed that benpyrine
tightly bound to TNF-α and blocked its interaction with TNFR1, with an IC50 value of
0.109 µM (Figure 2E). A drug affinity responsive target stability (DARTS) assay and
protein thermal shift (PTS) assay were performed using published methods to further
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
validate the targeting of TNF-α by benpyrine.17, 18 As shown in Figure 2F, most of the
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 47
1
2
3
4
5
6
7
8
9
TNF-α was degraded within 30 min after the pronase treatment, whereas the
degradation of TNF-α in benpyrine-treated samples was decreased in a
dose-dependent manner. And in Figure 2G, benpyrine increased the thermal stability
of TNF-α protein from tolerated temperature of 90 °C to 94 °C. Thus, benpyrine
inhibits TNF-α activity by directly binding to the protein.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Analysis of the binding mode. The binding pocket of the TNF-α dimer is
relatively large and featureless, lacking clearly defined binding crevices.19 The
binding site is mostly hydrophobic, consisting primarily of glycine, leucine,
isoleucine and tyrosine residues. Based on the generated docking model, as expected,
the binding of benpyrine to TNF-α was a relatively hydrophobic interaction driven by
the shape to prevent the binding of the third subunit, which forms the biologically
active trimer complex. As shown in Figure 2H and 2I, benpyrine adopted a
clamp-shaped conformation and formed several hydrophobic interactions with Leu57,
Tyr59, Tyr119, Gly121 and Tyr151 from both TNF-α monomers. No predicted
hydrogen bonding interactions were observed.
According to the predicted binding sites for benpyrine in TNF-α, several
site-directed mutations, including L57A, Y59A, and Y119A, were introduced. The MST
assay revealed a weaker binding affinity of benpyrine for the L57A and Y59A mutants
than wild type TNF-α, while no obvious changes in the binding affinity for the Y119A
mutant were observed. The efficacy and specificity of benpyrine-mediated inhibition
of TNF-α mutant-induced L929 cells death was further determined. As shown in
Figure 3A-3C, all TNF-α mutants significantly induced L929 cell death, with IC50
6
ACS Paragon Plus Environment

Page 7 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
values ranging from 3.21 to 6.36 ng/mL in combination with 1 μg/mL actinomycin D.
However, benpyrine only blocked cell death induced by TNF-αWT and Y119A and,
increased the cell survival rate up to 80%. Meanwhile, benpyrine did not obviously
affect L57A- and Y59L-induced cytotoxicity in L929 cells. Thus, Leu57 and Phe59 are
the key amino acids required for the binding of benpyrine, consistent with the docking
prediction.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benpyrine blocks TNF-α-induced signaling pathways. TNF-α-induced
inflammatory process is under the control of several sequences in gene promoters.20
In particular, the stimulation of the nuclear factor kappa-B (NF-κB) transcription
factor has attracted increasing attentions. NF-κB has recently been shown to induce
the expression of a variety of antiapoptotic factors, which are important for regulating
the TNFR1-mediated activation of apoptotic mechanisms.21 In the present study, we
investigated the effects of benpyrine on the activation of the NF-κB pathway in
RAW264.7 macrophages. After an incubation with different concentrations of
benpyrine for 12 h, TNF-α (10 ng/mL) or lipopolysaccharide (LPS, 1 µg/mL) was
added and incubated for another 2 h. Protein lysates were subjected to western
blotting with anti-IκBα and p-IκBα antibodies. In the absence of benpyrine, TNF-α
and LPS induced the phosphorylation of inhibitor of NF-κB α (IκBα). Encouragingly,
a pretreatment with benpyrine resulted in a dose-dependent decrease in the
phosphorylation of IκBα (Figure 3D and 3E). These observations were also consistent
with the result of the microscopy analysis shown in Figure 3F. In this experiment, the
addition of benpyrine abolished the TNF-α-induced nuclear translocation of
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 47
1
2
3
4
5
6
7
8
9
NF-κB/p65 in RAW264.7 cells. Furthermore, benpyrine exhibited low cytotoxicity
toward RAW264.7 cells (IC50 > 100 μM), indicating that benpyrine represents a
potentially hypotoxic TNF-α inhibitor.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Benpyrine inhibits the LPS-induced production of inflammatory cytokines
in vivo. LPS causes a systemic inflammatory response and acute tissue injury when
injected into mice as an endotoxin.22 Here, the effect of benpyrine on LPS-triggered
inflammatory responses was examined in vivo. Mice were preinjected with the vehicle
or benpyrine, followed by the administration of normal saline (NS) or LPS (1.5
mg/kg). As shown in Figure 4, serum levels of inflammatory cytokines, including
IL-6 and IL-1β, were substantially increased in mice treated with LPS compared with
the control group, while the increased levels of these cytokines was substantially
attenuated by pretreating mice with different concentrations of benpyrine.
The anti-TNF-α effects of benpyrine on an animal model of megadose
endotoxemia were further studied. The benpyrine pretreatment (25 mg/kg)
significantly increased the survival rate compared with the non-treated control group.
According to the histopathology, the benpyrine treatment significantly reduced the
liver and lung damage and emergence of hemorrhagic necrosis caused by the LPS
treatment. Based on these results, benpyrine has great potential to serve as an
anti-inflammatory agent.
8
ACS Paragon Plus Environment

Page 9 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Therapeutic effects of benpyrine on mice with type II collagen-induced
arthritis (CIA). Rheumatoid arthritis (RA) is the most common chronic
inflammatory autoimmune disorder affecting the synovial joints, with approximately
1% of the global population suffering from this disorder.23, 24 Inflammatory cytokines
such as TNF-α, interleukin (IL)-6, IL-1 and IL-17 play important roles in RA
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
pathogenesis, while an anti-TNF-α treatment effectively slows disease progression
26
among the various anti-rheumatic methods.25,
CIA is an autoimmune model
manifesting common immunological and pathological features associated with human
RA, including synovial hyperplasia, cartilage destruction, and immune hyperfunction.
In the present study, the benpyrine treatment significantly decreased the arthritic score
and spleen index of CIA mice compared with the untreated group (Figure 5A and 5B).
The regulation of inflammatory mediators was speculated to be important in the
pathogenesis and therapy of RA. Therefore, serum levels of IFN-γ, IL-1β IL-6, and
IL-10 were measured using ELISA analysis. As shown in Figure 5C-F, serum levels
of these cytokines were much higher in the non-treated CIA mice, suggesting the
induction of an obvious inflammatory response in the model group. Benpyrine
dose-dependently decreased the levels of proinflammatory cytokines, such as IFN-γ,
IL-1β and IL-6, and increased the concentration of the anti-inflammatory cytokine
IL-10.
The effects of benpyrine on the joints of rats were determined by performing
histochemical staining with H&E. As shown in Figure 5G, a normal joint histology
was observed in the control group. The ankle and toe joints of mice in the CIA group
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 47
1
2
3
4
5
6
7
8
9
showed significant histopathological changes related to severe arthritis, including
notable synovial hyperplasia, partial bone and cartilage destruction, inflammatory cell
infiltration into the synovium, and narrowing of the joint space. In contrast, benpyrine
and prednisone (Pro, 5 mg/kg) significantly ameliorated the degree of cartilage and
bone destruction and inflammation compared with the CIA group.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Therapeutic effects of benpyrine on imiquimod-induced psoriasiform
inflammation in mice. Psoriasis is a chronic inflammatory skin disease affecting
2–3% of the global population.27, 28 Despite the extensive studies, the detailed
mechanism underlying the pathogenesis of psoriasis remains to be elucidated.
Initially, psoriasis was described as a disease involving the excessive proliferation of
keratinocytes that subsequently triggered inflammation. Several neutralizing anti-TNF
agents, such as etanercept and infliximab, have been successfully applied to treat
psoriasis.29, 30 Indeed, the amelioration achieved with TNF antagonists confirms that
TNF-α is a pivotal proinflammatory mediator in psoriatic lesions.
The mouse model of imiquimod-induced psoriasiform inflammation (IPI) was
employed to evaluate the effects of benpyrine on psoriasis. Compared to the psoriatic
mouse models using a skin xenograft, gene knockout, or gene mutation, this model
represents a better tool for the investigation of the pathogenesis and therapeutic agents
of psoriasis because it can be elicited in a more natural immune state.31 The mice in
the model group displayed erythema, scales, and incrassation on their back skin
beginning on day 2, and these symptoms were most visible on day 5. After treatment
with benpyrine, differences in the skin were observed between all groups. Compared
10
ACS Paragon Plus Environment

Page 11 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
to the IPI model mice, the benpyrine treatment inhibited inflammation throughout the
period of imiquimod exposure and significantly decreased the adjusted scale scores
consisting of erythema, epidermal acanthosis, and the thickness of the lesions (Figure
6A-B). The benpyrine treatment also reduced the spleen index, which was distinctly
increased after the imiquimod application (Figure 6C). Proinflammatory cytokines,
such as IL-1β, IL-17F, IL-22, IL-6, etc., are crucially involved in psoriatic skin
inflammation. The serum levels of the proinflammatory cytokines IL-1β and IL-6
were further examined to obtain additional insights into the therapeutic effects of
benpyrine. As revealed by the ELISAs, the levels of IL-1β and IL-6 were significantly
increased in the IPI mice, while benpyrine dose-dependently decreased this increase
in the IL-1β and IL-6 levels after 7 days of treatment. Its efficacy was comparable to
prednisone at 5 mg/kg (Figure 6D-E). Additionally, histopathological changes
induced by the benpyrine group were obviously ameliorated compared to IPI mice
(Figure 6F).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
■ CONCLUSIONS
TNF-α is mainly secreted by active monocytes or macrophages and is a well-known
pleiotropic cytokine with crucial roles in the host immune system. Over-expression of
TNF-α is a hallmark of many inflammatory diseases, including rheumatoid arthritis,
psoriasis, inflammatory bowel disease and septic shock, making it an excellent
therapeutic target for clinical interventions.32-34 Significant advances have been made
in the development of biological agents targeting TNF-α and its signaling components.
Several well-known commercial TNF-α inhibitors, such as infliximab, adalimumab
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 47
1
2
3
4
5
6
7
8
9
and etanercept, are TNF-α antibodies or TNFR1-Fc chimeras.35, 36 The anti-TNF-α
drugs specifically bind to the TNF-α protein, thus preventing it from binding to the
receptors, TNFR1 and TNFR2.37 To date, those biomacromolecular agents have been
proven to be effective treatments for inflammatory bowel disease and rheumatoid
arthritis due to their high specificity. According to an industry analysis, TNF-α
antibodies account for over 30 billion dollars of the global antibody market. However,
several severe limitations, including poor stability, cost-ineffective commercial-scale
production, and exclusion by the blood/brain barrier, have also emerged. Instead,
small molecule compounds have been appreciated as appropriate alternatives for
overcoming most disadvantages associated with macromolecular inhibitors.
Furthermore, they offer additional clinical benefits, such as a simpler preparation for
oral medicine.38, 39
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To date, several selective small molecule antagonists of TNF-α activity have
been identified. However, none of these small molecule inhibitors have been reported
to reduce TNFα-induced inflammatory responses in vivo with high efficiency and low
toxicity. In this study, we applied the virtual screening technique to discover small
molecule antagonists of TNF-α, enabling us to identify the lead compound benpyrine
that potently inhibits TNF-α-induced cytotoxicity.
The formation of a trimer of TNF-α is a requirement for its binding to two
receptors, TNFR1 (also called CD120a) and TNFR2 (also called CD120b). The
engagement of TNF-α with TNFR1 and TNFR2 initiates signaling cascades that result
in inflammatory responses and control apoptosis. In this study, we took advantage of
12
ACS Paragon Plus Environment

Page 13 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the crystal structure of the TNFα/SPD304 complex and utilized it as the basis for the
virtual ligand screening. The molecular docking simulations suggested that benpyrine
formed the key hydrophobic interactions with Leu57 and Phe59, which blocked the
formation of the TNF-α trimer. This prediction was further confirmed by studying
mutant proteins. The L57A and Y59L mutations markedly attenuated the protective
effects of benpyrine on the death of L929 cells induced by these mutants. Additionally,
benpyrine displayed the decreased binding affinities for these mutants in the MST
assay. Meanwhile, benpyrine significantly inhibited TNF-α- and LPS-induced
phosphorylation of IκBα and abolished the TNF-α induced nuclear translocation of
p65 in RAW264.7 cells. Taken together, these results establish the function of
benpyrine as a potent TNF-α inhibitor.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In addition to its efficacy in vitro, benpyrine significantly attenuated
TNF-α-induced inflammation in vivo, and thus shows promise as a potential
therapeutic agent for TNF-α-mediated inflammatory diseases. In the mouse model of
LPS-induced endotoxemia, benpyrine remarkably decreased the death rate of mice.
Furthermore, benpyrine also exerted therapeutic effects on collagen-induced arthritis
and imiquimod-induced psoriasiform inflammation in mouse models. Benpyrine
showed superior therapeutic effects and obviously decreased the scale scores of
illnesses consisting of inflammatory and hyperactive immune reactions. Moreover,
the acute toxicity test confirmed the lack of toxicity of benpyrine at the dose of 2000
mg/kg (by gavage), thus validating its safety. Benpyrine is an effective small
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 47
1
2
3
4
5
molecule inhibitor of TNF-α that could be potentially useful as a therapeutic
6
7
intervention for various inflammatory diseases.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
■ EXPERIMENTAL SECTION
Reagents. The compound collection, including compounds 1-7, was obtained
from AnalytiCon Discovery (Molport, Latvia). The ZINC database
(http://zinc.docking.org/) is publicly available and can be accessed free of charge.
SPD304, LPS, and prednisone were purchased from Sigma-Aldrich (St. Louis, MO,
USA). The TNF-α cDNA was synthesized by GENEWIZ (Suzhou, China). TNFR-1
was obtained from Prospec (Rehovot, Israel). Mouse monoclonal antibodies against
IκBα, p-IκBα, and NF-κB/p65 were provided by Cell Signaling Technology (Boston,
MA, USA). The L929 and RAW264.7 cell lines were purchased from Boster
Bioengineering Company (Wuhan, China). For the further animal experimrnts, more
compound 7 was total systhesis as described in Scheme S1 of SI. Purity of compound
7 was determined to be higher than 95% by HPLC analysis which was performed on
Daicel CHIRALCEL IC column (250 mm × 10 mm, 5 μm) and method as: MeCN :
H2O = 30 : 70 by 20 min of isocratic hold, a flow rate of 2.0 mL/min, and plotted at
254 nm;
Animals. Balb/c mice (18–20 g) were purchased from the Laboratory Animal
Center of Tongji Medical College of Huazhong University of Science and
Technology (Wuhan, China). All animals were acclimated for 7 days before being
used in the experiment. Mice were housed under pathogen-free conditions at
14
ACS Paragon Plus Environment

Page 15 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
temperature of 22 ± 2 °C, humidity of 55 ± 5%, and a 12-h light/dark cycle. They had
free access to water and food under standard specific pathogen-free (SPF) conditions
throughout the study. All animal experiments were carried out in accordance with
protocols approved by the Institutional Animal Care and Use Committee at the Tongji
Medical College of Huazhong University of Science and Technology (NO.
2019-S972).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Virtual screening and molecular docking simulations. Lead-like compound
libraries from the ZINC database containing over 600,000 compounds were screened
in silico. Molecular docking was performed using the ICM-Pro 3.8.2 program
(MolSoftLLC, San Diego, CA).40 According to the internal coordinate mechanics
(ICM) method, the molecular system is based on the internal coordinates (IC)
representation of molecular objects that naturally reflects the covalent bond geometry
of molecules. For structure predictions and large-scale conformational sampling, ICM
employs a family of new global energy optimization techniques, including biased
probability Monte Carlo simulations, a pseudo-Brownian docking algorithm3 and
local deformation loop movements.
The initial model of TNF-α was built from the X-ray co-crystal structure of the
TNF-α dimer with SPD304 (PDB code: 2AZ5) using a previously reported procedure.
Hydrogens and missing heavy atoms were added to the receptor structure, followed
by local minimization by using the conjugate gradient algorithm and analytical
derivatives in the internal coordinates. In the docking analysis, the binding site was
assigned across the entire structure of the protein dimer. ICM docking was performed
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 47
1
2
3
4
5
6
7
8
9
to identify the most favorable orientation. The resulting trajectories of the complex
between the small molecules and TNF-α dimer were energy minimized, and the
interaction energies, which are expressed in kJ/mol, were computed. As a reference,
the molecular docking of the well-known TNF-α inhibitor SPD304 showed a score of
-32.9.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Expression and purification of recombinant wild type and mutant TNF-α.
The gene encoding TNF-α was cloned into the pET-28a vector (Novagen). After
verifying the sequence, the recombinant plasmid was transformed into E. coli BL21
(DE3) (Invitrogen) cells that were grown in LB medium at 37 °C to an OD600
(0.8-1.0),
and
expression
was
induced
with
0.4
mM
isopropyl-D-thiogalactopyranoside (IPTG) at 20 °C for 16 h. Bacterial cells were
collected and lysed by ultrasonication on ice in a buffer containing 20 mM Tris, pH
8.5, 200 mM NaCl, 5 mM mercaptoethanol, 0.1% TritonX-100, and 5% glycerol.
Soluble C-terminally hexa-histidine-tagged TNF-α was bound to Ni-agarose affinity
resin (Qiagen), washed with a buffer containing 20 mM Tris, pH 8.5, 200 mM NaCl,
and 10 mM imidazole, and eluted with a buffer containing 20 mM Tris, pH 8.5, 250
mM NaCl, and 150 mM imidazole. Thrombin (Roche) was added to the eluate at a
concentration of one unit per 4 mg of protein, which was then dialyzed against 20 mM
Tris, pH 8.5, and 100 mM NaCl at room temperature for overnight digestion. The
protein was further purified with anion exchange chromatography using a linear
gradient of 10 mM to 1 M NaCl and size exclusion chromatography in 20 mM Tris,
pH 8.5, and 200 mM NaCl.
16
ACS Paragon Plus Environment

Page 17 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Analysis of the direct binding of compounds 1-7 to TNF-α with MST. TNF-α
was labeled with the Monolith NT™ Protein Labeling Kit RED (Cat#L001) according
to manufacturer’s labeling protocol. Labeled TNF-α was maintained at a constant
concentration of 100 nM, while all tested samples were diluted in 20 mM HEPES (pH
7.5) containing 0.05% (v/v) Tween-20. Compounds were sequentially diluted
covering the range of appropriate concentrations. After a 10 min incubation at room
temperature, samples were loaded into MonolithTM standard-treated capillaries and the
thermophoresis was measured at 22 °C using 100% light-emitting diode (LED) and
20% MST power on a Monolith NT.115 instrument (NanoTemper Technologies,
München, Germany). The values of the dissociation constant KD were fitted using the
Nanotemper Analysis software v.1.2.101. The TNFR1 protein was also labeled as
described above, and the binding of TNFR1 to TNF-α or benpyrine was also tested.
Furthermore, after 10 or 100 μM benpyrine was preincubated with various
concentrations of TNF-α at room temperature for 15 min, the mixture was added to an
equivalent volume of labeled TNFR1 to determine the KD value.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Determination of the inhibitory effect of benpyrine on the interaction
between TNFR1 and TNF-α using an ELISA. Microtiter plates were coated with
TNFR1 (2.5 µg/mL) in PBS overnight at 4 °C. The wells were washed three times
with PBS/0.05% Tween 20 (PBST), blocked with 200 µL of PBST containing 1%
BSA for 60 min and washed as described above. Serial dilutions of benpyrine in 50
µL of PBS containing 2% DMSO were added to the wells, and the microtiter plates
were incubated with shaking for 20 min. TNF-α (0.01-10 µg/mL) in 50 µL of PBS
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 47
1
2
3
4
5
6
7
8
9
was added to the wells and the plates were incubated for an additional 120 min. The
plates were washed as described above and incubated with the TNF-α antibody
(1:1000) in 100 µL of PBST containing 1% BSA for 120 min. The plates were
washed five times with PBST and incubated with a horseradish peroxide-conjugated
secondary antibody for 120 min. The plates were washed as described above,
incubated with 100 µL of the TMB solution, quenched with 100 µL of 2 N sulfuric
acid, and the absorbance was measured at λ = 450 nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Evaluation of the cellular susceptibility to TNF-α proteins and neutralizing
bioactivity of benpyrine. Cytotoxicity toward L929 cells was analyzed using
previously described methods, with some modifications. A confluent monolayer of
L929 cells was trypsinized and resuspended in RPMI-1640 at a density of 1105
cells/mL. One hundred microliters of suspended cells (2105 cells/mL) were seeded in
each well of a 96-well tissue culture plate. The medium was discarded after an
overnight culture and replaced with RPMI-1640 containing various concentrations of
recombinant human TNF-α and 1 µg/mL actinomycin D. The CCK8 assay was
conducted after 24 h of culture at 37 °C. The sensitivity of L929 cells to TNF-α was
analyzed using a similar procedure without actinomycin D exposure.
In this experiment, 2.0 ng/mL TNF-α was incubated with various concentrations
of benpyrine for 2 h at 37 °C in a 96-well plate prior to the addition of 5  104 L929
cells/mL to measure the ability of the selected compounds to neutralize the bioactivity
of human TNF-α in L929 cells. The assay mixture in a total volume of 100 L was
incubated at 37 °C for 24 h. The inhibition rate of L929 proliferation was measured
18
ACS Paragon Plus Environment

Page 19 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
with a colorimetric assay using the CCK8 method. IC50 values were calculated
according to the Reed-Muench method.
8
9
Evaluation of the inhibitory effect of benpyrine on TNF-α-stimulated
activation of the NF-κB pathway. RAW264.7 cells (5×104 cells/mL) were cultured
overnight. Then, 5, 10 or 20 μM benpyrine was added and incubated for 12 h; 10
ng/mL TNF-α or 1 µg/mL LPS was added and incubated for another 2 h. Then, the
cellular proteins were extracted with RIPA lysis buffer (Beyotime, China). The
protein concentration was measured using the BCA assay (Beyotime, China). Equal
amounts of protein samples were separated by 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis and then transferred onto PVDF membranes. After
blocking with 5% non-fat milk for 2 h at room temperature, the PVDF membranes
were incubated with the primary antibodies at 4 °C overnight and subsequently with
HRP-conjugated secondary antibodies at room temperature for 2 h. The protein bands
were detected using ECL reagents. Chemiluminescent signals were detected and
analyzed using the ChemiDoc XRS imaging system (Bio-Rad, USA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RAW 264.7 cells were grown to 50–70% confluence in a glass chamber,
pretreated with DMSO or benpyrine for 12 h, and then stimulated with 10 ng/mL
TNF-α for 2 h. Cell treatments were terminated by washing the cells with PBS,
followed by fixation with freshly prepared 4% paraformaldehyde in PBS for 20 min.
The fixed cells were washed three times with PBS and then permeabilized with 0.25%
Triton X-100 in PBS for 10 min. After blocking with 2 mg/mL BSA for 1 h at room
temperature, we added the NF-κB/p65 antibody at a 1:1000 dilution at 4 °C overnight,
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 47
1
2
3
4
5
6
7
8
9
washed three times with PBS, then incubated with a 1:500 dilution of the secondary
antibody for 1 h at room temperature in dark. Finally, DAPI was used to stain the
nuclei at 37 °C for 30 min in dark. Images of the cells were captured and analyzed
using a Nikon eclipse microscope.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Acute toxicity test. Acute toxicity was tested using a variation of the method
described in a previous study.41 After a 12 h fast, Balb/c mice (18-22 g) were treated
with a dose of benpyrine (2000 mg/kg) by gavage. Animals administered 1%
CMC-Na served as controls. The animals were observed continuously for 1 h to
monitor any gross behavioral changes and death, intermittently for the next 6 h, and
then again one week after dosing with benpyrine.
Analysis of LPS-induced changes in the levels of proinflammatory cytokines.
Cytokines were assessed by measuring serum concentrations. Male Balb/c mice aged
7–8 weeks were randomly assigned into four groups: the control, LPS and benpyrine
(25 or 50 mg/kg) groups. Mice treated with the compound were orally administered
benpyrine for 90 min, and then the mice (LPS group, benpyrine-treated group) were
intraperitoneally injected with LPS (1.5 mg/kg). Mice in those groups were
euthanized and their serum was separated from clotted blood at 4 h after the
administration of LPS. Serum was stored at -80 °C, and concentrations of the
cytokines IL-1β and IL-6 were measured with a sandwich ELISA using commercially
available reagents according to the manufacturer’s instructions (Wuhan Boster
Bio-Technology). Serum samples were obtained from at least six mice in each group
and analyzed in duplicate.
20
ACS Paragon Plus Environment

Page 21 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Thirty mice were randomly divided into the following 3 groups (n=10 mice per
group) to further evaluate the effect of benpyrine on the survival of mice with
LPS-induced endotoxemia (15 mg/kg, i.p.): control (NS, i.p.), LPS (15 mg/kg, i.p.),
LPS + benpyrine (25 mg/kg, i.g.). Benpyrine (i.g.) was administrated 2 h before the
LPS injection. Saline or benpyrine was administrated 2 h before the injection of LPS
and at 12, 24, 48, 72 and 96 h after the injection of LPS. Survival was monitored daily
for up to 2 weeks. Liver and lung tissues were collected and fixed with 10% formalin,
and sections were stained with hematoxylin and eosin.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Induction of CIA and drug administration. Briefly, the normal group
consisting of 10 mice was randomly assigned before the experiment and did not
undergo immunization, while the model and treatment groups were immunized as
described below. Bovine type II collagen (Chondrex, USA) was dissolved in 0.1 M
acetic acid at a concentration of 2 mg/mL and stored at 4 °C overnight, and then it
was emulsified with an equal volume of Freund’s complete adjuvant (CFA) (Sigma,
USA) to a final concentration of 1 mg/mL. On the 1st day of the experiment, all mice
were intradermally injected with the CII emulsion at several sites at the base of the
tail, with a total volume of 0.1 mL per mouse, as the primary immunization. Two
weeks after the primary immunization (on the 14th day), the mice were challenged
again by injecting the same volume of the CII emulsion at the same location. The
mice in the benpyrine groups (i.g. 25, 50 mg/kg) and prednisone (Pro) group (i.g. 5
mg/kg) were administrated the drugs for 2 weeks.
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 47
1
2
3
4
5
6
7
8
9
Assessment of arthritis severity in mice with collagen-induced arthritis.
Arthritis severity was assessed by determining the clinical arthritis grade in all four
paws of the mice with a triple blind test. The results were assessed according to a
previously described method. Briefly, the severity was scored as follows: 0, normal; 1,
mild, apparent swelling limited to individual digits; 2, moderate, redness and swelling
of the ankle; 3, redness and swelling in the paw and in the digits; and 4, maximally
inflamed leg with the involvement of multiple joints. The arthritis score for each
mouse was the sum of arthritis severity scores recorded in all four paws, with 16
points representing the highest score.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Measurement of the spleen index and detection of serum cytokine levels. At
the end of the experiment, all mice were sacrificed by cervical dislocation after serum
samples were collected. Their spleens were weighed immediately after dissection. The
spleen indexes were calculated using the following formula: Spleen Index = Spleen
weight / Body weight. The levels of IL-1β and IL-6 in serum samples were measured
using commercially available enzyme-linked immunosorbent assay (ELISA) kits
according to the standard protocol provided with the kit.
Analysis of histopathology. For the histological analysis of knee joints, the right
hind limbs of mice were removed postmortem, fixed with 4% paraformaldehyde for
24 h, decalcified with 12.5% ethylene diamine tetraacetic acid (EDTA, pH 7.0) for 30
days, embedded in paraffin, and then sectioned at a thickness of 6–8 µm. Tissue
sections were stained with hematoxylin and eosin prior to observation with a light
microscope (Leica, DM2500, Germany). The infiltration of inflammatory cells,
22
ACS Paragon Plus Environment

Page 23 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
proliferation of synoviocytes, pannus formation, changes in the joint space, cartilage
hyperplasia and/or erosion, and bone destruction were blindly graded by a pathologist
who assigned scores ranging from 0 to 3 points based on the following criteria: 0, no
changes; 1, mild changes; 2, moderate changes; and 3, severe changes.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Imiquimod-induced psoriasis-like skin lesions and scoring of symptoms. Mice
were briefly sedated with isoflurane and an area of 5 cm × 4 cm of the back skin of
mice was shaved. Two days later, the mice in the control group were administrated a
topical treatment with a simple imiquimod base cream (i.e., vehicle), and the mice in
the benpyrine groups (i.g. 25, 50 mg/kg) and Pro group (i.g. 5 mg/kg) were
administrated the 5% imiquimod cream. Mice were scored daily based on a modified
psoriasis area and severity index scoring system, as described previously. Briefly,
erythema (redness of the skin) and scaling were scored in a blinded manner on a scale
ranging from 0 to 4 points to describe the severity of psoriasis: 0, none; 1, slight; 2,
moderate; 3, marked; and 4, very marked. Photos were taken daily to observe the
changes in the local skin. At the end-point of the experiment, all mice were
euthanized by cervical dislocation. Samples of blood, skin and spleen were collected
for future experiments. The skin samples from back lesions were collected in 10%
formalin and embedded in paraffin. For the histopathological examination, 3 µm
sections were stained with hematoxylin–eosin and observed under a light microscope
(Olympus, Tokyo, Japan). Serum were isolated after mice were euthanized. The
serum levels of IL-1β and IL-6 in mice were assayed using the ELISA kit as described
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 47
1
2
3
4
5
6
7
above. All procedures were strictly performed according to the manufacturers’
instructions.
8
9
Statistical methods. Results are presented as means ± standard deviations and
were analyzed using SPSS ver. 17.0 software. ANOVA followed by the
Student–Newman–Keuls or Wilcoxon rank sum test were utilized for the statistical
analyses. P < 0.05 was considered a statistically significant difference.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
■ AUTHOR INFORMATION
Corresponding Authors
E-mail: zhouyirong@hust.edu.cn (Y. Zhou), zhangyh@mails.tjmu.edu.cn (Y. Zhang),
syzyclx@163.com (L. Chen), and li_hua@hust.edu.cn (H. Li).
Authors’ Contributions
# Weiguang Sun, Yanli Wu and Mengzhu Zheng contributed equally to this work.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We acknowledge support from the Program for Changjiang Scholars of Ministry of
Education of the People’s Republic of China (No. T2016088), the National Science
Fund for Distinguished Young Scholars (No. 81725021), the National Natural Science
Foundation of China (Nos. 81703580, 81773637, and 81473254) and the
24
ACS Paragon Plus Environment

Page 25 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
Fundamental Research Fund for the Central Universities (HUST COVID-19 Rapid
Response Call, No. 2020kfyXGYJ037, China).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
■ ABBREVIATIONS USED
TNF-α, tumor necrosis factor α; RA, rheumatoid arthritis; TNFR, tumor necrosis
factor receptor; TACE, TNF-α converting enzyme; ICM, internal coordinate
mechanics; MST, microscale thermophoresis; DARTS, drug affinity responsive target
stability; NF-κB, nuclear factor kappa-B; LPS, lipopolysaccharide; IκBα, inhibitor of
NF-κB α; CIA, collagen-induced arthritis; ELISA, enzyme-linked immunosorbent
assay; IL-6, interleukin 6; IPI, imiquimod-induced psoriasiform inflammation
■ ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website.
Experimental details and spectra for important compounds including NMR spectra
HPLC chromatogram as well as computation results (PDF)
■ REFERENCES
1．Wajant, H.; Pfizenmaier, K.; Scheurich, P. Tumor necrosis factor signaling. Cell
Death Differ. 2003, 10, 45–65.
2．Aggarwal, B. B. Signalling pathways of the TNF superfamily: a double-edged
sword. Nat. Rev. Immunol. 2003, 3, 745–756.
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 47
1
2
3
4
5
6
7
3．Lewis; J. D. Anti-TNF antibodies for Crohn's disease--in pursuit of the perfect
clinical trial. N. Engl. J. Med. 2007, 357, 296–298.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4．Palladino, M. A.; Bahjat, F. R.; Theodorakis, E. A.; Moldawer, L. L.
Anti-TNF-alpha therapies: the next generation. Nat. Rev. Drug. Discov. 2003, 2,
736–746.
5．Chaparro, M.; Guerra, I.; Muñoz-Linares, P.; Gisbert, J. P. Systematic review:
antibodies and anti-TNF-α levels in inflammatory bowel disease. Aliment. Pharmacol.
Ther. 2012, 35, 971–986.
6．Chen, H. A.; Lin, K. C.; Chen, C. H.; Liao, H. T.; Wang, H. P.; Chang, H. N.; Tsai,
C. Y.; Chou, C. T. The effect of etanercept on anti-cyclic citrullinated peptide
antibodies and rheumatoid factor in patients with rheumatoid arthritis. Ann. Rheum.
Dis. 2006, 65, 35–39.
7．Vermeire, S.; Noman, M.; Van Assche, G.; Baert, F.; D'Haens, G.; Rutgeerts, P.
Effectiveness of concomitant immunosuppressive therapy in suppressing the
formation of antibodies to infliximab in Crohn's disease. Gut. 2007, 56, 1226–1231.
8．Alzani, R.; Corti, A.; Grazioli, L.; Cozzi, E.; Ghezzi, P.; Marcucci, F. Suramin
induces deoligomerization of human tumor necrosis factor alpha. J. Biol. Chem. 1993,
268, 12526–12529.
9．Mancini, F.; Toro, C. M.; Mabilia, M.; Giannangeli, M.; Pinza, M.; Milanese, C.
Inhibition of tumor necrosis factor-alpha (TNF-alpha)/TNF-alpha receptor binding by
structural analogues of suramin. Biochem. Pharmacol. 1999, 58, 851–859.
26
ACS Paragon Plus Environment

Page 27 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10．He, M. M.; Smith, A. S.; Oslob, J. D.; Flanagan, W. M.; Braisted, A. C.; Whitty,
A.; Cancilla, M. T.; Wang, J.; Lugovskoy, A. A.; Yoburn, J. C.; Fung, A. D.;
Farrington, G.; Eldredge, J. K.; Day, E. S.; Cruz, L. A.; Cachero, T. G.; Miller, S. K.;
Friedman, J. E.; Choong, I. C.; Cunningham, B. C. Small-molecule inhibition of
TNF-alpha. Science 2005, 310, 1022–1025.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11．Sun, H.; Yost, G. S. Metabolic activation of a novel 3-substituted
indole-containing TNF-alpha inhibitor: dehydrogenation and inactivation of CYP3A4.
Chem. Res. Toxicol. 2008, 21, 374–385.
12．Deighton, C.; Hyrich, K. International guidelines on access to biologic therapy:
why the differences and which is best?. Nat. Clin. Pract. Rheumatol. 2008, 4,
520–521.
13．Korswagen, L. A.; Bartelds, G. M.; Krieckaert, C. L.; Turkstra, F.; Nurmohamed,
M. T.; van Schaardenburg, D.; Wijbrandts, C. A.; Tak, P. P.; Lems, W. F.; Dijkmans,
B. A.; van Vugt, R. M.; Wolbink, G. J. Venous and arterial thromboembolic events in
adalimumab-treated patients with antiadalimumab antibodies: a case series and cohort
study. Arthritis. Rheum. 201, 63, 877–883.
14．McGeary, R. P.; Bennett, A. J.; Tran, Q. B.; Cosgrove, K. L.; Ross, B. P.
Suramin: clinical uses and structure-activity relationships. Mini. Rev. Med. Chem.
2008, 8, 1384–1394.
15．Seidel, S. A.; Dijkman, P. M.; Lea, W. A.; van den Bogaart,
G.; Jerabek-Willemsen, M.; Lazic, A.; Joseph, J. S.; Srinivasan, P.; Baaske,
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 47
1
2
3
4
5
6
7
8
9
P.; Simeonov, A.; Katritch, I.; Melo, F. A.; Ladbury, J. E.; Schreiber, G.; Watts,
A.; Braun, D.; Duhr, S. Microscale thermophoresis quantifies biomolecular
interactions under previously challenging conditions. Methods. 2013, 59, 301–315.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
16．Toogood, P. L. Inhibition of protein-protein association by small molecules:
approaches and progress. J. Med. Chem. 2002, 45, 1543–1558.
17．Pai, M. Y.; Lomenick, B.; Hwang, H.; Schiestl, R.; McBride, W.; Loo, J. A.;
Huang, J. Drug affinity responsive target stability (DARTS) for small-molecule target
identification. Methods. Mol. Biol. 2015, 1263, 287–298.
18．Martinez, M.D.; Jafari, R.; Ignatushchenko, M.; Seki, T.; Larsson, E.A.; Dan, C.;
Sreekumar, L.; Cao, Y.; Nordlund, P. Monitoring drug target engagement in cells and
tissues using the cellular thermal shift assay. Science 2013, 341, 84-87.
19．Chan, D. S.; Lee, H. M.; Yang, F.; Che, C. M.; Wong, C. C.; Abagyan, R.;
Leung, C. H.; Ma, D. L. Structure-based discovery of natural-product-like TNF-α
inhibitors. Angew. Chem. Int. Ed. 2010, 49, 2860–2864.
20．Hayden, M. S.; Ghosh, S. Shared principles in NF-kappaB signaling. Cell 2008,
132, 344–362.
21．Karin, M. Nuclear factor-kappaB in cancer development and progression. Nature
2006, 441, 431–436.
22．Dong, T.; Li, C.; Wang, X.; Dian, L. Y.; Zhang, X. G.; Li, L.; Chen, S.; Cao,
R.; Li, L.; Huang, N.; He, S.; Lei, X. G. Ainsliadimer A selectively inhibits IKKα/β
by covalently binding a conserved cysteine. Nat. Commun. 2015, 6, 6522.
28
ACS Paragon Plus Environment

Page 29 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
23．Mcinnes, I. B.; Schett, G. Cytokines in the pathogenesis of rheumatoid arthritis.
Nat. Rev. Immunol. 2007, 7, 429–442.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
24．Simsek, I. TNF inhibitors - new and old agents for rheumatoid arthritis. Bulletin of
the NYU Hospital for Joint Diseases. 2010, 68, 204–210.
25．Okroj, M.; Heinegård, D.; Holmdahl, R.; Blom, A. M. Rheumatoid arthritis and
the complement system. Ann. Med. 2007, 39, 517–530.
26．Vincent, F. B.; Morand, E. F.; Murphy, K.; Mackay, F.; Mariette, X.; Marcelli, C.
Antidrug antibodies (ADAb) to tumour necrosis factor (TNF)-specific neutralising
agents in chronic inflammatory diseases: a real issue, a clinical perspective. Ann.
Rheum. Dis. 2013, 72, 165–178.
27．Perera, G. K.; Di Meglio, P.; Nestle, F. O. Psoriasis. Annu. Rev. Pathol. 2012, 7,
385-422.
28．Elder, J. T.; Bruce, A. T.; Gudjonsson, J. E.; Johnston, A.; Stuart, P. E.; Tejasvi,
T.; Voorhees, J. J.; Abecasis, G. R.; Nair, R. P. Molecular dissection of psoriasis:
integrating genetics and biology. J. Invest. Dermatol. 2010, 130, 1213–1226.
29．Leonardi, C. L.; Powers, J. L.; Matheson, R. T.; Goffe, B. S.; Zitnik, R.; Wang,
A.; Gottlieb, A. B.; EtanerceptꢀPsoriasisꢀStudy Group. Etanercept as monotherapy in
patients with psoriasis. N. Engl. J. Med. 2003, 349, 2014–2022.
29
ACS Paragon Plus Environment