Structure-based discovery of a specific TLR1-TLR2 small molecule agonist from the ZINC drug library database

Article abstract of DOI:10.1039/c8cc06618c

We report herein the identification of urea structure-like small molecules by structure-based virtual screening of 10.5 million compounds. Based on a variety of HEK-Blue hTLRs reporter cell assay results, we validated a TLR1/2-specific small molecule agonist, ZINC666243 (SMU127), with EC₅₀ of 0.55 ± 0.01 μM. SMU127 stimulates NF-κB activation and promotes TNFα secretion in human macrophages and mononuclear cells. Moreover, the in vivo assay indicated that SMU127 could inhibit the growth of breast cancer tumors in BABL/c mice. This work has shown for the first time that a small molecule TLR1/2 agonist can inhibit breast cancer in vivo.

Full text of DOI:10.1039/c8cc06618c

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Structure-based discovery of a specific TLR1-TLR2 small molecule
agonist from ZINC drug library database
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
b
a
Zhipeng Chen, Xiaohong Cen, Junjie Yang, Xiaoshan Tang, Kai Cui, Kui Cheng*
DOI: 10.1039/x0xx00000x
www.rsc.org/
8
, 9
We report herein the identification of the urea structure-like small and tumor rejection. During the submission of our manuscript,
molecules from structure-based virtual screening of 10.5 million Beutler and Boger discovered a new TLR1/2 activator, Diprovocim,
compounds. Base on variety of HEK-Blue hTLRs reporter cells which was 100% cured against mouse melanoma in combination
1
0
assay results, we validated a TLR1/2-specific small molecule with the PD-L1 antibody. These potential applications and exciting
agonist ZINC666243 (SMU127) with EC50 of 0.55±0.01 μM. SMU127 results motivated us to discover and develop novel TLR2-selective
stimulates the NF-κB activation and promotes TNFα secretion in agonists. This task is particularly challenging, for TLR2 recognizes a
human macrophage and mononuclear cells. Moreover, the in vivo wide range of ligands, many of which are from Gram-positive
1
1
assay indicated that SMU127 could inhibit the breast cancer bacteria, and it signals as a heterodimer with either TLR1 or TLR6,
tumors growth in BABL/c mice. This work has shown for the first raising questions about the specificity of small molecule agents.
time that the small molecule TLR1/2 agonist can inhibit breast Second, until today, none of the TLR2 agonists in clinical or
cancer in vivo.
preclinical development are small molecules, and some contain
mixtures with more than one active ingredient. Our group has
focused on small molecules that target the TLR2 heterodimer
The ability of toll-like receptors (TLRs) to activate both immune
systems has made them desirable targets both for vaccine
adjuvants and/or cancer treatment. For example, the modified TLR4
agonist, monophosphoryl lipid A (MPLA), is used as an adjuvant in
1
2, 13
protein-protein interaction,
specifically modulating the TLR1/2
In this study we aim to discover new TLR1/2 small
molecule modulating agents through computer-aided drug design
14-16
response.
1
1
0
the FDA approved adult cervical cancer vaccine. Imiquimod (INN) is
which we have successfully employed in our previous research. In
search of small molecule probes, the 10,519,614 compounds from
a FDA approved drug which functions through TLR7 and used for
the treatment of genital warts, superficial basal cell carcinoma and
O
O
S
O
O
2
N
S
NH
S
actinic keratosis. Other TLR agonists are also being investigated for
NH
NH
NH
O
NH
S
F
NH
O
S
NH
vaccine adjuvant uses including the TLR9 agonist CpG-ODN in HBV
vaccines, TLR2 agonist (rLP208-A05 and rLP2086-B01) in the FDA-
O
S
F
ZINC13933877
ZINC4113504
ZINC13812294
ZINC6283936
3
approved Trumenba vaccine.
O
O
O
O
O
O
O
O
Recently, a TLR2 agonist, polysaccharide krestin (PSK), extracted
from mushroom is being tested in a clinical phase II trial now for the
NH
NH
NH
NH
S
S
N
N
S
NH
S
NH
O
O
S
O
4
treatment of breast cancer. Accumulated evidences have proven
ZINC695420
ZINC1927525
ZINC6662436
ZINC758177890
5
, 6
that TLR2 agonists could be used directly,
or as structurally
O
O
O
O
7
O
O
O
modified forms in cancer treatment, suggesting that TLR2 agonists
can be potential effective enhancers for cancer immunotherapies.
Administration of TLR2 agonists could enhance effector and
memory T cell responses, leading to elevate efficacy of vaccination
NH
H
N
NH
O
O
NH
S
N
NH
NH
S
S
S
O
O
ZINC146117
ZINC9013548
ZINC1200091564
ZINC442271
O
O
CF
3
O
O
O
N
NH
S
NH
H
N
H
N
S
N
Ph
ZINC939403
N
O
O
O
O
N
N
HN
Ph
O
NH
HN
O
Ph
HN
Ph
S
O
2
O
ZINC45194
CU-T12-9
Diprovocim
1
0
Scheme. 1 Chemical structure of the 14 top hits, Diprovocim
1
3
and CU-T12-9
.
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(
Scheme 1), and then verified their activity in vitro. Interestingly,
almost all of the hits identified, with ZINC6662436 as
a
representative structure, from the in silico screening generally share
the common motif of an amine conjugated with an acid
(
isocyanic/isothiocyanic/carboxylic) substituent, implying a novel
pharmacophore to target the Pam CSK -binding site of TLR1/2.
3
4
These initial hits were first evaluated using our previously
established cell assay of TLR2 activation. HEK-Blue hTLR2 cells
were employed to selectively activate TLR2 signaling, through
the adaptor proteins MyD88/TRAF/IKK NF-κB pathway to
active the NF-κB signaling. This triggers the secreted alkaline
Fig. 1. Comparison of the initial hits in the TLR2 related phosphatase (SEAP) promoter to secrete alkaline phosphatase,
or induces production of pro-inflammatory cytokines. We
monitored the SEAP level as an indicator of small molecule-
induced TLR2 activation to evaluate the compound’s activity
secreted alkaline phosphatase (SEAP) gene activation. HEK-
Blue hTLR2 cells were incubated with indicated concentrations
(0-100μM) of the initial hits for 24 hours, and the activation
(
Fig. 1). Fortunately, two of fourteen compounds
was measured using Quanti-Blue reagent by determining
alkaline phosphatase secretion in the culture supernatants at
A620. Data presented are mean ± SD and the figures shown
are representative of three independent experiments.
demonstrated relatively higher TLR2 activation effects in the
whole cell assay (Scheme 1 and Fig. 1), with ZINC6662436
(SMU127) being particularly excellent (EC50 at 0.55± 0.02 μM).
Therefore, SMU127 was selected out and synthesized (Scheme
S1, Fig. S3-S6) for further target validation and potency
evaluation.
To test whether the SMU127 can specifically activate the TLR2
signaling pathway, HEK-Blue hTLRs cells transfected with SEAP
reporter genes were used, including hTLR3, 4, 7 and 8. Cell
culture supernatant was collected to test the induction of NF-
κB signaling pathway. The representative dose-dependent
curve showed SMU127 had similar activation mode as the
well-studied TLR1/2 agonist Pam CSK . As shown in Figure 2B
3
4
to 2F, SMU127 specifically activates TLR2 rather than other
TLRs, including TLR3, TLR4, TLR5, TLR7 and 8. Next, we tested
whether SMU127 binds to TLR2 on the cell surface and induces
TLR2 expression. HEK-Blue hTLR2 cells, which overexpress the
human TLR2, were stimulated with SMU127. Cell lysates were
subjected to immunoprecipitation and immuno-detected using
TLR2 antibody. As shown in the Figure 3A, as the dose of
SMU127 increased, the expression of TLR2 was gradually
increased, showing that SMU127 interacts with TLR2 in a
whole-cell environment. HEK-Blue hTLR2 cells can
endogenously express TLR1 and TLR6, therefore whether
SMU127 is an activator of TLR1/2 or TLR2/6 still remains
unclear. Here, we performed an antibody inhibition
experiment to test its selectivity. SMU127 can efficiently
activate SEAP signaling at the concentration of 0.5 µM, and
addition of anti-hTLR1 and anti-hTLR2 but not anti-hTLR6
antibodies changed such activation in a dose-dependent way
Fig. 2. Specificity test for SMU127 in different HEK-Blue
hTLRs. (A) SMU127 stimulates signaling of SEAP in a dose-
dependent manner. (B to F) HEK-Blue Human TLR3 (B), TLR4
(C), TLR5 (D), TLR7 (E), and TLR8 (F) cells were incubated with
SMU127 (0 to 100 μM) with the control of TLR-specific
agonists separately for 24 hours, and activation was evaluated
by the optical density at 620nm. Agonist: TLR3, poly(I:C) (5
μg/ml); TLR4, LPS (10 ng/ml); TLR5, FLA-BS (5 μg/ml); TLR7 and
TLR8, R848 (4 μg/ml). Data presented are mean ± SD and the
figures shown are representative of three independent
experiments.
(
Fig. 3B). To confirm whether the anti-hTLR6 antibodies are
functional, we used a TLR2/6 agonist Pam CSK , and tested the
SEAP activation in the same cell line, and found that both anti-
4
hTLR2 and anti-hTLR6 antibodies can restrain Pam CSK -
2
4
2
induced SEAP signaling, whereas anti-hTLR1 antibodies did not
block the activity (Fig. 3C). Moreover, one oxygen and two
nitrogen form urea skeleton of SMU127 have very distinct
hydrogen bonding with the key amino acid residues Phe312,
3 4
ZINC database was screened against the Pam CSK -binding domain
1
1
of TLR1/2 (crystal structure PDB: 2Z7X) using the Glide 7.4
program (Fig. S1 and S2). Base on the computer scoring results,
including shape, chemical-feature, drug-like properties, 14
compounds were finally selected out and purchased from ZINC
11
Gln316 and Gly313 in TLR1 , and hydrophobic interaction
between cyclopentathiophene ring and Phe349 in TLR2 also
2
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been observed (Fig. 3C). These observations further confirmed
that SMU127 acts as an agonist of TLR1/2.
Fig. 4. SMU127 works through NF-κB pathway to activate the
downstream signaling. Representative flow graphs of the
TLR2-sensitive U937 human macrophage cell line with a GFP-
labeled NF-κB reporter treated with (A) 0, (B) 0.1, (C) 1, (D) 10,
Fig. 3. SMU127 was proved to be specific ligand of TLR1/2,
not TLR2/6. (A) SMU127 promoted the TLR2 protein
expression in HEK-Blue hTLR2 cells. (B) HEK-Blue hTLR2 cells
were treated with SMU127 and antibodies of anti-hTLR1, anti-
hTLR2, or anti-hTLR6 for 24 hours. Anti-hTLR1-IgG and anti-
hTLR2-IgA antibodies can inhibit SMU127-triggered SEAP
signaling on a dose-dependently manner, whereas anti-hTLR6-
IgG has no influence to the SEAP signaling. (C) The positive
control of Pam CSK , a TLR2/6 agonist, showed different
3 4
and (E) 100 ng/mL Pam CSK for 24 hours before
determination by flow cytometry. (F) Summarized the results
from A to E, and the result demonstrated SMU127 had
excellent NF-κB activation compared with the positive control
3 4
Pam CSK . (G) The NF-κB activation of SMU127 can be
inhibited by a NF-κB inhibitor, triptolide, in HEK-Blue hTLR2
cells. (H) SMU127 triggers TNF-α production after the TLR1/2
activation in human peripheral blood mononuclear cells
2
4
responses to TLR1, 2 and 6 antibodies comparing with
SMU127. Data are means ± SD of triplicate and representative
of three independent experiments. (D) Low-energy binding
conformations of SMU127 and Pam CSK bound to the TLR1–
(PBMC). Data are means ± SD of triplicate and representative
of three independent experiments. *P<0.01.
3
4
Previous reports suggest that TLR1/2 agonist BLP (bacterial
lipoprotein) can effectively inhibit lung cancer, leukemia,
melanoma, and lead to a long-lasting protective response
TLR2 interface as generated by virtual ligand docking.
In order to investigate the cellular and molecular mechanisms
of SMU127 with NF-κB, we developed a TLR2-sensitive U937
human macrophage cell line with a GFP-labeled NF-κB
reporter. Flow cytometry experiments demonstrated that
SMU127 activated NF-κB signaling in a dose-dependent
manner. At the dose of 1 μM, SMU127 showed comparable
activation to 100 ng/ml of Pam CSK , and at a higher dose (10
1
7
against tumor rechallenge. Moreover, TLR1/2's agonist
polysaccharide krestin (PSK) has shown amazing effects in the
inhibition of both implanted and spontaneous breast tumors
4
and is already in phase II clinical trial. SMU127 has a good
TLR1/2 activation effect at the cellular level, and does it also
have antitumor activity in vivo? In this report, we evaluated its
anti-breast cancer activity in the BABL/c mice model. The
results indicated that SMU127 decreased the progression
speed of breast cancer volume by compared with PBS as blank
control in BABL/c mice. Generally, groups of female BABL/c
3
4
μM) even had a better effect (Fig. 4A-4F). To investigate the
cell signaling of SMU127 induced activation, we employed a
known NF-κB inhibitor, triptolide. Triptolide was able to
efficiently inhibit the SMU127 induced SEAP signaling (Fig. 4G),
which further implies that SMU127 works through the NF-κB
signaling pathway. A key output from this activation is tumor
necrosis factor-alpha (TNF-α), which is proven to be directly
relevant to inflammatory diseases and cancer. To ascertain
whether the NF-κB activity induced by SMU127 reflected
upregulation of TNF-α, we assessed the ability of SMU127 to
activate TNF-α in human PBMC using an ELISA experiment. The
experiment showed SMU127 possesses the ability to activate
TNF-α signaling in a dose-dependent manner (Fig.4H).
mice were implanted with syngeneic 4T1 breast cancer cells (3
5
10 cells/mice, ~100 µl in PBS); on day 7 when tumors were
×
palpable, mice were treated with intraperitoneal injection of
SMU127 (0.1 mg per mice) or PBS and repeated every two
days. Tumor size and survival in these mice were monitored
regularly. As shown in Figure 5, SMU127 treatment
significantly inhibits the growth of breast tumor in BABL/c
mice. The tumor size after 24 days of treatment was 1035±196
3
3
mm in the SMU127 group and 2256±150 mm in the PBS
group. In addition, treatment with SMU127 significantly
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reduced the tumor volume and weight (Figure 5A, 5C, 5D), but potential therapeutic applications in vaccine adjuvants and
tumor immunity therapies.
no reduction in body weight as compared with the control
Figure 5B). Although SMU127, as the first TLR1/2 small
This work was supported by National Natural Science
Foundation of China (No. 81773558), start-up support in
Southern Medical University of China (No. C1033269), and
Youth Pearl River Scholar Program of Guangdong Province (No.
C1034007).
(
molecule ligand that inhibits breast cancer in vivo, there is still
a clear room for further efficacy improvement. In view of the
synergistic anti-melanoma effect of TLR1/2 activator
1
0
Diprovocim and PD-L1 antibody, combined SMU127 with PD-
L1 antibody or other immunological checkpoint inhibitors may
be a new direction to improve the tumor immunity. During the
tumor immunization, IL-10 needs to be considered for it will
Conflicts of interest
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5
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4
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