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Discovering Therapeutic agonists to stimulate TLR3-mediated
signaling in regeneration

Yukyung Park¹, Jiyeon Moon¹, Wooyoung Lee¹, Minji Kim¹, Garyung Kwon¹, Mina Ma¹, Dongwon Kim¹*
1 Bio-chemical Engineering, Dongseo University, Busan 47011, Korea
(** Presenting Author : Yukyung Park , *Corresponding Author: dwkim@dongseo.ac.kr)
¹
Introduction Results

Toll-Like-Receptor 3 (TLR3) is a family of Toll-Like-Receptor (TLR) A 8
which recognize pathogen associated molecular patterns (PAMPs)
from microbes and its associated Innate immune response. A Recent 6
study demonstrated that damaged tissue induced noncoding dsRNA 4
which activates TLR3 and promotes retinoic acid in the body. this TLR3-mRNA 0 (Fold increase) 2
mechanism induces skin regeneration. ¹˒² There is the only compound
Poly I:C to stimulate TLR3, but it has severe cytotoxicity because of CON PIC 3A 3B 3C 3D 3E 3F 3G 3H 4A 4B 4C 4D 4E 4F 4G 4H 5A 5B 5C 5D 5E 5F 5G 5H
inaccurate structural. To solve this problem, it is important to discover B 10
other agonists to stimulate TLR3 through chemical compound libraries.
Developed biologically active substances can promote skin KRT15-mRNA (Fold increase) 5
regeneration also it may offer new therapy for skin diseases in
intractable patients who can not get operation. 0 PIC 3A 3B 3C 3D 3E 3F 3G 3H 4A 4B 4C 4D 4E 4F 4G 4H 5A 5B 5C 5D 5E 5F 5G 5H
Representation of Wound-Induced CON
Hair Neogenesis (WIHN). Damaged C 10
tissues release dsRNA to activate TLR3
signaling. Further, it activates KRT19-mRNA (Fold increase) 5
endogenous RA synthesis and signaling
to regulate regeneration 0 PIC 3A 3B 3C 3D 3E 3F 3G 3H 4A 4B 4C 4D 4E 4F 4G 4H 5A 5B 5C 5D 5E 5F 5G 5H
CON
Result Goal Figure 1. (A) Effect of 24 compounds on expression of TLR3. After treatment for 48h.
(B) and (C) Effect of 24 compounds on expression of KRT15 and 19. After treatment
Poly I:C mediated TLR3 signaling but it occurs cytotoxicity. In this
for 48h. KRT15 and KRT19 are biomarker of the hair follicle stem cells. Three
study, we wonder not only how new lead compound stimulate biomarkers were evaluated by qRT-PCR in Normal Human Epidermal Keratinocytes
TLR3-signalling, but also TLR3-mediated signaling helpful skin (NHEK) stimulated with Poly I:C (1µg/ml) and same concentration 5µM of 24
regeneration without cytotoxicity. compounds. Each figure compared with the DMSO 5µM treated control set. Results
Poly I:C shown are averages of three independent experiments.

Skin
regeneration
D
Table 2. EC50 value on 3H (235-G 11) using Cell-based readout system.
E
Methods 200 40
35
KRT 10 mRNA (Fold increase) 100 KRT 1 mRNA (Fold increase) 20
25
In vitro validation – Cell based readout system 150 30
15
50
10
5
0 0
CON PIC 3H CON PIC 3H
Figure 2. (D) and (E) effect of 3H(235-G 11) on expression of KRT1 and KRT10.
SEAP Measurement After treatment for 48h. KRT1 and KRT10 are biomarker of differentiation.
Results shown are averages of two independent experiments.
In vitro experiments – Gene expression qRT-PCR
Conclusions
DMSO, Poly I:C and
24 lead compounds
1. We used a cell-based readout system to screen TLR3 agonists.
2. selected 24 compounds were treated on NHEK cells, the
Results discovery of compound 235-G 11 as a TLR3 agonist.
3. 235G- 11 also potent agonist to expression about
Chemical libraries screening differentiation markers.
The human embryonic kidney 293 hTLR3 4. we are able to develop the potential therapeutic agonist to
(HEK293-hTLR3) cell lines are designed to regeneration skin and hair follicles.
study stimulation of human TLR3 by
monitoring the activation of NF-kB.
HEK293-hTLR cell lines were introduced References
with a transcription-controlled Secreted
embryonic alkaline phosphatase (SEAP) 1. Nelson et al. (2016). Cell Stem Cell. 17(2):139-151
reporter gene placed under the control of
an NF-KB promoter. Cells were treated by 2. Kim et al. (2019). Nat Commun. 10:2811
25 µM of each library compound, DMSO
was used as the negative control and Poly Funding
I:C was used as the positive control. 6,696
chemical compounds were treated on
HEK293-hTLR3 cell lines, SEAP secretion 1. Laboratory specific start-up leading university project
measured using a Microplate reader. 24 2. The chemical library used in this study was kindly provided by
compounds with more than half the effect Korea Chemical Bank (www.chembank.org) of Korea Research
compared with the positive controls were Table 1. EC50 value about chemical Compounds Institute of Chemical Technology.
the high rank group using Cell-based readout
selected.
system. 3. Supported by BB21+ project in 2021