This course required us to present an article which prof gave us randomly. And my article is a review paper related to TLR signaling! I upload here just hope that it can be useful for someone who is interested in this approach for studyding TLR signaling dynamics based on Synthetic ligands! Many thanks for your look at my presentation and leave some comments if I got mistakes inside!

2. • Toll-like receptors (TLRs): key family of the
innate immune system which recognizes
invading pathogens through pattern-
recognition receptors (PRRs) based on
pathogen-associated molecular patterns
(PAMPs).
• Domains: N-terminal ectodomain, single
transmembrane, C-terminal cytosolic Toll-
interleukin-1 receptor (TIR)
• Classification: based on predominant
cellular location; extracellular (TLR1, 2, 4, 6)
and intracellular (TLR3, 5, 7, 8, 9)
Type of TLR Specific binding
TLR2, TLR4 Bacterial Cell wall
components
TLR5 Flaggellin
TLR3 Viral double-stranded
RNA
TLR7, TLR8 Viral single-stranded
RNA
TLR9 Unmethylated DNA
containing CpG motif
Innate Immune Signaling via Toll-like Receptors
2

3. • Immune mechanisim: TLRs binding to conserved microbial structures (such as cell
wall components)
 Induction of a variety of signaling pathways
 Induction of pro-inflammatory cytokines and type I interferons (IFN-I)
Innate Immune Signaling via Toll-like Receptors
Pro-inflammatory
cytokine
Type I interferon (IFN-I)
- Mediated through
NF-κB
(transcription
factor nuclear
factor κB)
- Induce maturation
of innate immune
cells
 Adaptive immune
responses
- Induced by interferon
regulatory transcription
factor (IRF)
- Interacts with IFN-α/β
receptors in autocrine +
paracrine manner
 Inducing the
transcription of
hundreds of IFN-
stimulated genes (ISGs)
 Antimicrobial activities

4. Synthetic ligands
• Helped to eclucidated TLRs function
 Ligands served as activation for TLRs signaling in immune systems. (Important!)
• Developing defined synthetic ligands
 Specific TLRs could be activated selectively
• Chemistry is combined with structural biology
 Clarify specific interactions between TLRs and adaptors in signaling
4

5. Synthetic ligands
 Pam3CSK4
• Lipopeptide acts as lipoprotein mimic in activating TLR2/1
signaling pathways
• Still large molecule containing 3 highly lipophilic tails + 6 a.a
• Palmitoyl tail on N-terminus of cysteine
 Dispensable moiety for activation
 Removal of this tail makes highly potent Pam2CSK4
 Pam2CSK4
5

6. Synthetic ligands
Figure 2. Structures and Relative
Activities of Synthetic TLR2 Ligands
Depicted are TLR2 agonists that have
resulted from several SAR studies. These
studies aimed to simplify the ligand
structure, while maintaining their
activity and increasing their solubility in
aqueous medium. Highlighted in red are
alterations applied within the molecule.
Relative activities (R.A.) are based on
EC50 and IC50 values as reported in the
biological evaluation of the respective
compound in the cited study are 1, 2>3 ,
4, 7, 9 > 8 >> 6; relative activity of 5 is
unknown
- Using Pam2CSK4 as a benchmark, several Structure-activity relationship (SAR) studies were conducted
with Cys-Ser lipodipeptide (3) as the minimal structure for TLR2 activation
6

7. (S)
Palmytoyl tail
Cys-Ser
Cys-Gly
Reduce activities Ether bridge => reduce 8 times magnitude
No difference
Remove palmitoyl ester and methylene
=> Monoacylated PamCS methyl ester lipopeptide
=> Inactive in murine, but human
Small enhance
Pam2CSK4
Pam3CSK4
Synthetic ligands-SAR studies
Little impact
7

8. Using Synthetic ligands to Elucidate TLR Signaling
• Using ligands as to discover binding sites between TLRs and ligands which server as
initiating signal or signaling complexes
 Studying signaling pathway through interactions or stimulation
 Developing signaling capacity of TLRs through ligands for applications in pharmaceutical field
in the future
• Disadvantages in elucidating TLRs signaling
- Antibodies against many TLRs is lacking
- The delicacy of TLRs signaling results in cell type-specific outcomes
 Difficult to access (TLRs activation should address in their native cellular context)
• However, 2 subsets of dendritic cells (DCs) ( cell type-specific of TLRs activation) have
resonses to synthetic TLR ligands
- Plasmacytoid DCs (pDCs) produces large amount of INF-I in response to TLR7 and TLR9
ligands
- Conventional DCs (cDCs) mainly produce pro-inflammatory cytokines
• Detailed characterization of the synthetic ligands is utmost importance, as small changes in
ligand structures can affect the interaction with TLRs
 Take caution when translating functional results with synthetic TLR ligands to a more nature
mode of activation
• Small-molecule imidazoquinoline ligands and natural single-stranded RNA
ligands occupy different binding pockets within TLR7 and TLR8
 Different functional outcomes
8

9. Using Synthetic ligands to Elucidate TLR Signaling
• Crystal structure of the imidazoquinoline ligand
resiquimod in complex with human TLR8
• In solution, recombinant TLR8 ptoteins exist as
dimers inactive form. Ligand binding induces
changes of conformation leading to shortening of
the gap between C terminus from 53Å to 30Å
 Dimerization of TIR domains
 Downstream signaling
 Explain: Hydrophilic interaction with carboxylate
functionality in the side chain of an aspartic acid
residue (TLR8), which form hydrogen bonds with
the amidine functionality of the ligand in almost
perfect geometry
• In recent years, chemists have developed more
advanced chemical tools based on synthetic TLR
ligands
• Bi- and tri-functional TLR ligands
 Induce ligation of multiple TLRs near plasma
memberane
9

10. Surface-Expressed TLRs Induce NF-κB and IRF Activation
from Different Locations within Cells
translocation
• TLR4’s capacity to signal from endosomes was identified using chemical inhibitors of clathrin-mediated
endocytosis; dynasore and chlorpromazine; selectively inhibited the synthesis of IFN-I, not pro-
inflammatory cytokines
• Translocation of ligand-engaged TLR4 from cell surface to endosomes actually induced by accessory
protein, dynamics of these processes is difficult to visualize
 Microscopes have enabled mobility studies of TLR4 and its accessory and adaptor proteins through
fluorescence recovery after photo-bleaching experiments 10

11. Surface-Expressed TLRs Induce NF-κB and IRF Activation
from Different Locations within Cells
• In summary, both TLR4 and TLR2 assemble
distinct signaling platforms at plasma
membrane and endosomal vesicles
 Induce the activation of NF-KB and members of
IRF family
• Controlled translocation of TLR4
 Critical for shaping the induced innate response
 Facilitate studying processes regulate TLR2-
induced responses in the future
• Potential candidates include accessory for TLR2
ligand binding: CD36, CD14, and mannose
binding lectin
11

12. Photocaged TLR2 and TLR4 Ligands as Tools to Study Signaling Dynamics
• Photocaging: the protection of ligands to a non-binding state using photolabile protecting
group
• TLR2, TLR4 ligands yielding tools by which TLR activation can be conditionally controlled using
light
According to SAR studies of TLR2
ligands Pam2CSK4
• Substitution at N terminus
cysteine renders the ligand
inactivate
 Photocageing attatched this site
of Pam2CSK4
• Moreover, pure steric hindrance of the
protecting group present on cysteine amine
 Hydrophobic moiety capable of interacting with
hydrophobic wall, potentially preventing the
LRR11 loop shift from occurring  absence of
hydrogen bond from proximity F319
 Increase interaction of ligands to only TLR2
12

13. Photocaged TLR2 and TLR4 Ligands as Tools to Study Signaling Dynamics
• Ortho-nitrobenzyl (NB)-derived
photocleavable group
• Incubation TLR2-expressing cells with
photocaged TLR2 ligand
 Pre-existing pool of ligand-bound
TLR2 molecules without activation,
distribution on both plasma
membrane and endosomes
 Induce nuclear translocation of p65,
subunit of NF-KB and release TNF-α
after UV irradiation
• Based on synthetic agonist pyrimido[5,4-b]-indole by
alkylating critical nitrogen with nitroveratryloxycarbonyl
(NVOC)-group
• Using UV/Vis spectroscopy and HPLC analysis,
conversion into free TLR4 ligand following UV
irradiation was detected
• Induce p65 nuclear translocation and NF-KB-dependent
transcriptional activities only after UV expose
 NPPOC
 NVOC
• Improve NB derivatives backward: low
efficiency for TPE (Two Photon Excitation)
 Enhance efficiency by increasing size of
conjugated system, the amount of strong
donor/acceptor couples, etc
 Nitrodibenzofuran (NDBF)
• Photolyzes 16~160 times faster than NB
derivatives
 NDBF
13

14. The Dynamics of Intracellular NA-sensing TLRs
• TLR7, TLR8, and TLR9 induce signaling pathways
that activate NF-KB and IRF7 from functionally
distinct intracellular vesicles
• Synthetic TLR9 ligands: CpG-A and CpG-B
oligodeoxynucleotides (ODNs)
 In mouse, pDCs produce IFN-I in response to
multimeric CpG-A, monomeric CpG-B only induce
pro-inflammatory cytokines
 Immune outputs rely on the distinct intracellular
trafficking properties of ligands
• In pDCs CpG-A was specifically retained in early
endosomes for a prolonged time while CpG-B
rapidly translocated to late endosomes and
lysosomes
• In cDCs, CpG-A and CpG-B both ligands induce
pro-inflammatory cytokines
• While not normally released by cDCs, IFN-I are
produced in reponses to CpG-A complexed with
liposomal transfection agent DOTAP which
enhances endosomal retention of CpG-A
 Assembly of signaling platform capable of
activating IRF7 14

15. Caging Strategies for NA-sensing TLRs
• Conditionally control the activation of NA-sensing TLRs using UV light as external trigger
encompass the development of photocaged ligand for TLR7, TLR8 and TLR9
• Based on crystallographic data, C4 amine of
the dual TLR7 andTLR8 agonist resiquimod
can be critical for its activation
 NPPOC group was introduced at this position,
rendering ligan inactive
 A mouse macrophage cell line with NF-KB
reporter (RAW-blue) confirming production
of UV-irradiated NPPOC-resiquimod induced
TLR7, TLR8 activation
• Bone marrow-derived DCs (BMDCs) treated with photocaged ligands secreted the cytokines
IL-6, IL-12 ND TNF-a only after UV irradiation
• For TLR9, several CpG ODNs synthesized carrying photocage nitropiperonyloxymethyl, by
incorporating pre-protected thymidine phosphotamidite building blocks. These thymidine
residues either distributed over ODN or concentrated at both termini
 Both caging strategies prevented activation of TLR9
 Photoactivation for ODN was observed by measuring IL-6
 Central section of ligand is able to bind TLR9 without inducing activation before UV irradiation15

16. Outlook
 The previous static model of TLR signaling has undergone major
revisions in recent years
• TLR signaling is regulated dynamically within cells
• New reagents, techniques, and chemistries are appearing fast
 Spatial and temporal control over TLR activation
• These chemicals are expected to become valuable tools for complex dynamic
nature of TLR signaling
• Controlled activation of TLRs is essential for potent antimicrobial immune
responses. At the same time, undesired TLR signaling may induce deleterious
immune responses in the case of misregulation or overactivation toward
infection, or auto-immunity toward self-agonists.
 A better understanding of the dynamic cellular processes that regulate TLR
signaling will guide the rational design of novel therapeutics to effectively
prevent these instances of immune pathogenesis
16

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18
Two-photon excitation microscopy is
a fluorescence imaging technique that allows
imaging of living tissue up to about one
millimeter in depth. It differs from
traditional fluorescence microscopy, in which the
excitation wavelength is shorter than the emission
wavelength, as the wavelengths of the two
exciting photons are longer than the wavelength
of the resulting emitted light. Two-photon
excitation microscopy typically uses near-infrared
excitation light which can also excite fluorescent
dyes. However, for each excitation, two photons
of infrared light are absorbed. Using infrared light
minimizes scattering in the tissue. Due to the
multiphoton absorption, the background signal is
strongly suppressed. Both effects lead to an
increased penetration depth for these
microscopes. Two-photon excitation can be a
superior alternative to confocal microscopy due to
its deeper tissue penetration, efficient light
detection, and reduced phototoxicity.[1]
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