1. TOLL-LIKE RECEPTORS SIGNALING PATHWAY:
Alma Mater Studiorum – University of Bologna
Department of Pharmcy and Biotecnology (FaBiT)
TRAINING LABORATORIES
TOLL-LIKE RECEPTORS SIGNALING PATHWAY:
DUAL-LUCIFERASE APPROACH IN THE DISCOVERY OF NOVEL THERAPEUTIC OPPORTUNITIES
Dr. Andrea Bedini

2. TOLL-LIKE RECEPTORS: CRITICAL IMMUNE SENSORS
Immediate first line of defense against a diverse repertoire of invading microbial pathogens
TLRs ability to engage different intracellular signalling molecules and cross-talk with other regulatory pathways is crucial in shaping TYPE, MAGNITUDE, DURATION of inflammatory response

3. TOLL-LIKE RECEPTORS: STRUCTURAL FEATURES
TOLL-LIKE RECEPTORS (TLRs):
Type I integral transmembrane glycoproteins
Extracellular Leucine-rich Repeats
Toll/IL-1 receptor (TIR) signalling domains
To date more than a dozen of TLRs
TLR 1 -9 conserved among humans and mice
TLR10 selectively expressed in humans

4. TOLL-LIKE RECEPTORS IN HEALTH AND DISEASE
TLRs recognize conserved structures of microbes and endogenous (host-derived) molecules
TLRs = PPR pattern recognition receptor
TLRs act as SENTINELS against a wide range of Pathogens-Associated Molecular Patterns (PAMPs) and Danger-Associated Molecuar Pattern molecules (DAMPs)
TLRs are both KEY HOST DEFENCE MECHANISMS and RAPID RESPONSE MECHANISM TO LOCAL TISSUE DAMAGE
WIDE RANGING IMPACT ON SEVERAL DISEASE SETTINGS

5. TOLL-LIKE RECEPTORS: CELLULAR LOCALIZATION AND LIGAND SELECTIVITY
TLRs are located on the outer cell membrane or on endosomes
Each TLR detect a specific set of ligands

6. TOLL-LIKE RECEPTORS: INTRACELLULAR SIGNALING PATHWAYS
All TLRs mediate the production of inflammatory cytokines
TLR3, TLR4, TLR7, TLR8 and TLR9 stimulate the production of type I Interferons

7. TOLL-LIKE RECEPTORS: LIGAND RECEPTOR INTERACTIONS

8. TARGETING TOLL-LIKE RECEPTORS: NOVEL POTENTIAL THERAPEUTICS
INFECTION, INFLAMMATION, AUTOIMMUNE DISEASES AND CANCER involve complex signalling pathways that contain several possible drug targets
Despite this complexity it has proven possible to target A SINGLE PROTEIN (e.g.: TNFa) and obtain a SIGNIFICANT THERAPEUTIC EFFECT!
Toll-like RECEPTORS

9. TARGETING TOLL-LIKE RECEPTORS: NOVEL POTENTIAL THERAPEUTICS
TOLL-LIKE RECEPTORS (TLRs) fullfill many of the criteria that are required to be considered potential THERAPEUTIC TARGETS
TLRs are over-expressed in several diseases
TLRs knock-out mice are RESISTANT to disease in disease models
Genetic differences in TLRs or their SIGNALLING PROTEINS correlate with increased risk of disease
Many polymorphisms in genes that encode TLRs and their signaling molecules have been associated with human disease progression and susceptibility
Hennessey EJ et al. Nature Reviews Drug Discovery 2010

10. TARGETING TOLL-LIKE RECEPTORS:
WHEN COULD IT BE USEFUL?
TLRs activation promotes maturation of APCs which in turn direct the induction of adaptive immune responses => TLRs AGONISTS (cancer, infections)
TLRs activation promotes inflammatory cytokines production and play a pathogenic role in many diseases with inflammatory basis => TLRs ANTAGONISTS (asthma, atherosclerosis, multiple sclerosis, rheumatoid arthritis, systemic lupus eritematosus, diabetes)
TLRs activation is reported in different pahtological states within CNS => MODULATION OF TLRs (opioid-resistant chronic pain, neurodegenerative diseases)
Xenobiotics and drugs may mis-activate TLRs => ADVERSE EFFECTS (Opioids, Antidepressants) 10

11. TLR AGONISTS AND CANCER: Drugs presently under investigation11

12. TLR AGONISTS AND CANCER: Perspectives and drawbacks
POLY-TLR AGONISTS
Cadi-05 (autoclaved mycobacteria targeting several TLRs) is being evaluated for the treatment of advanced melanoma, prostate and bladder cancer => in phase I trial 5/9 became asymptomatic and showed no disease recurrence at 2 years
Although there is empirical evidence for a given TLR as a good target for cancer therapy, a compelling rationale is often lacking in human clinical trials. Many cancer patients are immunosuppressed due to traditional antitumour therapies, and it is difficult to produce a strong positive innate immune response. Nevertheless, there is optimism that the combination of innate immune stimulatory compounds and anticancer agents will ultimately lead to a successful anticancer therapy.
LACKING OF COMPELLING RATIONALE IN HUMAN CLINICAL TRIALS
MANY CANCER PATIENTS ARE IMMUNOSUPPRESSED 12

13. GREAT POTENTIAL OF TLRs AGONISTS COMBINED TO STANDARD THERAPIES IN THE TREATMENT OF INFECTIONS13

14. Inappropriately overactive immune system
TLR ANTAGONISTS
Inappropriately overactive immune system
TLR ANTAGONISTS
HCQ (TLR9 antagonist, TLR7 and TLR8 weak antagonist) is the current treatment for SLE
CPG (TLR7, TLR8, TLR9 antagonist) is being evaluated in a Phase I clinical trial
TLR7 – TLR9 dual antagonists are being developed for the treatment of SLE, rheumatoid arthritis, multiple sclerosis, colitis, psoriasis.
Eritoran (TLR4 antagonist) decreased by 6.4% the mortality due to sepsis in a Phase II clinical trial; Phase III is presently under initiation
Ibudilast (TLR4 antagonist) is being studied in advanced clinical trials for the treatment of chronic pain states and addiction withdrawal and TLR4 antagonistic antibodies are very promising anti-inflammatory drugs in animal models of colitis 14

15. TOLL-LIKE RECEPTORS AND CENTRAL NERVOUS SYSTEM
TLRs are differentially expressed by the various cell type within the CNS
TLRs in fact are differentially expressed by the various cell types that constitute CNS 15

16. NEURON-TO-GLIA SIGNALS IN NEUROPATHIC PAIN:
Maladaptive plasticity within the nociceptive system
Periferal tissue or nerve injury
Cytokines, chemokines, neurotrasmitters, neuromodulators, alarmins, xenobiotics released by neurons
Cross-talk neuron-glia
Pro-inflammatory cytokines
(IL-1, TNF, IL-6), chemokines, prostaglandins, ATP, NO…
Elevated neuron excitability, pain enhancement
CHRONIC PAIN
Maladaptive plasticity and activation of glial cells
Neuropathic pain originates from peripheral tissue or nerve fiber injury which induce neurons to release neuromodulators and neurotransmitters that can be detected as “endogenous danger signal” by glia (activate astrocytes and microglia). Under such conditions, subsequent glial responses to classic neurotransmitters and glial–neuronal interactions are altered.
On release of these danger signals, the innate immune pattern recognition receptors TLR4 and TLR2 detect the presence of the danger signal resulting in the activation of TLR-expressing cells.
Upon activation, glia release a variety of neuroexcitatory, pain-enhancing substances, key amongst these being proinflammatory cytokines.
These altered cellular dynamics significantly contribute to the development of chronic pain and to the ineffectiveness of analgesics (such as opioids); therefore, a better understanding of the cellular and molecular processes underlying pathological pain is necessary to find novel therapeutic targets and approaches to successfully treat such chronic pain states. 16

17. TLRs AND CENTRAL NERVOUS SYSTEM
THE ROLE OF TLRs IN CNS IS ONLY STARTING TO BE UNCOVERED
When resting low TLR expression (TLR4); upon activation TLR expression is strongly induced (TLR2, TLR4, TLR9)
Microglia
STANDARDIZED CYTOKINES AND CHEMOKINES RESPONSE TO RECRUIT ASSISTANCE BY OTHER CELLS

18. TLRs AND CENTRAL NERVOUS SYSTEM
THE ROLE OF TLRs IN CNS IS ONLY STARTING TO BE UNCOVERED
Astrocytes express detectable amounts of TLR1-4 (TLR3 the highest); upon activation TLR expression is strongly induced (TLR3 levels much higher than TLR2 and TLR4)
Astrocytes
STRONGER TLR3 MEDIATED RESPONSE TO PRODUCE A VARIETY OF NEURO-PROTECTIVE AND ANTI-INFLAMMATORY FACTORS
activated astrocytes

19. 19

20. TLRs AND CENTRAL NERVOUS SYSTEM
TLRs in the CNS not only control host-defense responses but play also important roles in tissue development, cellular migration and differentiation, limiting inflammation and mounting repair processes following trauma
Type, duration and strength of TLRs activation
Neuroprotection
Neurodegeneration

21. TLR4: A NOVEL TARGET FOR NEUROPATHIC PAIN THERAPY?
TLR4 ANTAGONISTS combined with opioids
OPIOID AGONISTS that do not bind to TLR4 21

22. TLRs MIS-ACTIVATION: A SOURCE OF DRUG-MEDIATED SIDE EFFECTS?
Xenobiotics and drugs may mis-activate TLRs
OPIOIDS
TLRs as novel drug targets:
Finding AGONISTS
Finding ANTAGONISTS
Checking if drugs not directed to TLRs may activate them
ANTIDEPRESSANTS
OTHERS ? 22

23. TARGETING TOLL-LIKE RECEPTORS: EXPERIMENTAL STRATEGIES
Screening molecules to evaluate their activity towards TLRs
Library of putative antagonists
Library of putative agonists
TLR agonist
TLR agonist
Library of miRNA or small molecules
RECEPTOR ACTIVATION
BLOCK OF RECEPTOR ACTIVATION
BLOCK SPECIFIC SIGNALLING
CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

24. IS THERE IN TLRs INTRACELLULAR SIGNALLING AN EFFECTOR WICH IS COMMONLY ACTIVATED?
NF-kB

25. SCREENING OF TLR LIGANDS: Measuring NF-kB activation
Screening molecules to evaluate their activity towards TLRs
Library of putative antagonists
Library of putative agonists
TLR agonist
TLR agonist
Library of miRNA or small molecules
MODULATION OF NF-kB ACTIVATION
NF-kB ACTIVATION
BLOCK OF NF-kB ACTIVATION

26. EVALUATING NF-kB ACTIVATION: Traditional approaches
Nuclear translocation by WB
INDIRECT, NON HOMOGENEOUS, TIME CONSUMING; BETTER FOR RESEARCH THAN FOR SCREENING OF LIBRARIES
Binding to responsive elements by EMSA
IkB phosphorylation by WB

27. EVALUATING NF-kB ACTIVATION: Innovative strategies
QUICK, SENSITIVE, OMOGENEOUS
SUITABLE FOR AUTOMATION AND HTS APPLICATIONS
NF-kB reporter vectors

28. REPORTER VECTORS: General mechanism of action
ENZYME
STIMULUS TF TF
Promoter
REPORTER GENE
NF-kB RE + Minimal Promoter
NF-kB
RNApol

29. S TLR Nucleus Reporter Reporter Drug IkB NFkB Enzyme RNApol NF-kB RE

30. EVALUATING TLRs LIGANDS: NF-kB different reporter systems
PRO
Fixed expression levels of TLR and reporter system
Reporter enzyme readily detectable in cell supernatant => OMOGENEUS
CONS
Reporter enzyme accumulates => less sensitivity and kinetics hardly measurable
HEK-293 STABLY EXPRESSING SPECIFIC TLRs
AND NF-kB/SEAP REPORTER VECTOR

31. EVALUATING TLRs LIGANDS: NF-kB different reporter systems
NF-kB RE
PRO AND CONS DEPEND ON THE REPORTER SYSTEM EMPLOYED!
CELL LINES EXPRESSING TLRs TO BE TRANSFECTED WITH THE REPORTER SYSTEM OF INTEREST
SECOND REPORTER TO NORMALIZE RESULTS

32. Normalising results - Dual Reporters
First reporter (e.g. luc)
Experimental plasmid
Cells
Control
plasmid
Second reporter (e.g. CAT, ß-Gal)
Normalised = Experimental reporter response Control reporter

33. Dual-Luciferase Reporter Assay
Experimental plasmid
FF LUC
Cells
Control
plasmid
R LUC
Two luciferase reporter enzymes
Sequential quantification of Firefly luciferase and Renilla luciferase
Distinct evolutionary origin

34. Firefly luciferase reaction Oxyluciferin +AMP + PPi + CO2 + light
Photinus pyralis
Luciferase
Oxyluciferin +AMP + PPi + CO2 + light
Luciferin + ATP + O2
Luciferases:
Co-substrates:
Luciferin:
Monomeric, 61 kDa
ATPMg2+, CoA

35. Renilla luciferase reaction
Sea Pansy (Renilla reniformis)
Luciferase
Coelenteramide
+ CO2 + light
Coelenterazine + O2
Luciferases:
-Monomeric, 36 kDa (Renilla)
Coelenterazine:

36. Dual-Luciferase Assay Format Luciferase assay reagent4.
Precise (both measurements from a single sample)
Light
Output
(Renilla) 3.
Linear
Stop & GloTM
Reagent
Light
Output
(Firefly) 2.
Sensitive 1.
Rapid (30s manual assay, 7s automated assay)
Luciferase assay reagent
Sample

37. TARGETING TOLL-LIKE RECEPTORS: NF-kB Dual Luciferase systems
Firefly LUC
NF-kB RE
Renilla LUC TK
PRO
Physiological TLR expression levels
Specific cell-type features
Reporter enzyme sequentially detectable in lysates => OMOGENEUS
CELL LINES EXPRESSING TLRs TO BE TRANSFECTED WITH THE REPORTER SYSTEM OF INTEREST
CONS
Reporter enzyme accumulates => less sensitivity and kinetics hardly measurable

38. Destabilized Luciferase Genes
Greater induction
Reporter intracellular
reporter pool
Induction detection sooner
Destabilized Reporter
intracellular reporter pool
Cloned cAMP Response Elements (CREs) into the various Rapid Response Reporter Vectors and the appropriate controls
Transfected HEK 293 cells
24 hours post-transfection, to Inducted wells added isoproterenol. To non-induced wells added nothing
Collected samples
Fold Induction was calculated by dividing the induced samples by non-induced samples

39. SEARCH OF OPIOID ANALGESICS THAT DO NOT ACTIVATE TLR4 IN GLIAL CELLS
CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School
Chronic pain condition in which opiod analgesics are ineffective or detrimental because of opioid-mediated TLR4 activation!!!
SEARCH OF OPIOID ANALGESICS THAT DO NOT ACTIVATE TLR4 IN GLIAL CELLS

40. OUR CELL MODEL AND REPORTER VECTORS
CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School
OUR CELL MODEL AND REPORTER VECTORS
U-87 MG HUMAN GLIOBLASTOMA CELL LINE
Physiological expression levels of TLR4
Good model of human glia

41. OUR TRANSFECTION SYSTEM
CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School
OUR TRANSFECTION SYSTEM
Cationic polymer reagent
Non liposomic
Every 3rd atom is a protonable nitrogen
Stable small POLYMER/DNA complexes ( nM)

42. CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School
OUR TREATMENTS
LPS from E. Coli
(1 ng/ml; 6 h)
TLR4 agonist
POSITIVE CONTROL
Morphine (10 mM; 6 h)
The prototypical OPIOID ANALGESIC B C A
DAMGO – peptidic opioid agonist
(10 mM; 6h)

43. OUR DUAL-LUCIFERASE ASSAY
CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School
OUR DUAL-LUCIFERASE ASSAY

44. CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School
OUR EXPECTED RESULTS
LPS
DAMGO
Morphine
TLR4 ?

45. LUMINESCENCE DATA Output
Firefly Luminescence
754
942
283
423 47
27,6
678
765
194
374 50
618
550
322
325 53
510
528
109
103 51
FIRST subtract basal luminescence from luminescence signals of each well for both Firefly and Renilla
Renilla Luminescence 34 43 37 42
25,475 41 35 33 28 32 30 40 36
CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

46. LUMINESCENCE DATA Calculation of Specific Luminescence
Firefly Specific Luminescence
726
914
256
395 19
650
737
166
346 22
590
523
294
298 26
482
500 81 76 23
Use specific luminescence to calculate FF-Luc/R-luc ratio for each well
Renilla Specific Luminescence 8 18 12 17 9 16 15 10 7 2 6 4 11 5
CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

47. LUMINESCENCE DATA Result normalization and RLU calculation
Library of putative agonists
Normalized Luminescence => NF-kB Activation Levels
87,21922
52,17689
21,97849
23,64126
2,200573
41,6192
47,79903
17,28831
49,28114
10,06742
61,28831
34,77537
43,12088
47,05138
6,206061
32,95043
45,79405
10,377
16,38919
4,766839
NF-kB ACTIVATION
CALCULATE MEAN AND STANDARD DEVIATION WITHIN EACH EXPERIMENTAL GROUP
EXPRESS DATA AS Relative Luminescence Units (RLU)
CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

48. DUAL LUCIFERASE ASSAY Representative results
LPS and MORPHINE, but not DAMGO, could significantly induce NF-kB activation
CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

49. DUAL LUCIFERASE ASSAY Summer School 2016
LPS and MORPHINE, but not DAMGO, could significantly induce NF-kB activation
CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

50. DUAL LUCIFERASE ASSAY Previous data vs Summer School 2017

51. TARGETING TOLL-LIKE RECEPTORS: NOVEL POTENTIAL THERAPEUTICS
TLRs are crucial for both host defense against pathogens and for a rapid response to local tissue damages.
Because of TLRs involvement in immune responses as well as in several diseases related to inflammation, targeting TLRs may represent a novel and effective therapeutic opportunity.
NF-kB Dual Luciferase Reporter systems represent an useful experimental tool to screen library of putative TLRs ligands in a rapid, sensitive, linear and accurate way.