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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
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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
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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
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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
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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
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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
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TOLL-LIKE RECEPTORS: LIGAND RECEPTOR INTERACTIONS
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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
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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
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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
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TLR AGONISTS AND CANCER: Drugs presently under investigation
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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
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GREAT POTENTIAL OF TLRs AGONISTS COMBINED TO STANDARD THERAPIES IN THE TREATMENT OF INFECTIONS
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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
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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
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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
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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
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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
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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
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TLR4: A NOVEL TARGET FOR NEUROPATHIC PAIN THERAPY?
TLR4 ANTAGONISTS combined with opioids OPIOID AGONISTS that do not bind to TLR4 21
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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
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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
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IS THERE IN TLRs INTRACELLULAR SIGNALLING AN EFFECTOR WICH IS COMMONLY ACTIVATED?
NF-kB
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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
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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
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EVALUATING NF-kB ACTIVATION: Innovative strategies
QUICK, SENSITIVE, OMOGENEOUS SUITABLE FOR AUTOMATION AND HTS APPLICATIONS NF-kB reporter vectors
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REPORTER VECTORS: General mechanism of action
ENZYME STIMULUS TF TF Promoter REPORTER GENE NF-kB RE + Minimal Promoter NF-kB RNApol
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S TLR Nucleus Reporter Reporter Drug IkB NFkB Enzyme RNApol NF-kB RE
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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
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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
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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
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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
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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 ATPMg2+, CoA
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Renilla luciferase reaction
Sea Pansy (Renilla reniformis) Luciferase Coelenteramide + CO2 + light Coelenterazine + O2 Luciferases: -Monomeric, 36 kDa (Renilla) Coelenterazine:
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Dual-Luciferase Assay Format Luciferase assay reagent
4. 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
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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
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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
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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
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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
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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)
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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)
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OUR DUAL-LUCIFERASE ASSAY
CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School OUR DUAL-LUCIFERASE ASSAY
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CHRONIC PAIN, TLRs AND DRUG DISCOVERY: The practical training at the Summer School
OUR EXPECTED RESULTS LPS DAMGO Morphine TLR4 ?
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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
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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
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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
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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
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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
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DUAL LUCIFERASE ASSAY Previous data vs Summer School 2017
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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.
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