570 likes | 711 Views
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.
E N D
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
INNATE IMMUNE RESPONSE AND TOLL-LIKE RECEPTORS Innate immunity is evolutionarily ancient and selected mechanisms are conserved TLRs play a cucial role in innate immune responses to infection
INNATE IMMUNE RESPONSE AND TOLL-LIKE RECEPTORS Different cell types may express a diverse set of TLRs The selective engagement of adaptors defines the mode of signal transduction
IMMUNE RESPONSE IN HEALTH AND DISEASE MOLECULAR COMPONENTS INNATE IMMUNE SYSTEM INFLAMMATION
TOLL-LIKE RECEPTORS IN HEALTH AND DISEASE TLRsrecognizeconservedstructuresofmicrobes and endogenous (host-derived) molecules TLRs = PPR pattern recognitionreceptor 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
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
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
TOLL-LIKE RECEPTORS: STRUCTURAL FEATURES Extracellular Leucine-rich Repeats Toll/IL-1 receptor (TIR) signalling domains TOLL-LIKE RECEPTORS (TLRs): Type I integral transmembrane glycoproteins To date more than a dozen of TLRs TLR 1 -9 conserved among humans and mice TLR10 selectively expressed in humans
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
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
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
TOLL-LIKE RECEPTORS: LIGAND RECEPTOR INTERACTIONS
TARGETINGTOLL-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)
TLR AGONISTS AND CANCER TLR-mediatedstimulationofadaptiveimmunity and activationofNKs and cytotoxicT-cellsmediatesantitumoureffects • In the 19th Century William Coley observed antitumour effects of repeated injections of toxins from dead Gram+ and Gram- bacteria => activation of TLR4 and TLR9 • Imiquimod, one of the first and most successful drugs targeting TLRs, determines a TLR7-dependent secretion of the antiviral and antitumour cytokine IFNa • Individuals with breast cancer carrying a loss-of-function allele of TLR4 relapsed much quicker following radiotherapy and chemotherapy than their wild-type counterparts • Hennessey EJ et al. Nature Reviews Drug Discovery 2010 QUEST OF TLRs AGONISTS FOR NOVEL ANTICANCER THERAPIES
TLR AGONISTS AND CANCER: Drugs presently under investigation
TLR AGONISTS AND CANCER: Perspectives and drawbacks POLY-TLR AGONISTS Cadi-05 (autoclavedmycobacteriatargetingseveralTLRs) isbeingevaluatedfor the treatment ofadvanced melanoma, prostate and bladdercancer => in phase I trial 5/9 becameasymptomatic and showed no diseaserecurrence at 2 years LACKING OF COMPELLING RATIONALE IN HUMAN CLINICAL TRIALS MANY CANCER PATIENTS ARE IMMUNOSUPPRESSED
GREAT POTENTIAL OF TLRs AGONISTS COMBINED TO STANDARD THERAPIES IN THE TREATMENT OF INFECTIONS
TLR ANTAGONISTS Inappropriately overactive immune system TLR ANTAGONISTS • HCQ (TLR9 antagonist, TLR7 and TLR8 weak antagonist) is the current treatment for SLE • CPG-52364 (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 • Poly I:C (dsRNA molecule; TLR3 agonist) => treatment of Hepatitis B, Hepatitis C, HIV, influenza
TOLL-LIKE RECEPTORS AND CENTRAL NERVOUS SYSTEM TLRs are differentially expressed by the various cell type within the CNS
NEURON-GLIA INTERACTIONS Glia crucial role in the maintenance of neuronal homeostasis Neurogenesis Myelination Synaptic plasticity Neuronal migration, proliferation and differentiation ASTROCYTES Trophic support to neurons Clearing cell debris Taking up released transmitters MICROGLIA Active immune surveillance
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
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
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
NEUROPATHIC PAIN Chronic pain condition in which opiod analgesics are ineffective or detrimental PAIN MORPHINE-MEDIATED ACTIVATION OF TLR4!!! FIRST STEP: damage to neuronal fibers • SECOND STEP: • Glial cell activation (crucial role of Toll-like receptors) • Release of pro-inflammatory mediators which acts on neurons and glial cells
NEURON-TO-GLIA SIGNALS IN NEUROPATHIC PAIN: Maladaptive plasticity within the nociceptive system Maladaptiveplasticity and activationofglialcells
TLR4: A NOVEL TARGET FOR NEUROPATHIC PAIN THERAPY? TLR4 ANTAGONISTS combined with opioids OPIOID AGONISTS that do not bind to TLR4
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 ?
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 BLOCK OF RECEPTOR ACTIVATION BLOCK SPECIFIC SIGNALLING RECEPTOR ACTIVATION CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS
TARGETING TOLL-LIKE RECEPTORS: EXPERIMENTAL STRATEGIES DIFFERENT CELL MODELS • PRO • Expression of single TLR • High expression levels • No interference with any other signalling pathway • CONS • Sovraphysiological TLR expression • Not reproducing desired cell type (glia) HEK-293 STABLY EXPRESSING SPECIFIC TLRs CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS
TARGETING TOLL-LIKE RECEPTORS: EXPERIMENTAL STRATEGIES DIFFERENT CELL MODELS • PRO • Physiological TLR expression levels • Specific cell-type features • CONS • Expression of more than one TLR • Presence of other receptors and signalling pathways that may interact/interfere with TLRs CELL LINES EXPRESSING TLRs AND REPRODUCING SPECIFIC CELL TYPES CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS
IS THERE IN TLRs INTRACELLULAR SIGNALLING AN EFFECTOR WICH IS COMMONLY ACTIVATED? NF-kB
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 BLOCK OF NF-kB ACTIVATION NF-kB ACTIVATION
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
EVALUATING NF-kB ACTIVATION: Innovative strategies QUICK, SENSITIVE, OMOGENEOUS SUITABLE FOR AUTOMATION AND HTS APPLICATIONS NF-kB reporter vectors
ENZYME Promoter RNApol STIMULUS NF-kB NF-kB RE + Minimal Promoter REPORTER VECTORS: General mechanism of action TF TF REPORTER GENE
Reporter Reporter Drug NF-kB RE NF-kB RE NFkB Enzyme RNApol IkB S TLR Nucleus
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
EVALUATING TLRs LIGANDS: NF-kB different reporter systems Reporter 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
Normalising results - Dual Reporters First reporter (e.g. luc) Experimentalplasmid Cells Control plasmid Second reporter(e.g. CAT, ß-Gal) Normalised = Experimental reporterresponse Control reporter
Current Dual Reporter Systems • Cells may have endogenous -Gal activity. • CAT, -Gal assays are endpoint assays. • Can’t combine luciferase, CAT, -Gal in a single-tube format that is equally sensitive and rapid for both reporter assays.
Cells Dual-Luciferase Reporter Assay Experimentalplasmid FF LUC Control plasmid R LUC • Two luciferase reporter enzymes • Sequential quantification of Firefly luciferase and Renilla luciferase • Distinct evolutionary origin
Luciferases: Monomeric, 61 kDa Co-substrates: ATPMg2+, CoA Luciferin: Firefly luciferase reaction Photinus pyralis Luciferase Oxyluciferin +AMP+ PPi + CO2 + light Luciferin + ATP + O2
Luciferase Renilla luciferase reaction Sea Pansy (Renilla reniformis) Coelenteramide + CO2 + light Coelenterazine + O2 Luciferases: -Monomeric, 36 kDa (Renilla) Coelenterazine:
1. Rapid(30s manual assay,7s automated assay) 2. Sensitive 3. Linear 4. Precise(both measurementsfrom a single sample) Dual-Luciferase Assay Format Light Output (Renilla) Stop & GloTM Reagent Light Output (Firefly) Luciferase assay reagent Sample
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
Destabilized Luciferase Genes Intracellular Reporter Pool Non-destabilized Newly expressed Reporter Signal Destabilized Signal Newly expressed Reporter
Greater induction Reporter intracellular reporter pool Destabilized Reporter intracellular reporter pool Induction detection sooner Destabilized Luciferase Genes
Destabilized Luciferase Genes • Advantages: • Faster response to stimulus • Greater magnitude of response • Better temporal coupling • No secondary effect detected
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