2. Lecture 2 Sept 4 Glia, Comminication, Transmitters part A B, ion channels, modern drug CLASS NOTES.pptx

1 Glia cells have about a 1:1 ratio in the brain with neurons In this magnified image of brain tissue, neurons (blue) are surrounded by large numbers of glial cells, including astrocytes (red) and oligodendrocytes (green) 2 Supporting cells • Glia or neuroglia - supply nutrients, physical supp...

1 Glia cells have about a 1:1 ratio in the brain with neurons In this magnified image of brain tissue, neurons (blue) are surrounded by large numbers of glial cells, including astrocytes (red) and oligodendrocytes (green) 2 Supporting cells • Glia or neuroglia - supply nutrients, physical support and barrier, debris removal, transmitters) functional interaction (release 3 Glia Cells reuptake of neurotransmitters • • part of blood brain barrier 4 What do the Neurotransmitters do? Communication: transferring information/signals between groups of neurons - Emptied into synaptic cleft and can bind to 2 types of receptors - these receptors are located post-synaptic neuron, glia cells, and also on the same neuron that released them 5 Types of Receptors 1. Ionotropic receptor - also called “ligand gated receptor” - binding site and ion channel combined - a ligand must bind to the receptor in order to open up the ion channel - efficient = quick response (10-50 ms) - drugs can act at the receptor binding site or at the channel to affect its function - if acts at channel, can interfere with ion flow 6 Ionotropic receptor - Conformational change of ion channel protein once the ligand is bound to the receptor - results in ion channel flux (inflow or outflow of ions) 7 Types of Receptors 2. Metabotropic receptor: - sometimes called “G-coupled receptors” - only binding site (no channel) - triggers 2nd messenger system (thru G-proteins) - can open ion channels (indirectly) by intracellular processes - slower due to indirect action on ion channels - changing ion channel function is key to modulating neuron signaling - drugs can act at binding site, 2nd messenger system, or on ion channel 8 Metabotropic receptor: - 2nd messenger system - neurotransmitter binds to receptor and activates G-proteins triggering production of 2nd messengers - 2nd messengers can change conformation of nearby ion channels - effects other cell functions as well - there are different types of 2nd messenger systems - examples of 2nd messengers include cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP) 9 What do the Neurotransmitters do? • Neurotransmitters can bind to post-synaptic receptors • They can also bind to pre-synaptic receptors to give feedback to the originating neuron (pre-synaptic) • Autoreceptors and Heteroreceptors - receptors on terminals of presynaptic neuron - typically inhibit production of self (auto) or other (hetero) neurotransmitters (feedback inhibition) - also a site of drug action (to slow or increase production of neurotransmitters) 10 Regulating Neurotransmitter Communication • Neurotransmitter communication is brief and stopped by: - reuptake: transporters bring the neurotransmitters back to the neuron (then recycled or degraded) - deactivation: enzymes breakdown the neurotransmitters (in cleft, astrocytes, or within neuron itself) - ensures lack of build up of transmitters (reinstates dynamics of the communication) - able to react to new signaling/information (build-up would “clog” the cleft) - drugs are made to interrupt or aid these processes X 11 Ion channels on the Post-Synaptic Neurons The transmitters released from pre-synaptic neurons (multiple) determine excitation or inhibition of post-synaptic neuron • The “net” influx/outflux of ions through ion channels determines the outcome (excitation or inhibition) - Na+, Ca2+, Cl-, K+ - ion flow “in-out” follows same principles described in action potential (i.e., diffusion, electrostatic) - triggered directly from ionotropic and indirectly from metabotropic receptors • If positive ions flow in (e.g., Na+, Ca2+ ) – get an excitatory postsynaptic potential (EPSP) - makes the inside less (-): depolarized • If negative ions flow in (e.g., Cl-), or positive ions flow out (e.g., K+) – get an inhibitory postsynaptic potential (IPSP) - makes the inside more (-): hyperpolarized • The net effect of EPSPs and IPSPs contribute to firing of neuron: excitation or inhibition - if overwhelming EPSPs, signal hits the axon hillock (at -55 mV) to trigger an action potential - if more IPSPs, keeps the neuron hyperpolarized = no action potential 12 +40 mV -55 mV -70 mV • The more EPSPs, the more likely an action potential will be initiated • The more IPSPs, the more difficult it is to trigger an action potential 13 14 “Ya, I know that” Quiz... An IPSP is likely to get triggered if which of the below happens? Answer: D Cl- flows in 15 Ion Channels 16 Diversity of Ion Channels • Ion channels found throughout body and cells - all neurons have ion channels - more than 200 ion channels identified - animal toxins (venom) often work on ion channels - more than 60 “channelopathies” (mutation in channel triggers/prevents a disease/condition) - people lacking Nav1.7 channels do not feel pain 17 Ion channels as Drug Targets • Voltage Dependent (Gated) Channels - channel opening depends on membrane potential - Nav - Cav - Kv (v = voltage: these are all activated by specific voltage ranges) • Ligand Gated Receptors (Ionotropic Receptors) - channel opening depends on binding of molecule to receptor - P2X receptors - ionic glutamate receptors (iGluR ) - serotonin receptor 3 (5-HT3) - transient Receptor Potential (TRP) cation channels - -aminobutyric acid receptor A (GABAA) - nicotinic acetylcholine receptors (nAChR ) 18 Voltage Gated Channels • Voltage gated channel has 3 states: 1. closed 2. activated 3. inactivated All states are dependent on a specific voltage range for that particular channel 19 Voltage Dependent Channels: Nav • NaV channels - allow flow of Na+ into the neuron - activated by low (negative) voltages - 9 subtypes: Nav1.1-Nav1.9 - channels initially differentiated: if activated by a (tetrodotoxin, TTX) or not fish toxin - TTXr (r = resistant)- Nav1.5, Nav1.8, Nav1.9 - TTXs (s = sensitive) – all other Nav 20 Voltage Dependent Channels: Cav • CaV channels - allow flow of Ca2+ into the neuron - role in neurotransmitter release - 2 families - high/moderate voltage activated = during depolarization (in “+ mV” range) - low voltage activated (close to resting potential) - Cav1, Cav2, Cav3 with “sub-subtypes” - Cav1.1 – 1.4 - Cav2.1 - 2.3 - Cav3.1 - 3.3 (low voltage activated) 21 w many subtypes of human Kv channels are there??? Answer: – “38” 22 Voltage Dependent Channels: Kv • KV channels - allow flow of K+ out of the neuron (repolarization) - mainly activated by high (positive) voltages (with some exceptions) - Kv1 to Kv12 with many “sub-subtypes” - largest # of subtypes is 8 (Kv1.1 – 1.8) - most abundant and diverse 23 Ligand Gated Channels • Opening and closing depends on presence of ligand 24 Ligand Gated Channels: P2X • P2X receptors - activated by ATP (adenosine 5’-triphosphate) and analogs - when activated allows in-flow of Na+ and Ca2+ (depolarization) - also allows flow of K+ - 7 subtypes P2X1-P2X7 25 Ligand Gated Channels: iGlutamate • Ionotropic Glutamate receptors (iGluR) - when activated allows in-flow of Na+ (depolarization) - also allows flow of K+ - there are also metabotropic glutamate receptors • Ligands activate different 3 subtypes of iGluRs - these subtypes are named after a distinct ligand that activates them 1. NMDA (N-methyl-D-aspartate) 2. AMPA (α-amino-3-hydroxy-5-methyl-43. KA (kainic acid) subtypes isoxazolepropionic acid) Important notes: - each subtype is also activated by glutamate - glycine is needed with glutamate to activate NMDA receptors - NMDA related channels also allow in-flow of Ca2+ - iGluRs most studied because of the essential role of glutamate in neuron excitability - throughout the body and many functions inside and outside of the nervous system 26 Ligand Gated Channels: 5-HT3 • Serotonin receptor 3 (5-HT3) - activated by serotonin (5-HT) - 5 receptor subtypes (A-E) - when activated allows in-flow of Na+ and Ca2+ (depolarization) - also allows flow of K+ - most serotonin receptors are metabotropic 27 Ligand Gated Channels: Transient Receptor Potential (TRP) Cation Channels Transient Receptor Potential (TRP) Cation Channels - when activated allows in-flow of Na+ and Ca2+ (depolarization) - also allows flow of K+ - activated by numerous ligands (specific channels) - TRPV1 – heat (>43oC), acid, capsaicin, endogenous lipids (e.g., anandamide) - TRPA1 – allyl isothiocyanate (wasabi, mustard oil), eugenol (clove oil), cinnamaldehyde, gingerol, 4-HNE (endogenous), environmental toxins, mechanical transduction - TRPM8 – menthol, eucalyptol, cool/cold - TRPV3 – carvacrol (thyme, oregano), camphor, warm - TRPV4 – warm, anandamide - diverse functions inside and outside of the nervous system 28 Ligand Gated Channels:  -Aminobutyric Acid Receptor A (GABAA) •  -aminobutyric acid receptor A (GABAA) - when activated allows in-flow of Cl- (hyperpolarization) - most abundant inhibitory neurotransmitter - ~20% of all neurons - GABAC is also ligand gated, GABAB is not (metabotropic) - Activated by GABA - receptors are “omnipresent” in CNS 29 Ligand Gated Channels: Nicotinic Acetylcholine Receptors (nAChR ) • Nicotinic Acetylcholine Receptors (nAChR ) - when activated allows in-flow of Na+ and Ca2+ (depolarization) - also allows flow of K+ - activated by acetylcholine (ACh) and nicotine - many subtypes, most notable  7,  4β2 (though 11 family members) - can be both pre-synaptic and post-synaptic - needs 2 ACh molecules for its activation 30 Take home message • The release of transmitters facilitates neuron-to-neuron communication • Ion channels play a role in action potential generation and neuronal signaling - inhibitory and excitatory • Ion channel state (opened or closed) can be voltage or ligand dependent • There are diverse ion channels with multiple roles inside and outside of the nervous system • Receptors and channels are major mechanisms for drugs to change (facilitate OR impede) normal or pathological communication between neurons 31 32 Neurotransmitters Part A 33 Neurotransmitter: “Chemical substance packaged in a synaptic vesicle and released by a neuron to communicate across a synapse with another neuron, muscle cell, organ, or a hormone-producing cell in an endocrine gland” There are about 100 known neurotransmitters - 10 neurotransmitters are the most prevalent 34 Neurotransmitters: Monoamines • Dopamine (DA) • Norepinephrine/Noradrenaline (NE/NA) • Epinephrine/Adrenaline (closely related to NE) Catecholamines • Serotonin (5-HT) (synthesized from L-Tryptophan) • Histamine (synthesized from histidine) 35 Neurotransmitters: Dopamine • Dopamine (DA) - G-coupled (metabotropic) receptors - D1-D5 receptors - D1 and D5 excitatory (“D1-like”) - D2 (also autoreceptor), D3, and D4 inhibitory (“D2-like”) 36 Neurotransmitters: Dopamine - motor function, reward, cognition, learning • Dopamine (DA) - three main pathways: 1. Nigrostriatal 2. Mesolimbic 3. Mesocortical Substantia nigra to striatum Ventral tegmental area (VTA) to limbic area Ventral tegmental area (VTA) to cortical regions - reward/aversion, motivation, pleasure, cognition, fear, learning, emotion, executive functioning Dorsal striatum Prefrontal cortex “Reward” pathway 37 Neurotransmitters: Dopamine - DA destruction/reuptake - dopamine transporters (DAT) - monoamine oxidase (MAO-A and MAO-B) – enzyme that degrades DA in presynaptic - catechol-O-methyltransferase (COMT) – enzyme that degrades DA post-synaptic terminals and astrocytes neuron and astrocytes astrocyte G 38 Neurotransmitters: Noradrenaline • Noradrenaline or Norepinephrine (NA or NE) - also a hormone secreted by adrenal glands - adrenaline or epinephrine are closely related - G-coupled (metabotropic) receptors: also called “adrenoceptors” - α1(A,B,D), α2(A,B,C), β1, β2 receptors (both NE and epinephrine bind) - α2 (also autoreceptor) only one that is inhibitory 39 Neurotransmitters: Noradrenaline • Noradrenaline or Norepinephrine (NA or NE) - locus coeruleus (LC, A6) is origin site (branches from there): - to forebrain - to cerebellum - to spinal cord - LC: stress, arousal, sleep cycle, attention/memory, cognition, pain, mood, posture, balance - NE destruction/reuptake - noradrenaline transporters (NET) - monoamine oxidase (MAO-A) – destroys in pre-synaptic terminals - catechol-O-methyltransferase (COMT) – post-synaptic and astrocytes 40 Neurotransmitters: Serotonin • Serotonin (5-HT, 5-hydroxytryptamine) - ligand gated (ionotropic) and G-coupled (metabotropic) receptors - 14 receptor subtypes - 5-HT1A,B,D,F, 5-HT2A-2C, 5-HT3, 5-HT4, 5-HT5a-5B, 5-HT6, 5-HT7 - 5-HT1 and 5-HT5 (postsynaptic and autoreceptor) – inhibitory - all other metabotropic receptors are excitatory 41 Neurotransmitters: Serotonin • Serotonin (5-HT, 5-hydroxytryptamine) - 9 clusters of 5-HT neurons: most found along midline of brainstem - brainstem to forebrain - brainstem to spinal cord B6, B7: dorsal raphe B5, B8: median raphe B9: dorsal pontine tegmentum B4: dorsal raphe obscurus B1: raphe pallidus B2: raphe obscurus B3: raphe magnus - arousal, sleep (dreaming), aggression, cognition, pain, depression, anxiety 42 • Serotonin (5-HT, 5-hydroxytryptamine) - 5-HT destruction/reuptake - 5-HT transporters (SERT) - monoamine oxidase (MAO-A) MAO-A 43 Neurotransmitters: Histamine • Histamine - G-coupled (metabotropic) receptors - H1-H4 receptors - H3 are autoreceptors and heteroreceptors (inhibitory) - neurons originate in tuberomammillary nucleus (TMN, posterior hypothalamus): - to brain stem, spinal cord, and cerebral cortex 44 Neurotransmitters: Histamine • Histamine - also produced by mast cells and basophils (immune cells) - H4 is mainly on immune cells - histamine destruction/reuptake - no specific reuptake transporter - Histamine N-methyltransferase (HNMT) and neurons) Hnmt (astrocytes 45 46 “Ya, I know that” Quiz... Which of these degrades dopamine in both the postsynaptic neuron and in astrocytes? Answer: A COMT 47 Neurotransmitters Part B 48 Amino Acid Neurotransmitters: Glutamate • Glutamate - both ligand-gated (ionotropic) and G-coupled (metabotropic) receptors - metabotropic receptors mGluR1-mGluR8 - Group I (mGluR1, mGluR5) located postsynaptically: excitatory transmission - Group II (mGluR2, mGluR3) (postsynaptic too) autoreceptors presynaptic inhibitory - Group III (mGluR4, mGluR6, mGluR7, mGluR8 heteroreceptors & glia 49 Amino Acid Neurotransmitters: Glutamate • Glutamate - both ligand-gated (ionotropic) and G-coupled (metabotropic) receptors - metabotropic receptors mGluR1-mGluR8 - Group I (mGluR1, mGluR5) located postsynaptically: excitatory transmission autoreceptors - Group II (mGluR2, mGluR3) (postsynaptic too) presynaptic heteroreceptors - Group III (mGluR4, mGluR6, mGluR7, mGluR8 - throughout the nervous system - glutamate destruction/reuptake - glutamine synthase (enzyme) that breaks glutamate down to glutamine - removed by excitatory amino acid transporters (EAAT1-5) 50 Amino Acid Neurotransmitters: GABA • γ-aminobutyric acid (GABA) - both ligand-gated (ionotropic) and G-coupled (metabotropic) receptors - metabotropic receptors GabaB - synthesized from glutamate (via glutamate decarboxylase (GAD)) - post- and pre-synaptic - found throughout nervous system - main inhibitory transmitter - GABA destruction/reuptake - removed by GABA transporters GAT-1, GAT-2, GAT-3 - degraded by GABA aminotransferase (GABA-AT) 51 Neurotransmitters: Acetylcholine • Acetylcholine (ACh) - both ligand-gated (ionotropic) and G-coupled (metabotropic) receptors - metabotropic receptors: muscarinic mACh (most common ACh receptor) - activated by muscarine - ionotropic and metabotropic receptors both activated by ACh 52 Neurotransmitters: Acetylcholine • Acetylcholine (ACh) - 2 major pathways 1) Basal forebrain projects to cortical, thalamic, and hippocampus) (sleep, arousal, attention, memory, cognition) limbic sites (amygdala and 2) Pontine tegmental areas project to subcortical sites and spinal cord (arousal, reward, attention, pain, motor) Plus 2 other regions with localized ACh (motor, reward, cognition) - striatum - cerebellum 53 Neurotransmitters: Acetylcholine • Acetylcholine (ACh) - 5 receptor subtypes M1-M5 - M1, M3, M5 are excitatory - M2 (mainly heart), M4 are inhibitory - ACh destruction/reuptake - acetylcholinesterase (AChE) - no reuptake 54 What well-known “lifestyle” drug works through reducing ACh release??? 55 AC 56 Neurotransmitters: Peptides and Lipids • Peptides also released from vesicles and can act as neuromodulators and neurotransmitters - can act both through cleft and diffusion to other neurons or other cells (e.g., immune cells) - degraded by a variety of enzymes but no reuptake (depends on peptide) - examples: opioids (enkephalins, dynorphins, β-endorphins), substance P, calcitonin gene-related peptide (CGRP), somatostatin, neuropeptide Y • Lipids are released through the lipid membrane (not in vesicles), typically in the post-synaptic neuron - retrograde transmission (communication post-synaptic to pre-synaptic) - example lipds: endocannabinoids like anandamide (AEA), and 2arachidonyl glycerol (2-AG) - multiple enzymes for degradation and reuptake by different transporters (depends on lipid) 57 Take home message • Monoamines, GABA, glutamate, acetylcholine, various peptides, and lipids form the bulk of neurotransmitters in the CNS with diverse functions • Dysfunction of one of more of these are associated with the majority of neurological disorders 58 59 60 Answer C: Somira • • • • • Talimogene Laherparepvec - Melanoma Linzess – IBS Xgeva – Osteoporosis Farydak – Cancer Idebenone – Alzheimer's Disease 61 Ancient Drug Discovery • Observation • Trial and error • Spreading the word Modern Drug Discovery • Observation • Trial and error • Spreading the word Groups responsible for different elements of Drug Discovery/Development Preclinical Preclinical & Clinical Clinical After FDA approval 62 Step 1: Identify the disease you want to treat 63 Step 2: Target Identification • Target = find a mechanism that is involved in pathophysiology (cause) of disease - could be a receptor, neurotransmitter, re-uptake, cytokine, ion channel…. - need to understand the disease • Access to human samples to mine for targets that are relevant to the disease - nervous tissue (i.e., brain, spinal cord, peripheral nerves) - blood - cerebral spinal fluid 64 Step 3: Target Validation • Is your target really linked to the disease in question • Develop in vitro, ex vivo, in vivo models to interrogate the relationship between the target and disease GABA expression in “depressed“ mice • Confirm expression of the target in relevant diseased sites - also: is the target in other non-desired tissue (can be a problem) • Use pharmacological tools (if available) to test hypothesis - may not be that selective for target: Dirty Drug (have activity at other targets) - may not be ideal tool, but is a place to start • Genetic models - what happens when the gene expression for that target is changed - “knock-outs” (developmental or inducible in adults) where delete gene of - “knock-ins” where augment activity/quantity of gene of interest interest Optogenetic probe in rat 65 Step 4: Identifying early chemical matter: A “Hit” • Screen your compound library - pharmaceutical companies have millions of compound in their libraries • High Throughput Screen (HTS) - assay is developed that typically address a functional component of the drug/target - typically cell-based assay that must be amenable to large (96…6144 wells) “plate” platforms - do any compounds from your library activate or block the functional assay? Link to Robot video - automated (objective screen) • Chemist reviews “hits” (compounds that were effective in functional assay) for chemical properties (machines cannot do everything) - pick out most promising hits/compounds 66 Step 5: “Hit to Lead” (HTL) stage • Chemists go to work and improve on screen “hits” - chemistry efforts improve the potency, selectivity, and physiochemical (e.g., pharmacokinetics, solubility) properties of hits 67 Step 5: “Hit to Lead” (HTL) stage • A screening funnel is developed - primary cellular assay: potency and/or binding of compounds at desired target (activity at target) Pre-determined - secondary criteria to advance thru funnel cellular assays: - selectivity of compounds against other targets - druggability (e.g., pharmacokinetics, solubility) - primary in vivo model systems to mimic key components of the disease 68 Step 5: “Hit to Lead” (HtL) stage • If a compound does not pass the criteria at any stage of funnel, it does not progress to lower parts – start with many compounds at top of funnel - best compounds (called “leads”) survive down into the funnel 69 Step 6: Lead Optimization • Chemists work to improve on the “lead” or best compound identified from funnel • More limited screening funnel is developed -assay is focused on the key components you want to fix (e.g., potency, selectivity, pharmacokinetics) 70 Step 7: Preclinical Toxicology • Lead has advanced to be a “clinical candidate” (it is being considered for human testing) • Are the clinical candidates safe enough for humans - push the doses beyond “efficacious doses”: where do we start to see a side effect - rule of thumb: need at least 10-fold difference between efficacy causes a side dose and the lowest dose that - the acceptable “fold” difference between efficacy and side effects can depend on the disease - the more serious the disease (life threatening) the more willing to accept nonserious side effects 71 Step 8: Clinical Testing • Phase 1: primary assessment: is the compound is safe - usually healthy people - determine pharmacokinetic profile - may do a small safety and efficacy readout in the disease people: phase 1b) population (limited number of • Phase 2: proof of concept (PoC) - time to test your hypothesis in humans - relatively small number of patients with the disease of interest (bigger than phase 1) - still monitoring safety • Phase 3: confirm that you are doing benefit to your disease population - large number of patients - all over the world - safety still monitored • Approval by FDA or other country regulatory board 72 “Ya, I know that” Quiz... Which Drug Development Stage tries to improve on “best compound” for a specific disease by trying to fix things like potency, selectivity, and pharmacokinetics? Answer B: “Lead optimization” stage 73 The Long Road to Developing a Drug 1-4 years 2-4 years 1-2 years 6-8 years Only 1 out of 10 drugs make it all the way once in the clinic Cost - $700 million-3 billion (depending disease and number of failures) 74 Take Home Points • The modern development of new drugs is a multi-stage endeavor involving the most basic understanding of biology for the disease and target, chemical science, and pre-clinical and clinical evaluation of the drug’s efficacy and safety 75 To do for Next Week • Read text chapters 4 (LO 4.1, 4.3, 4.4, 4.6-4.9) • Pharmacodynamics • Types of Drug Modulation (agonist, antagonist interactions) • Read Journal Club papers: Olivia + Lorena “TRPA1 participation in behavioral impairment induced by chronic corticosterone administration” by Pereira et al., 2023. Leenah + Andrea + Val “Cannabinoid receptor 1 positive allosteric modulator (GAT229) attenuates cisplatin-induced neuropathic pain in mice” by Bagher et al., 2023. 76 Sept 7 Seminar Teams Link 77 Final thoughts… 78