Pharmacology Lecture 2 Notes on Glia, Communication and Transmitters

A 1 PHARMACOLOGY 170 Min 2 Glia cel 3  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...

A 1 PHARMACOLOGY 170 Min 2 Glia cel 3  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) 4 Supporting cells Glia or neuroglia - supply nutrients, physical support and barrier, debris removal, functional interaction (release transmitters) 5 Glia Cells reuptake of neurotransmitters Part of blood-brain barrier 6  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 7 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 8 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) 9 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 10 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) 11  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) 12 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 13  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 14 +40 mV The more EPSPs, the more likely an action -55 mV potential will be initiated The more IPSPs, the more difficult it is to trigger an action potential -70 mV 15 16 “Ya, I know that” Quiz... An IPSP is likely to get triggered if which of the below happens? Answer: D Cl- flows in 17 Ion Channels 18  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 19 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 ) 20  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 21  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 fish toxin (tetrodotoxin, TTX) or not - TTXr (r = resistant)- Nav1.5, Nav1.8, Nav1.9 - TTXs (s = sensitive) – all other Nav 22  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) c 23 w many subtypes of human Kv channels are there??? Answer: – “38” 24  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 25  Ligand Gated Channels Opening and closing depends on presence of ligand 26  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 27  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-4- isoxazolepropionic acid) 3. KA (kainic acid) subtypes 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 28  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 29 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 30  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 31  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 32  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 33 34 Neurotransmitters Part A 35 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 36  Neurotransmitters: Monoamines Dopamine (DA) Norepinephrine/Noradrenaline (NE/NA) Catecholamines Epinephrine/Adrenaline (closely related to NE) Serotonin (5-HT) (synthesized from L-Tryptophan) Histamine (synthesized from histidine) 37  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”) 38  Neurotransmitters: Dopamine - motor function, reward, cognition, learning Dopamine (DA) - three main pathways: - reward/aversion, motivation, pleasure, 1. Nigrostriatal Substantia nigra to striatum cognition, fear, learning, emotion, executive 2. Mesolimbic Ventral tegmental area (VTA) to limbic area functioning 3. Mesocortical Ventral tegmental area (VTA) to cortical regions Dorsal striatum Prefrontal cortex “Reward” pathway 39  Neurotransmitters: Dopamine - DA destruction/reuptake - dopamine transporters (DAT) - monoamine oxidase (MAO-A and MAO-B) – enzyme that degrades DA in presynaptic terminals and astrocytes - catechol-O-methyltransferase (COMT) – enzyme that degrades DA post-synaptic neuron and astrocytes astrocyte G 40  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 41  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 42  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 43  Neurotransmitters: Serotonin Serotonin (5-HT, 5-hydroxytryptamine) - 9 clusters of 5-HT neurons: most found along midline of brainstem B6, B7: dorsal raphe B5, B8: median raphe - brainstem to forebrain B9: dorsal pontine tegmentum B4: dorsal raphe obscurus B1: raphe pallidus - brainstem to spinal cord B2: raphe obscurus B3: raphe magnus - arousal, sleep (dreaming), aggression, cognition, pain, depression, anxiety 44  Serotonin (5-HT, 5-hydroxytryptamine) - 5-HT destruction/reuptake - 5-HT transporters (SERT) MAO-A - monoamine oxidase (MAO-A) 45  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 46  Neurotransmitters: Histamine Histamine - also produced by mast cells and basophils Hnmt (immune cells) - H4 is mainly on immune cells - histamine destruction/reuptake - no specific reuptake transporter - Histamine N-methyltransferase (HNMT) (astrocytes and neurons) 47 48 “Ya, I know that” Quiz... Which of these degrades dopamine in both the postsynaptic neuron and in astrocytes? Answer: A COMT 49 Neurotransmitters Part B 50  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) presynaptic autoreceptors inhibitory - Group III (mGluR4, mGluR6, mGluR7, mGluR8 & glia heteroreceptors 51  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 - Group III (mGluR4, mGluR6, mGluR7, mGluR8 heteroreceptors - throughout the nervous system - glutamate destruction/reuptake - glutamine synthase (enzyme) that breaks glutamate down to glutamine - removed by excitatory amino acid transporters (EAAT1-5) 52  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) 53  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 54  Neurotransmitters: Acetylcholine Acetylcholine (ACh) - 2 major pathways 1) Basal forebrain projects to cortical, thalamic, and limbic sites (amygdala and hippocampus) (sleep, arousal, attention, memory, cognition) 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 55  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 56 What well-known “lifestyle” drug works through reducing ACh release??? 57 AC 58  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 2- arachidonyl glycerol (2-AG) - multiple enzymes for degradation and reuptake by different transporters (depends on lipid) 59  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 60 61 62 Answer C: Somira Talimogene Laherparepvec - Melanoma Linzess – IBS Xgeva – Osteoporosis Farydak – Cancer Idebenone – Alzheimer's Disease 63 Ancient Drug Discovery Modern Drug Discovery Observation Observation Trial and error Trial and error Spreading the word Spreading the word Groups responsible for different elements of Drug Discovery/Development Preclinical Preclinical & Clinical Clinical After FDA approval 64 Step 1: Identify the disease you want to treat 65  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 66  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 Confirm expression of the target in relevant diseased sites GABA expression in “depressed“ mice - 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 interest - “knock-ins” where augment activity/quantity of gene of interest Optogenetic probe in rat 67  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 Robots - typically cell-based assay that must be amenable to large “plate” platforms (96…6144 wells) - do any compounds from your library activate or block the functional assay? - 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 68  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 69  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 cellular assays: - selectivity of compounds against other targets criteria to advance thru funnel - druggability (e.g., pharmacokinetics, solubility) - primary in vivo model systems to mimic key components of the disease 70  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 71  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) 72  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 - 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 non-serious side effects 73  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 population (limited number of people: phase 1b) 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 74 75 “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 76  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) 77  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 78  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: Megha + Monique + Daniel T. “Melatonin alleviates PTSD-like behaviors and restores serum GABA and cortisol levels in mice” by Xu et al., 2023. Nilu + Michelle + Julie “5-HT2A receptors are involved in the pharmaco-toxicological effects of the synthetic cannabinoids JWH-018 and 5F-PB22: In vivo studies in mice” by Corli et al., 2024. 79 80 Final thoughts… 81