Pharmacology (Drug Targets)

Pharmacology (Drug Targets) 4PY019 Dr Ayman Antoun Reyad What is a drug? A drug is a natural or synthetic substance that alters the physiological state of a living organism Most drugs are intended to have a selective action, however, this is rarely occurs and may result in side effects Understanding a drug’s characteristics makes it more predicable to determine its likely benefits and drawbacks of its administration How do drugs work? Drugs work by inducing physiological changes by interacting with the body at a chemical level Most drugs produce their effects by acting on specific targets Some drugs work by a ‘non-specific’ mechanism Drugs acting on specific targets The majority of drugs act on target proteins:     Receptors Enzymes Carrier molecules Ion channels Receptors: • receptors are specific protein molecules expressed by cells • Most located on the cell membrane • Function: interact with chemical messengers and initiate cellular responses Receptor Signalling • Receptors relay external signals to the inside of cells to produce physiological responses. • Although there are many types/subtypes of receptor (nicotinic, muscarinic or α- and β-adrenoceptors etc.), there are only four known mechanisms by which receptors actually work • Each type of mechanism places receptors together into super-families:  Ligand gated ion channel (ionotropic) receptors  G-Protein coupled (metabotropic) receptors  Kinase-linked receptors  Nuclear receptors vesicle neurotransmitter terminal action potential CNS neurone synaptic cleft vesicle fusion postsynaptic membrane agonist receptor binding receptor receptor activation Tissue response Ligand Gated Ion Channels • Plasma membrane proteins • Similar structure to ion channels however they contain a ligand binding site • This ligand binding site forms the receptor site • Therefore the receptor site is directly coupled to the ion channel. •Very quick acting (responses are measured in milliseconds). •Typically involved with neurotransmission. • Examples include: ganglionic and neuromuscular junction nicotinic acetylcholine receptors, gammaaminobutyric acid type A (GABAA) receptor and glutamate receptors Ligand Gated Ion Channels - Molecule Structure Nicotinic Acetylcholine receptor (nAChR): •five protein subunits (2 α, β, γ and δ) • Each subunit contains a membrane spanning segment. • When clustered together the subunits Central form a pore. pore •~ 0.7 nm in diameter. β δ • Each receptor has two αsubunits and the junctions α α γ between them and neighbours form ACh binding sites. ACh binding sites Each receptor has two ACh binding sites. nAChR •Both sites must bind ACh in order to activate the receptor. The Gating Mechanism of the nAChR cytosol gate forming region Cross section view of the nAChR 2 nm • When ACh binds, these kinks are swivelled out of the way, so opening the channel. a b d a 3 nm • These kinks constrict the pore, stopping any ions from crossing the membrane. ACh binding sites 6 nm • The proteins forming the subunits have domains that line the channel of the pore, which are sharply kinked inward. The Gating Mechanism of the nAChR • Like other ligand gated ion channel receptors, the nAChR is responsible for transiently increasing membrane permeability for particular ions. • When triggered, the nAChR increases membrane permeability to Na+ and K+ ions, which can result in cellular depolarization. large number of negatively charged amino residues a a • The lining of the pore is made up from a large number of negatively charged amino acid residues. •This lining makes the pore cation (+ve ion) selective. The Gating Mechanism of the nAChR • The extracellular Na+ ion concentration (150 mM) is much higher than the intracellular levels (15 mM). • The intracellular K+ ion concentration is much higher (150 mM) than the extracellular concentration (5 mM). • So, when the nAChR opens, both Na+ and K+ ions flow down their concentration gradients, however it is the inward rush of Na+ ions mostly responsible for cellular depolarization. • Since the intracellular side of a plasma membrane is negatively charged compared to the extracellular surface, adding positive charge to this negative charge reduces the polarization. •Therefore, if this effect is big enough (enough nAChRs are being triggered), cellular depolarization will occur. • This produces an “all-or-nothing” response + + α α _ _ Na+ D repolarization ACh Na+ + + α _ _ Na+ Na+ Na+ + + + + α _ _ _ _ pore closed Na+ Na+ α _ _ pore closing ACh Na+ + + Na+ Na+ Na+ + _ α _ + ACh B _ _ Na+ Na+ ACh ++ α Na+ depolarization A Na+ nAChR Mechanism of Activation Overview Na+ Na+ Na+ Na+ ACh Na+ Na+ Na+ C bound ACh ACh α _ + _ + pore open So, if enough nAChRs are stimulated on postsynaptic sarcolemmal membranes in neuromuscular junctions, this will cause the depolarization of muscle cell membranes (resulting in muscle contraction) In autonomic ganglia, if enough nAChRs are stimulated on the membranes of the postsynaptic neurones, this will cause neuronal depolarization, with the result of either ACh or noradrenaline being released at nerve terminals. G-Protein Coupled Receptors • This superfamily includes many different types of receptors, e.g. muscarinic ACh, adrenoceptors, dopamine, some 5-hydroxytryptamine (5-HT), purine (adenosine, ATP), opiate, peptide receptors and those used in olfaction. • G-Protein coupled receptors (GPCR) are constructed from a single protein (1,100 residues) and found in the plasma membrane (7 transmembrane spanning receptors). • GPCR are NOT directly coupled to the processes they govern (slower acting: seconds not ms). • These receptors use “G-proteins” to form the link between the receptor and the target process Y R G E D L G D ECI W T G N C R F E F K D G D Y T D N W I H R W P F S ECII A Y V Y Y L P L L A A S D A L Y A V S M F H L T T Y S A C V L L P V S Y G V V C V L G L C L N A V A L Y L F L C R L N K T W Transmembrane Intracellular I R G D G M A A V L K L V R F L F Y T N L Y C S I L F L T C I S V H R C L G V L R R S L Q M D T R S V L G S L R I V T C H D T Extracellular S D L S C S H P R T E ECIII ECIV F L L S N A F Y A S S T R I Y T R T L Y F V F V A N M A F H V T Y S S V K V A P V L F L P A C Q M L G L L T R P L L V L F A V P F A L C A S A F A V W V L A V N S C L I L V C G A V I A V V D P V Y V L V A K S V R T L Y F L A R M R R G A K R A Q A R Y R R L R L P A L L V R K G R G P G F S A R W Y G T A R D A D S R R L G L R K R R A P P T A P S P G T P T E E D S R R S T P A D K T N E S G R G G R T P L spanning region P L D A L G-Proteins • May be thought of as “go-between”proteins, as they form the link between GPCRs and their targets. • G-Proteins acquired their name from their ability to bind GDP and GTP. • Made up of α, β and γ subunits. • The β and γ subunits form a complex together. • These complexes are hydrophobic and reside in the intracellular surface of plasma membranes. • Able to freely diffuse across the plane of the membrane. • The α subunit has three properties: i. Binds guanine nucleotides ii. Forms a loose association with the βγ complex. iii. Catalyse GTP hydrolysis to GDP. GPCR Mechanism of Activation GPCR Resting State. • The subunits of G-proteins are associated together. receptor • In the resting state, the GPCR binding site remains unoccupied β γ α GDP The α subunit binds GDP. The target is inactive. G-protein target GPCR Mechanism of Activation GPCR Agonist Binding. • When an agonist binds to a GPCR, a conformation change occurs This allows the α subunit of a G-protein to bind. The α subunit then exchanges its GDP for intracellular GTP. α GDP GTP β γ target GPCR Mechanism of Activation G-Protein Activation of Target • The activated α subunit dissociates from the receptor and diffuses across the surface plane of the internal plasma membrane. • α subunit interacts with the target protein, activating it. β γ target α GTP target response • The target response will induce a cellular response. GPCR Mechanism of Activation Termination of G-protein Activation • α subunit, by associating with the target, increases its GTPase activity. • Its bound GTP is hydrolysed to GDP and a phosphate group. • This terminates α subunit’s activation, and terminates the target’s activation. β γ target α GDP + P • The α subunit can now re-associate with the βγ complex and if the receptor is still occupied by an agonist, the whole process is ready to be repeated. GPCR Mechanism of Activation Overview receptor A β γ α GDP target G-protein β γ α B target GDP GTP D C β γ target β γ α α GTP GDP + P target target response Additional GPCR Information • Once activated by binding an agonist, a GPCR can activate several a subunits - this amplifies the signal. • It is not just the activated α subunit that can interact with targets, the βγ complex may also activate targets too. • Not all G-proteins are the same, there are several types of G-protein that interact with different receptors and control different targets. • There are three main types of G-protein:  Gs - stimulatory.  Gi - inhibitory.  Gq - activate phospholipase C. GPCR and G-Protein Targets • There are three main targets for activated G-proteins: Ion channels Adenylate cyclase/cyclic AMP system (inhibited by α2, but stimulated by β1+2 adrenoceptors). The phospholipase C/inositol phosphate system (stimulated by α1 adrenoceptors). GPCR Controlled Ion Channels • Muscarinic receptors are GPCRs that bind ACh and on cardiac muscle cells, they will increase membrane permeability to K+ ions. • Activated G-proteins from cardiac muscarinic receptors, cause K+ channels to open. • K+ ions then diffuse out, resulting in cardiac muscle cell hyperpolarization • This therefore makes it harder for cells to depolarize and contract. • Therefore heart rate and force of contraction decreases. Muscarinic Receptor Control of Ion Channels ACh K+ α GTP ion channel ACh β γ K+ channel open K β γ K + K K+ Muscarinic receptor + α GTP + K + K+ K+ + K K+K+ • Currently it is still unresolved whether it is the α or the βγ complex that actually interacts with the ion channel. Adenylate cyclase/cyclic AMP N • Cyclic AMP (3’, 5’-adenosine monophosphate or cAMP) is a nucleotide second messenger. N • Adenylate cyclase (membrane bound) H C 2 O synthesizes cAMP from ATP. O • cAMP is a “second messenger”, whose O concentration varies in response to P O OH Oreceptor activation. NH2 N N • Receptors linked to Gs-proteins stimulate adenylate cyclase, so increase intracellular cAMP levels. • Receptors linked to Gi-proteins inhibit adenylate cyclase, so reduces cAMP levels. • Thus, [cAMP]i can be tightly controlled, which therefore tightly regulate the function of cellular components. • cAMP regulates many cellular functions by activating various protein kinases. • A kinase is an enzyme that catalyses phosphorylation reactions. • ATP is used to power these reactions and to provide the inorganic phosphate groups. • The phosphorylation of cellular inactive components regulates their function. protein kinase Adenylate cyclase cAMP ATP phosphorylation/regulation of cellular components activated protein kinase ATP ADP • cAMP is inactivated by hydrolysis to 5’-AMP by phosphodiesterase enzymes (this enzyme is inhibited by methylxanthines such as caffeine and theophylline). The phospholipase C/inositol phosphate system. • Phosphatidylinositols are a class phospholipids found in membranes. Ptd Ins Inositol ATP Ptd Ins 4-P Ins (1)-P ATP of Ins (1, 4)-P2 ATP Gq-protein PIP2 intracellular phosphatases phosphatidic acid agonist/receptor complex InsP3 phospholipase C release of intracellular Ca2+ DAG • Phosphadtidylinositol -(4, 5)-diphosphate (PIP2) is cleaved by phospholipase C. protein kinase C activation • Both diacylglyerol (DAG) and inositol-(1, 4, 5)-triphosphate (InsP3) are second messengers. • Both DAG and InsP3 are recycled back to PIP2, which takes time (leads to depletion and desensitization during prolonged receptor stimulation). • InsP3 binds to receptors on the endoplasmic reticulum, which opens Ca2+ ion channels, so increasing the cytoplasmic concentration of free Ca2+. • DAG binds to protein kinase C, which controls the phosphorylation (and hence regulation) of proteins such as enzymes etc. Kinase-linked receptors These receptors are linked to tyrosine Kinase enzymes and other kinase enzymes. The most well known ligand that binds to this class of receptor is insulin. Other ligands that bind to kinase-linked receptors include cytokines (autocrine or paracrine protein hormones produced by antigen-presenting cells, endothelial cells, fibroblasts and lymphocytes), growth factors. Kinase-linked receptor basic structure Just like ionotropic and metabotropic receptors, kinaselinked receptors are found in membranes. Each receptor possesses an extracellular and intracellular domain. Each domain contains 400700 amino acid residues. intracellular domain The extracellular and intracellular domains are connected together by a single a-helix that spans the membrane. extracellular domain The extracellular domain contains the receptor’s ligand binding site. The amino acid sequences of these extracellular domains are very variable between kinaseagonist linked receptors. This variability extracellular therefore confers ligand domain selectivity. Intracellular domains are much more ‘conserved’, especially noticeable around the region closest to the membrane. intracellular domain ATP ATP binding site This region is reported to contain a binding site for ATP. Kinase enzymes are responsible for catalyzing the phosphorylation (addition of phosphate groups) to various cellular components. Since these receptors are ‘kinase-linked’ receptors, they therefore require a source of phosphate groups to enable them to work. The ATP molecules, bound to the intracellular domains, provides a source of phosphate groups: ATP ADP + phosphate Ligand binding and receptor activation Due to the receptor’s structure, ligand binding to the extracellular domain does not result in a conformation change through the rest of the receptor molecule (as seen with ionotropic and metabotropic receptors). This is because only a very simple α-helix connects these two domains together. Instead a process of ‘dimerization’ of receptors occurs (a process in which two receptors pair up to form a dimer). Ligand binding and receptor activation agonist binding In the resting state, receptors are not coupled together in a dimer. In order for receptor activation, agonists have to bind to pairs of receptors. ATP ATP Ligand binding results in a conformation change. Receptors can then pair up, forming dimers. ATP ATP The intracellular domain contains tyrosine residues. When a dimer forms, a process of ‘autophosphorylation’ occurs, in which the tyrosine residues become phosphorylated. P T P T ATP ATP T P T P The phosphate groups for this process are donated by the bound ATP. These ‘phosphotryosine’ residues now become high affinity binding sites for various intracellular proteins. The proteins that interact with these receptors all contain highly conserved ‘SH2’ domains (~100 amino acid residues). SH2 domains get their name from ‘Src-homology’ (initial identified in Src oncogens). The bound protein now becomes activated, eliciting a response. P T P T ATP ATP T P T P The Insulin Receptor The insulin receptor is a complex of two α- and two βsubunits. insulin binding Held together by disulphide site bridges. The α-subunits are α-subunits extracellular. The βsubunits are Cys S transmembrane cysteine spanning proteins. residue Cys S Cys S S disulphide bridge Cys Cys S S Cys plasma membrane β-subunits The α-subunits form the insulin binding site. The intracellular portion of the β-units possess tyrosine kinase activity. When insulin binds, the β-subunits autophosphorylate, which enhances the kinase activity of other target proteins. Insulin is a polypeptide hormone (6,000 MW) that is produced in the islets of Langerhans in the pancreas. Insulin is important in regulating blood glucose levels and high blood sugar levels stimulate its secretion. Less than 10% of insulin receptors have to be occupied in order to evoke a maximum response. When insulin binds, clusters of ligand/receptor complexes form, which are then internalized in vesicles. This results in a loss of insulin receptors from the cell surface, which may contribute to the condition of insulin resistance. Cellular events triggered by activated insulin receptors have both short- and long-term actions. The short-term actions are the immediate metabolic effects (recruiting glucose receptors, increasing glucose uptake, increasing glycogen synthesis and reducing both gluconeogenesis and glycogen breakdown). These effects are generally governed by regulating kinase and phospholipase enzymes. Insulin’s long-term actions involve regulating the synthesis of specific mRNA molecules, thus controlling the synthesis of structures such as enzymes, which in turn regulates cellular processes. Nuclear receptors These receptors are not expressed on membranes, instead they are intracellular and involved with regulating DNA transcription (synthesis of RNA). Ligands for nuclear receptors include steroid hormones, thyroid hormone and agents such as vitamin D and retinoic acid. Hormones are produced by endocrine glands, which are then passed into the blood and distributed around the body, eventually binding to their receptors. Examples of hormones: sex hormones and adrenaline Basic nuclear receptor structure These receptors are large monomeric proteins containing 400-1000 amino acid residues. The middle portion of ~60 residues is highly conserved. COOH Zn2+ It contains two finger shaped loops of peptide chain, each being 15 residues long. Zn2+ NH2 These are referred to as ‘zinc fingers’ because each contains a Zn2+ ion. The receptor’s carboxyl end contains the steroid binding domain. The zinc finger region forms the DNA binding region, as the fingers are believed to be able to wrap around DNA helixes. COOH ligand binding domain ‘zinc finger’ domain Zn2+ Zn2+ regulatory domain NH2 The amino end is the receptor’s regulatory domain that can activate the transcription of specific genes. Ligand binding and mechanism of action Steroid hormones and drugs all operate by triggering the transcription of specific genes, which leads to the synthesis of proteins. Since nuclear receptors are intracellular, their ligands have to be lipid soluble to pass through plasma membrane. steroid receptor steroid cytoplasm nucleus nuclear Steroid molecules are membrane highly lipid soluble and nuclear easily gain access to the pore intracellular compartment, where they can bind to their receptors in the nucleus. plasma membrane When a steroid binds to its receptor, it induces a conformation change in which the receptor unfolds to expose the zinc finger domain. The ligand/receptor complex can now bind to specific portions of DNA. These binding positions are well defined regions of the DNA that contain ‘hormone responsive elements’. steroid/receptor complex hormone responsive nuclear elements membrane DNA double helix These hormone responsive elements are ~200 DNA base pairs upstream from the genes that are regulated. Within minutes of ligand/receptor complex binding to DNA, there is an increase in RNA polymerase activity. This results in the production of mRNA . This mRNA leaves the nucleus to form a complex with ribosomes. steroid/receptor complex binding to DNA new mediator protein translation ribosome transcription mRNA This enables the synthesis of new protein Therefore the actual physiological responses maybe delayed by several hours or even days, as the whole process is relying on protein synthesis. Glucocorticoids, mineralocorticiods, sex hormones, thyroid hormones, vitamin D3 and retinoic acid are all agents that bind to nuclear receptors. Interacts with DNA and modify gene transcription. Almost every type of tissue contains receptors that can bind to glucocorticoids. Depending on cell type, it is thought that between 10-100 genes of each cell are steroid responsive.