Drug Targets Student Study Guide

4PY019 Drug Targets TBL Pack Drug Targets Student Study Guide Facilitators Required References Names Required Dr Ayman Antoun Reyad This reading pack Dr Waseem Kaialy Drug targets Ligand-gated ion channels as drug targets G protein coupled receptors as drug targets Tyrosine kinase receptors as drug targets Nuclear hormone receptors as drug targets Contact Details a.antounreyad@wlv.ac.uk w.kaialy@wlv.ac.uk Optional Pharmacology 9th edition 2018 eds Rang, Dale, Ritter, Flower, Henderson. Chs 1-5 Learning Outcomes This learning pack considers drug targets and how they initiate changes in cell function when occupied by agonist molecules (neurotransmitters, hormones, growth factors). On completion of this topic, you should be able to explain: Concept of ligand-gated channels, and describe the steps of neurotransmission at the mammalian neuromuscular junction The structural features of G Protein coupled receptors and their mechanism of action The structural features of tyrosine kinase receptors and their mechanism of action The structural features of nuclear receptors and their mechanism of action Various receptor-effector mechanisms; receptors linked to ion channels, to G-proteins or to protein kinases. Team-based Learning 1 4PY019 Drug Targets TBL Pack Content 1. Ligand gated ion channels 3 2. G protein coupled receptors 8 3. Kinase Receptors 13 4. Nuclear receptors 18 5. Enzymes as drug targets 22 6. Carrier molecules as drug targets 25 2 4PY019 Drug Targets TBL Pack Drug Targets Receptors are molecular structures within a cell or on its surface membrane, which are characterised by the selective binding of specific substances (agonists). Such binding evokes a specific physiologic effect. Examples include receptors for neurotransmitters, peptideand steroid hormones. Receptors therefore enable extracellular signals to be relayed to the inside. There are four main types of receptors (each type is known as a superfamily): 1. ligand gated ion channels, 2. G protein coupled receptors, 3. kinase receptors, 4. nuclear/intracellular receptors 1. Ligand gated ion channels Ligand gated ion channels are also known as ionotropic receptors. These receptors are directly coupled to ion channels. Examples of these receptors include nicotinic (studied in more detail below), GABAA, GABAC and glutamate receptors, which are all fast acting and involved with neurotransmission. The endogenous agonist for the nicotinic receptor is acetylcholine (ACh). This receptor gets its name from the fact that nicotine was the first drug that was able to distinguish it from the other receptor (muscarinic) that ACh could bind too. Location Nicotinic receptors are found on the plasma membranes of cells and are one of the most numerous receptors in the body. They occur in the neuromuscular junction (NMJ), in autonomic ganglia and in the central nervous system (CNS). Structure of the Nicotinic Receptor One of the most studied nicotinic receptor is the one that occurs in the electroplax organ of the Torpedo California electric fish. This receptor is made up of a pentamer of four different subunits (Fig. 1): 2 x α (40KD) β (49 KD) ε (57 KD) δ β Fig. 1. Structure of the δ (65 KD) extracellular domain of a nicotinic receptor, viewed from above. Each α-subunit can bind α α one ACh molecule ACh binding sites ε The molecular structures of the nicotinic receptors that occur in different locations of a human body are very similar. The differences are accounted for by the existence of several variants of the α-, β- and δ- subunits. In addition to this, neuronal and NMJ 3 4PY019 Drug Targets TBL Pack receptors can have more diversity by including different α- subunits. ACh actually binds to the α-subunits in order to trigger the receptor. Although agonists show little selectivity between nicotinic receptors that occur in different locations, they can be distinguished from each other pharmacologically. The drug decamethonium is an exception to this rule, since it is a potent agonist at the NMJ, but is a weak antagonist on autonomic ganglia. Antagonists, however, show strong selectivities since very low concentrations of βbungarotoxin blocks NMJ nicotinic receptors, but has no effect on autonomic ganglia. Mecamylamine is an antagonist in the CNS and on autonomic ganglia, however it has no effect on the NMJ. Function As with all ionotropic receptors, ligand binding and channel opening occur on a millisecond time scale. So, when ACh binds to a nicotinic receptor, it causes the ion channel to open very quickly, which increases the plasma membrane’s Na+ and K+ permeability, so depolarizing the cell and increasing the probability that an action potential will be generated (Fig. 2). The inward rush of Na+ ions, down its concentration gradient, is mostly responsible for this depolarization. The intracellular side of a plasma membrane is negatively charged compared to the extracellular surface (mostly due to the uneven distribution of Na+ and K+ ions set up by the Na/K ATPase pump), so an inward rush of positively charged Na+ ions will reduce this negative membrane potential, depolarizing the cell. It has been found that motor endplate nicotinic receptor channels are only open for 12 milliseconds, which is equivalent to an influx of 1x107 ions per second per channel. Fig. 2. Schematic A diagram of a nicotinic receptor in a postsynaptic membrane. ‘A’ shows the receptor in the unbound state with its ion channel closed. ‘B’ and ‘C’ represents the binding of ACh. ‘D’ shows the conformation change in the receptor opening the ion channel, so increasing membrane permeability to Na+ and K+ ions. Each transition state is completely reversible. B + ACh binding ACh Na site extracellular ACh Na+ ACh lipid bilayer intracellular + K D channel closed ACh ACh + K The NMJ Na+ C Na+ channel open K+ ACh channel closed ACh + K channel closed The function of the NMJ is to transmit action potentials, conducted along motor (somatic) nerves, across the synaptic clefts that occur in motor endplates. Unlike ganglia synapses, the postsynaptic membrane is not part of another neurone, but instead it is the sarcolemma of a skeletal muscle fibre. The postsynaptic region of the sarcolemma 4 4PY019 Drug Targets TBL Pack contains many invaginations, which increases the total surface area of the membrane in this region, thus increasing its exposure to ACh. ACh synthesis occurs in the motor neurone terminal and is catalysed by the cytosolic enzyme choline acetyltransferase (C.A.T.). Acetyl groups are donated by acetylcoenzyme A (acetyl CoA), which are then covalently bonded to choline to form ACh. The ACh is then stored in vesicles, ready for release. When action potentials conducted along the motor nerves reach their nerve terminals, depolarization causes an influx of Ca2+ ions, which triggers ACh release into the synaptic cleft, as vesicles now fuse with the terminal membrane (Fig. 3). n-type 2+ Ca channel motor neurone terminal CNS axon post synaptic membrane Ca2+ Acetyl CoA + choline synaptic cleft mitochondrion C.A.T. ACh choline uptake process ACh ACh choline + acetic acid sarcolemma invagination ACh ACh containing vesicle fusing to membrane Na A SI + nicotinic receptor acetylcholinesterase Fig. 3. Schematic diagram of the NMJ. Released ACh can now diffuse across the synaptic cleft and bind to nicotinic receptors on the sarcolemma, causing a depolarization, which if large enough can stimulate an action potential in the muscle fibre, causing it to contract (referred to as the endplate potential or EPP). ACh in the synaptic cleft also interacts with the enzyme acetylcholinesterase, which hydrolyses it to choline and acetic acid. This process occurs the whole time ACh is present in the synaptic cleft. So, when no more ACh is being released from the neurone terminal, acetylcholinesterase reduces the synaptic concentration of ACh, terminating the response evoked. Approximately 50% of the choline produced is then recaptured by the nerve terminal, which is then synthesized into ACh again. Nicotinic Receptor Pharmacological agents The following two tables list some of the drugs that affect nicotinic receptors and ACh neurotransmission. 5 4PY019 Drug Targets TBL Pack Table 1. Nicotinic agonists and antagonists. Agonists Antagonists Neuromuscular junction Autonomic ganglia Tubocurarine Pancuronium -Bungarotoxin Atracurium Vecuronium Trimetaphan Hexamethonium ACh Nicotine Suxamethonium Decamethonium ACh Nicotine Lobeline Epibatidine Table 2. Drugs affecting ACh neurotransmission. Agents reducing ACh release: Transmission potentiating agents: Hemicholinium Anticholinesterases: Botulinum toxin Pyridostigmine Mg2+, Mn2+ and Co2+ ions Neostigmine Aminoglcoside antibiotics Edrophonium Distigmine Competitive Neuromuscular blocking Ambenonium Physostigmine (eserine) agents: Tubocurarine Dyflos Gallamine Pancuronium Choline carrier inhibiting agent (synthesis Alcuronium inhibitor): Atracurium Hemicholinium Vecuronium ACh release enhancing agent: 4-aminopyridine Depolarizing: Neuromuscular blocking agents: Suxamethonium Decamethonium Note: Suxamethonium and decamethonium appears in both tables 1 and 2. Tubocurarine is found as a mixture of alkaloid substances called ‘curare’, extracted from the Chondrodendron tomentosum plant that grows in the rain forests of South America. Native Indians use curare to treat their arrow tips before using them for hunting. Myasthenia gravis Myasthenia gravis is a chronic disease characterized by an abnormal fatigability and weakness of skeletal muscles, which chiefly affects adolescents and young adults (under 40 years old). Other symptoms include ptosis (drooping of the upper eyelid), double vision and dysarthria (a speech disorder in which pronunciation is unclear). It is an autoimmune 6 4PY019 Drug Targets TBL Pack disease affecting neuromuscular transmission. IgG antibodies cause the loss of nicotinic receptors in the NMJ. The fatigue experienced might become so extreme that muscles become temporarily paralysed. Treatment for this condition include: administering anticholinesterases, using prednisolone or azathioprine, performing plasmaphoresis and surgically removing the thymus. Review ‘Ligand-gated ion channels as drug targets’ and make brief notes here. 7 4PY019 Drug Targets TBL Pack G-Protein Coupled Receptors G-protein coupled receptors (GPCR) can also be called metabotropic, 7-transmembrane spanning receptors; examples include: muscarinic, adrenergic, dopaminergic, 5hydroxytryptamine (5-HT), purine (adenosine, ATP) and opiate receptors. In addition to these, many receptors for peptides and olfaction are also GPCRs. Unlike ionotropic receptors, which are directly connected to their ion channel, GPCRs are indirectly connected to the processes that they govern. So, these receptors use G-proteins to form the link between the receptor itself and the ‘target’ process that is under control. GPCR structure GPCRs have a similar basic structure, consisting of seven transmembrane spanning regions that are connected by extracellular and intracellular loops. These receptors are three dimensional structures that arrange their extracellular loops and some of the transmembrane regions into pockets that can accept ligands. The extracellular domains of GPCRs are very varied, so meaning that a large number of diverse receptors exist, all with different affinities for ligands. The one thing that these domains have in common is the fact that they contain many disulphide bonds, which maintains the three dimensional structure. G-proteins You can think of G-proteins as being ‘go’-between proteins, however they actually get their name from the guanine nucleotides GDP and GTP. G-proteins made up of alpha-, betaand gamma-subunits. The beta- and gamma-subunits form a complex together and, since they are very hydrophobic, they are found residing in a cell’s plasma membrane, where they are able to freely move about. The alpha-subunit has three properties: i) it can bind to guanine nucleotides, ii) it forms a loose association with the beta/gamma complex and iii) it has the ability of catalysing the hydrolysis of GTP to GDP. When an agonist binds to its receptor, a conformation change is induced within the receptor that enables alpha-subunit binding (Fig. 4). Once bound, the alpha-subunit releases its GDP and binds instead to GTP. This activated alpha-subunit now completely dissociates from the receptor and its beta/gamma complex, allowing it to diffuse across the membrane surface to stimulate its target. By associating with the target the alphasubunit’s GTPase activity is increased, so the bound GTP is hydrolysed to GDP and an inorganic phosphate group, terminating the alpha-subunit’s activation. The alpha-subunit can now re-associate with the beta/gamma complex and the whole process is now ready to be repeated. Although GPCRs are not as rapidly acting as ionotropic receptors (they work on a second time scale, rather than a millisecond scale), their advantage is that they can amplify a signal. Basically this means that one agonist/receptor complex can activate several alphasubunits and these in turn can go on to stimulate several targets, thus producing amplification. Stimulation of a GPCRs can produce either stimulatory or inhibitory effects, and this is determined by what sort of receptor is controlling what type of target. Therefore it is apparent that not all alpha-subunits are the same, otherwise activated alpha-subunits from completely different agonist/receptor complexes would interfere with each other’s targets. To date over twenty different alpha-subunits have been identified, however, the 8 4PY019 Drug Targets TBL Pack actual G-proteins fit into three categories: Gs, Gi and Gq. Gs and Gi proteins produce either stimulation or inhibition of their targets respectively. Activated Gq proteins activate phospholipase C (see below). A B extracellular target β γ α GDP unbound receptor conformation agonist α G-protein complex bound GTP receptor conformation D β γ target GDP C β γ A SI phosphate target A β γ GDP target α GTP Fig. 4. The G-protein cycle. ‘A’ represents the resting state. ‘B’ shows agonist/receptor binding, with the receptor conformation change allowing alpha-subunit binding and activation. ‘C’ shows an activated alpha-subunit interacting with its target and ‘D’ represents the cessation of activation when GTP is hydrolysed into GDP and a phosphate group. G-protein targets The three main targets of G-proteins are: • Ion channels. • The adenylate cyclase/cyclic AMP (cAMP) system. • The phospholipase C/inositol phosphate system. GPCRs Commonly Encountered in the Cardiovascular System Muscarinic receptors, named after the drug muscarine (extracted from the Amantia muscaria mushroom), are found on cells and tissues innervated by the parasympathetic nervous system. Like nicotinic receptors, ACh also binds to muscarinic receptors. The word ‘cholinergic’ refers to ACh, so cholinergic receptors can be divided into nicotinic and muscarinic subtypes. These different receptors can be distinguished from each other by the binding of different drugs (Tables 2 & 3). The parasympathetic nervous system can be thought of as the body’s “resting and digesting” system and so when the muscarinic receptors of the heart are stimulated (either by the vagus nerve releasing ACh or by drugs), this reduces heart rate and force of contraction. 9 4PY019 Drug Targets TBL Pack Table 3. Drugs acting on muscarinic receptors. Agonists ACh Carbachol Pilocarpin e Antagonist s Atropine Hyoscine Ipratropiu m Muscarinic receptors found on cardiac muscle cells are known to increase membrane permeability to K+ ions, which reduces the rate and force of contraction. So, once ACh binds to muscarinic receptors, activated G-proteins can open K+ channels (Fig.5). As K+ ions diffuse out of the cell, down their concentration gradient (~150 mM intracellular concentration/~5 mM extracellular concentration), the intracellular compartment looses positive charge and so becomes more negatively charged. This hyperpolarization therefore makes it harder for cells to depolarize and contract. + ACh K ion channel A GTP K+ + K muscarinic G-protein receptor B K+ K+ channel open K+ GTP K+ K+ K+ Fig. 5. A schematic diagram of a muscarinic receptor. In the resting state the K+ ion channel is closed. Once ACh binds to the receptor and the G-protein becomes activated (A), the G-protein can open the K+ ion channel, thus hyperpolarizing the cell (B). The exact mechanism behind the coupling of GPCRs and ion channels is still unresolved. In the above description and diagram, it seems that activated alpha-subunit links the receptor and ion channel together, however this is still a matter for debate as it is possible that beta/gamma complex maybe the important link. It is however believed that similar mechanisms operate for opiate receptors, since it is known that opiate analgesics open K+ channels to reduce neurone excitability. Adrenergic receptors acquired their name from the fact that the neurotransmitter noradrenaline and the hormone adrenaline can bind to them. The British convention still uses the names noradrenaline and adrenaline. The recommended international nonproprietary names are now, norepinephrine and epinephrine. This class can be divided into α and β- subtypes, which can then be further divided into α1, α2, β1 and β2 subtypes. These receptor subtypes can be distinguished from one another by the different levels of responses obtained by different drugs selectively binding to them (Table 4). 10 4PY019 Drug Targets TBL Pack Table 4. Adrenergic receptor specificities for (A) agonists and (B) antagonists. (A) Agonists Adrenoceptors α1 α2 β1 β2 Noradrenaline +++ +++ ++ + Adrenaline ++ ++ +++ +++ Phenylephrine ++ Clonidine +++ Isoprenaline +++ +++ Dobutamine +++ + Salbutamol + +++ (B) Antagonists α1 +++ +++ +++ + Adrenoceptors β1 2 α + ++ +++ β2 ++ Labetalol Phentolamine Prazosin Yohimbine +++ Propranolol +++ +++ Atenolol +++ + Butoxamine + +++ Note: See how some drugs can have effects at more than one receptor subtype. In the body, adrenergic receptors are generally considered to be involved with the sympathetic nervous system. The effects of sympathetic stimulation can most easily be remembered by thinking about the “fright or flight” response. So when the adrenergic receptors on the heart are stimulated, rate and force of contraction increase. The distribution and function of the subtypes of these adrenergic receptors is different around the body. For instance, α1-adrenoceptors are generally stimulatory and in blood vessels will cause a vasoconstriction. α2-adrenoceptors are generally found on presynaptic nerve terminals and negatively feedback on neurotransmitter release. β1adrenoceptors are mostly concentrated in cardiac tissue and stimulate the heart to beat faster and with more force. β2-adrenoceptors generally induce tissues to relax e.g., bronchodilation in lung airways and vasodilatation in blood vessels that supply muscles. Like cardiac muscarinic receptors, adrenoceptors send signals to parts of cells to induce a response. Unlike cardiac muscarinic receptors, which are fairly closely coupled to their ion channels, adrenoceptors use a system of “second messengers”. Second messengers are agents whose intracellular concentrations vary, depending on the levels of receptor stimulation. These agents, once produced or released, can diffuse away from the cell membrane to alter the rate at which cellular processes occur within the cell. Common second messengers include Ca2+ ions, cyclic adenosine monophosphate (cAMP), inositol1, 4, 5-triphosphate (InsP3) and diacylglycerol (DG). So, for example, when βadrenoceptors are stimulated, cAMP is formed from ATP by the enzyme adenylate cyclase. cAMP then activates the enzyme protein kinase A, which then phosphorylates proteins (enzymes, muscle fibres, ion channels etc.) to produce a physiological effect. 11 4PY019 Drug Targets TBL Pack Review ‘G protein coupled receptors as drug targets’ and make brief notes here. 12 4PY019 Drug Targets TBL Pack Kinase Linked Receptors These receptors are often linked to tyrosine kinase. The most well known ligand that binds to a kinase receptor is insulin. Other ligands for this class of receptor include various different growth factors. Kinase-linked receptors are found bound to membranes. Basic structure of kinase-linked receptors Kinas-linked receptors possess extracellular and intracellular domains, with each domain containing between 400-700 amino acid residues. These two domains are connected together by a single alpha helix that spans the thickness of the membrane (Fig. 6). Not all kinase receptors are constructed in the same way. It is known, for example, that the extracellular domain of an insulin receptor is made from a single polypeptide chain that is then attached, via disulphide bridges, to a second polypeptide chain that forms the transmembrane spanning alpha helix and the intracellular domain. Alternatively, the kinase receptors that bind to growth factors are constructed from a single long polypeptide chain, which can be over 1,000 residues long. Extracellular domains form the receptors’ ligand binding sites, whilst the intracellular portions form the tyrosine kinase domains. extracellular ligand binding site extracellular domain membrane intracellular A SI tyrosine kinase domain transmembrane alpha helix T tyrosine residue T ATP binding site Fig. 6. A generalized schematic diagram of a kinase-linked receptor, showing the extracellular and intracellular domains connected together by a single membrane spanning alpha helix. Since the extracellular domains contain a ligand binding site, the amino acid residue sequences of these regions are very variable between different receptor subgroups, thus producing ligand selectivity. Intracellular domains, are much more ‘conserved’ (much more sequence homology between subgroups), which is noticeable around the region closest to the membrane. This region is reported to contain a binding site for ATP (Fig. 6). Kinase enzymes are responsible for catalyzing the phosphorylation of various cellular components. This process requires a source of phosphate groups, which the bound ATP can provide. Ligand binding Due to the receptor’s structure, ligand binding to the extracellular domain does not result in a conformation change in the intracellular domain, since only a very simple alpha helix connects these two together. Instead ligand binding results in a ‘dimerization’ of receptors (a process in which two receptors pair up together to form a dimer). When this happens (Fig. 7), a process of ‘autophosphorylation’ occurs, which basically means that the tyrosine 13 4PY019 Drug Targets TBL Pack residues of the intracellular domains become automatically phosphorylated, with the phosphate groups being donated by the bound ATP. The phosphorylated tyrosines, which are sometimes referred to as phosphotyrosine residues, now become high affinity binding sites for various intracellular proteins. The various intracellular proteins that interact with kinase receptors all have one feature in common, which is that they contain highly conserved ‘SH2’ domains. These special domains contain about 100 amino acid residues and their name is derived from “Srchomology”, since it was first identified in products obtained from Src oncogenes. An oncogene is a mutated and/or over-expressed version of a normal gene in a cell, which is free from normal growth restraints, thus converting the cell into a tumour cell. Src oncogenes are the transforming (sarcoma inducing) gene of the Rous sarcoma virus. The gene’s product is a cytoplasmic protein with specific tyrosine kinase activity, which associates with the cytoplasmic face of the plasma membrane. With great selectivity, the various cellular proteins containing SH2 domains bind to the intracellular domains of the kinase-linked receptors and it is these substrate binding sites that incorporates the phosphotyrosine residues (Fig. 7). These bound SH2 domain containing proteins now become activated. The SH2 proteins that bind to kinase-linked receptors include enzymes such as other protein kinases and phospholipases. Some growth factors are known to trigger the activation of phospholipase C, which stimulates the production of diacylglycerol and inositol triphosphate. Other SH2 containing proteins behave like adapter molecules that form a link between activated receptors and a wide variety of cellular proteins. Many of these proteins play a role in controlling cell division and differentiation, and they do this by stimulating the transcription of particular genes. agonist conformation change A B and dimer formation T ATP ATP type 3 receptors T T T T T T C T T RESPONSE ATP T D A SI ATP ATP ATP P P T P T SH2 domain containing protein 14 P T P T ATP ATP T P T P tyrosine autophosphorylation 4PY019 Drug Targets TBL Pack Fig. 7. Ligand/receptor interactions. ‘A’ represents receptors in their resting state before ligand binding. ‘B’ demonstrates a conformation change occurring in the external domains, once ligands have bound, and the formation of a dimer. Autophosphorylation of the dimer’s tyrosine residues then occur (C), followed by the binding of a protein containing a SH2 domain (D). Cytokines are small proteins or biological factors that are released by cells and have specific effects on cell-to-cell interactions. So, cytokines are important for co-ordinating cellular communication and behaviour. Cytokines are not really different from hormones, but the term is convenient to use for agents such as interleukins and several related signalling molecules such as tissue necrosis factor (TNF) and interferons. Many cytokines act on receptors that have a fairly similar structure, although they lack the intracellular tyrosine kinase domain. The operation of these receptors, however, still utilizes a tyrosine phosphorylation mechanism. Experimental evidence suggests that when cytokine ligands bind to these receptors, receptor dimerization once again occurs, after which cytosolic tyrosine kinase units are then able to bind. Following this, SH2 containing proteins bind and become activated, so producing the cellular response. Guanylate cyclase/cyclic guanosine monophosphate system Just like cAMP, cyclic guanosine monophosphate (cGMP) is also a second messenger. The most famous activator of guanylate cyclase, the enzyme that catalyses the synthesis of cGMP, is nitric oxide. The membrane bound form of this enzyme, however, has a structure similar to the tyrosine kinase family. It has therefore been hypothesized that when peptide agonists, such as atrial natriuretic peptide binds to its receptor, the production of cGMP occurs via a tyrosine phosphorylation process. The insulin receptor Insulin is a polypeptide hormone (6,000 MW) that is produced by the beta cells of the islets of Langerhans in the pancreas. Insulin is very important in regulating blood glucose concentration, with high blood sugar levels stimulating its secretion. Insulin binds to specific receptors on the surface of its target cells. The insulin receptor comprises of four glycoprotein molecules, made up of two α- and two β- subunits, and has a total molecular weight of about 350,000 (Fig. 8). The subunits contain cysteine residues that enable the formation of disulphide bridges, which is important for keeping the complex bound together. insulin α-subunits Cys Cys cysteine residue A SI S S S S Cys Cys disulphide bridge Cys S S Cys plasma membrane β-subunits 15 4PY019 Drug Targets TBL Pack Fig. 8. The insulin receptor, which is a complex of two α- and two β-subunits held together by disulphide bridges. Insulin binds to the extracellular α-subunits. The β-subunits are transmembrane proteins. The α-subunits of the insulin receptor are extracellular and form the insulin binding site. The β-subunits have both extracellular and intracellular domains, and it is their intracellular portion that possesses tyrosine kinase activity. When insulin binds to its receptor, the βsubunits autophosphorylate, which enhances the kinase activity of other target proteins. Insulin receptor binding Less than 10% of insulin receptors have to be occupied to evoke the 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 go some way to explain insulin resistance (see below). The cellular events triggered by activating insulin receptors are very varied and involve several diverse pathways, since insulin can evoke both short- and long-term actions. Insulin’s short-term actions are generally considered to be its immediate metabolic effects, which are governed by regulating kinase and phospholipase enzymes, via the tyrosine kinase activity of the receptor. Insulin triggers the recruitment of glucose transporters, which increases glucose uptake by cells, as well as increasing glycogen synthesis, reducing both gluconeogenesis and glycogen breakdown. Activated insulin receptors may also trigger a specific phospholipase C, which cleaves glycosylphosphatidylinositol (NOTE: this is different to phosphatidylinositol (4, 5)-diphosphate). Cleavage forms DAG, but instead of inositol triphosphate being produced, inositol phosphoglycan (IPG) is formed instead. DAG activates protein kinase C, whilst IPG regulates the activity of kinases enzymes. Insulin’s long-term actions involve regulating the synthesis of specific messenger RNA molecules, thus controlling the synthesis of important molecules such as enzymes, which in turn regulates cellular processes such as cell replication/proliferation. 16 4PY019 Drug Targets TBL Pack Review ‘Kinase linked receptors as drug targets’ and make brief notes here. 17 4PY019 Drug Targets TBL Pack Nuclear Receptors Nuclear receptors are very different from the receptors mentioned previously, since they are not expressed on cell membranes. Instead, these receptors are intracellular and are involved with regulating DNA transcription. Steroid hormones, thyroid hormone and agents such as vitamin D and retinoic acid can bind to them. Neurotransmitters or hormones? Since both neurotransmitters and hormones act on receptors, why are they distinguished apart and not simply lumped together in a single category? Well, basically this is because neurotransmitters and hormones are distributed differently. Neurotransmitters are released from neurone terminals and transmit action potential signals across synapses. Such synapses are minute and occur between neurone terminals and a wide variety of postsynaptic cells such as other neurones, muscle fibres and glands. Hormones, however, are substances that are produced by endocrine glands, which are then passed into the blood, where they are distributed around the body to modify the structure or function of specific organs or tissues. However, this method of dividing up substances into either neurotransmitters or hormones is not very precise, since it now appears that a spectrum of agents exists, with ‘classical’ hormones (sex hormones) at one end and ‘classical’ neurotransmitters (acetylcholine) at the other. NOTE: Adrenaline (called epinephrine in American textbooks) is classed as a hormone. It is the chief hormone produced by the medulla of the adrenal glands. It has the function of preparing the body for ‘flight or fight’ responses and has widespread circulatory, muscular and metabolic effects. Adrenaline will induce tachycardia and has a positive ionotropic effect on the heart. It also makes breathing easier, raises the body’s metabolic rate, enhances muscular force of contraction and delays the onset of fatigue. In addition to this, blood is diverted away from non-essential areas such as the bladder and intestines (their muscular walls relax and their sphincters contract). Originally nerves of the sympathetic autonomic nervous system were thought to act by releasing adrenaline at their terminals, and were therefore called ‘adrenergic’ nerves. It is in fact noradrenaline that is the main substance released from adrenergic neurones. Since adrenaline is an agonist for α- and β-adrenoceptors, it is very easy to start thinking of adrenaline as a neurotransmitter instead of a hormone! Basic receptor structure Nuclear receptors are large monomeric (consisting of a single component) proteins, containing between 400-1,000 amino acid residues (Fig. 1). The middle portion of the molecule contains a highly conserved region of approximately 60 residues. It contains two finger shaped loops of peptide chain, each being 15 residues long, which forms what are termed ‘zinc fingers’. This is because each finger contains a zinc ion, which is held in place by four cysteine residues. This zinc finger domain forms the receptor’s DNA binding region, since the fingers are believed to be able to wrap around DNA helixes. The carboxyl (-COOH) end of the protein contains the steroid binding domain, whilst the amino (-NH2) end is the receptor’s regulatory domain that can activate the transcription of specific genes. 18 4PY019 Drug Targets TBL Pack COOH Fig. 1. A generalized structure of a steroid receptor. The receptor, Zn2+ made from a single protein can be divided into three domains: a Zn2+ ligand binding domain, a zinc finger domain and a regulatory domain. The zinc finger domain contains two NH2 loops of the peptide chain and forms the fingers, which surrounds the zinc ions (as emphasized in the enlarged insert). ligand binding domain & hinge region ‘zinc finger’ domain regulatory domain Ligand binding and mechanism of action Steroid drugs and hormones all operate by the same basic mechanism i.e.; they trigger the transcription of specific genes, which leads to the synthesis of proteins (Fig. 2). Since nuclear receptors are intracellular, this means that their ligands have to be lipid soluble in order to pass through a cell’s plasma membrane. Steroid molecules are highly lipid soluble and easily gain access to the intracellular compartment, where they can then bind to their receptors located in the nucleus. When a steroid binds to its receptor, it induces a conformation change in which the receptor unfolds to expose the zinc finger domain. Ligand/receptor complexes can now bind to specific portions of DNA. These binding positions are well defined regions of the DNA that contain ‘hormone responsive elements’ or ‘transcription factor activator proteins’, which occur approximately 200 base pairs upstream from the genes that are regulated. Within minutes there is an increased RNA polymerase activity, resulting in the production of messenger RNA (mRNA). This mRNA leaves the nucleus to form a complex with ribosomes, enabling the synthesis of protein. Therefore actual physiological responses maybe delayed by several hours or even days, since the whole process is relying on protein synthesis. 19 4PY019 Drug Targets TBL Pack Fig. 2. Direct gene activation by steroids. (A) Lipid soluble steroids diffuse through the plasma membrane and bind to nuclear receptors. (B) The activated steroid/receptor complex can now bind to DNA at sites determined by hormone responsive elements. (C) Transcribed mRNA passes out of the nucleus, binds to ribosomes and new proteins are synthesized. Glucorocorticoids, mineralocorticoids, sex hormones, thyroid hormones, vitamin D3 and retinoic acid are all agents that bind to nuclear receptors, interact with DNA and modify gene transcription. Almost every type of tissue contains receptors that can bind to glucocorticoids and, depending on cell type, it is thought that between 10-100 genes of each cell are steroid responsive. Glucocorticoids bind to their receptors with very high affinity and each cell may contain between 3,000-10,000 of these receptors. Different steroid hormones bind to different receptors so activating different genes, which obviously results in the synthesis of different proteins, thus producing different physiological effects. So, for example, glucocorticoids are known to stimulate the production of annexin-1 (lipocortin-1), which inhibits the activity of phospholipase A2, thus reducing the formation of arachidonic acid. This may account for the anti-inflammatory effects of glucocorticoids, since arachidonic acid is the precursor of 20 4PY019 Drug Targets TBL Pack the prostanoids, leukotrienes and platelet activating factor. Mineralocorticoids, however, stimulate the production of various transport proteins that are involved in renal tubular function. Not all steroid/receptor complexes bind to DNA to initiate mRNA transcription. Some complexes, when bound to DNA repress the transcription of particular genes, thus inhibiting the synthesis of certain proteins, so producing the physiological effect. 21 4PY019 Drug Targets TBL Pack Enzymes Not all drugs, however, exert their effects by acting via receptors; in fact many drugs act on enzymes or cell transporter molecules to produce their effect. You already encountered an example of this type of drug. This was a class of drugs called the anticholinesterases, which can be used to treat myasthenia gravis. You will learn in more detail about drugs that target enzymes as well as study transporter molecule modifying agents. An introduction to enzymes Enzymes are globular proteins that are the catalysts of biological systems. A catalyst is a substance that alters the rate at which a chemical reaction occurs, but is itself not used up or changed by the end of the reaction. Catalysts and enzymes do not alter the equilibrium position of a reaction nor increase the product yield; they can only alter the rate at which the equilibrium is achieved. Also, catalysts cannot force reactions to occur between molecules that are normally unable to react together. A good analogy is to think of enzymes as being ‘molecular bellows’, which can be used to ‘fan the flames’ of slow molecular reactions into roaring activity! Without enzymes, biochemical reactions could still take place, but they would proceed at a pace that would be too slow to sustain life. So, for all intents and purposes the rule ‘no enzyme – no reaction’ applies. Enzymes are highly specific and will only catalyze a single chemical reaction or a small group of reactions. Therefore the presence of enzymes in a biological system not only speeds up a reaction, but ensures that only particular reactions occur, removing the possibility of unwanted reactions and products being formed. Chemical reactions require a certain amount of energy in order to get underway and this is called the activation energy (Fig. 4). So, once this activation energy has been absorbed, it pushes the energy levels of the reactants up to a point at which their random collisions are forceful enough to cause a reaction. One obvious non-biological way of doing this in a laboratory is to increase the reactants’ temperature. In biological systems, this is not a practical solution, since raising temperatures too much will result in denatured proteins. Enzymes allow reactions to occur without any abnormal increases in temperature and they achieve this by lowering the amount of activation energy that is required. This is accomplished by two ways: • Reducing the randomness of reactions by temporarily binding the reactants to the enzyme. • Presenting the reactants to each other in the proper position for the reaction to occur. A e n e r g y B non-catalyzed reaction e n e r g y activation energy exothermic energy 22 catalyzed reaction activation energy exothermic energy 4PY019 Drug Targets TBL Pack Fig 4. A comparison of the activation energies required in non-catalyzed and catalyzed reactions. The amount of activation energy that must be absorbed by the non-catalyzed reaction (A) is far more than when a catalyst is included (B). Generally enzymes work in a three-step process: 1. The reactants, which are referred to as substrates, must bind to the enzyme. This substrate binding occurs at a particular site of the enzyme known as the ‘active site’. 2. The enzyme-substrate complex undergoes an internal rearrangement to form the product. 3. The products of the reaction are then released. As shown in Fig. 5, enzymes are left unaltered by the catalyzation process and so are able to catalyze the reaction again and again. The actual active site takes up only a relatively small part of an enzyme and it is a three dimensional unit, which is made up from groups that appear in different parts of the enzyme’s linear amino acid sequence. Substrates can initially bind to enzymes by weak attractions such as hydrogen bonds, van der Waals forces, electrostatic attractions and hydrophobic interactions. A B active site enzymesubstrate complex substrate substrate enzyme D C product energy internal rearrangement (bond formation) free enzyme Fig. 5. Schematic diagram representing an enzyme’s mechanism of action. Initially substrate molecules bind to the active site of an enzyme (A). This brings the substrates together in close proximity, with the correct orientation, which enables a reaction to take place once the required amount of activation energy has been absorbed (B and C). In this case a bond is formed between the two substrates. The enzyme is now free to release the product (D). Inhibition of enzymes Inhibition of enzymes can be reversible or irreversible, competitive and non-competitive (Fig. 6). As mentioned above, under normal circumstances, substrates temporally bind to the active sites of enzymes, forming an enzyme-substrate complex. The product then formed leaves the enzyme and fresh substrates can bind to the active site. Competitive 23 4PY019 Drug Targets TBL Pack inhibition involves substrate and inhibitor molecules competing for the occupancy of the enzymes’ active sites. Many competitive inhibitors resemble the substrate and when occupying the active site, they prevent the actual substrate from binding. Therefore a competitive inhibitor diminishes the rate of catalysis by reducing the proportion of substrate molecules binding to enzymes. Non-competitive inhibition, just like competitive inhibition, is reversible. Here, the inhibitor and substrate can bind simultaneously to an enzyme, because they have separate binding sites and so are not in competition with each other for occupancy of the enzyme. Therefore non-competitive inhibition cannot be overcome by increasing the substrate’s concentration. Once the inhibitor has bound to the enzyme, the conformation of the substrate’s active site alters. An irreversible inhibitor dissociates very slowly from its target enzyme because it is very tightly bound to the enzyme, which occurs by either covalent or non-covalent bonding. The bound inhibitor molecule hinders the interaction between the substrate and the active site. This type of inhibition can be overcome by either synthesizing new enzymes, in the absence of the inhibitor, or by administering an enzyme reactivating drug e.g., dyflos inhibition being overcome by using pralidoxime. B A substrate substrate enzyme C active site shape change substrate competitive inhibitor enzyme D enzyme enzyme covalent bond non-competitive inhibitor irreversible inhibitor substrate Fig. 6. The different types of enzyme inhibition. ‘A’ simply represents the normal function of an enzyme with substrates moving in and out of the active site. ‘B’ illustrates competitive inhibitors competing with substrate molecules for the occupancy of the active site. As in ‘A’, the inhibitor and the substrates can move in and out of the active site. ‘C’ represents non-competitive inhibition, since the inhibitor does not compete for the active site with the substrate, because it binds to a different site on the enzyme. Once the non-competitive inhibitor binds, a conformation change occurs in the enzyme, which alters the conformation of the active site, stopping product formation. ‘D’ shows an irreversible inhibitor covalently bonded to the enzyme, which hinders the substrate from entering the active site. 24 4PY019 Drug Targets TBL Pack Carrier Molecules As you already know, plasma membranes are made up of a lipid bilayer. This means that ions and many naturally occurring molecules and drugs are too polar (insufficiently lipid soluble) to cross lipid bilayers on their own. Therefore these polar particles require membrane carrier or transporter molecules to gain access to intracellular compartments. Examples of these carriers include ion, glucose, amino acid transporters and neurotransmitter re-uptake transporter molecules. These molecules obviously contain recognition sites that are specific for the particular particle that they have to transport. This therefore means that drugs can specifically target them too. Although there are many examples of drugs that target enzymes and transporter molecules, only a small selection will be looked at here. Inhibitors of acetylcholine synthesis These drugs work by principally inhibiting choline transport. Hemicholinium and triethylcholine are the two main drugs of this category. These drugs are very useful as experimental tools, but they have no real clinical application. Hemicholinium is a competitive inhibitor of choline uptake into the neurone terminal (Fig. 7). Triethylcholine also inhibits uptake, but is also transported into the neurone, where it is acetylated to form acetyltriethylcholine. Acetyltriethylcholine is then stored in vesicles, in the place of acetylcholine (ACh) and is released as a ‘false transmitter’, which means that it has no depolarizing effect on the postsynaptic membrane. neurone terminal acetyl CoA CAT CoA ACh carrier postsynaptic membrane vesamicol ACh - ACh botulinum choline empty vesicle - - ACh choline + acetate synaptic cleft nicotinic receptor choline carrier hemicholinium anticholinesterases acetylcholinesterase Na+ Fig. 7. Schematic diagram of a cholinergic synapse, showing the synthesis of ACh, its storage in vesicles and the various transporter molecules that are involved. Also included are the transporter molecules’ inhibiting drugs (written in italics). 25 4PY019 Drug Targets TBL Pack ACh is packed into empty vesicles via an active process that uses ACh carriers. A drug called vesamicol can inhibit this process (Fig. 7). This results in a neuromuscular blockade, which develops slowly. Botulinum toxin is a bacterial protein that is produced by the anaerobic Clostridium botulinum, which can contaminate preserved food, resulting in a very severe form of food poisoning (botulism). Botulinum toxin is a very potent neurotoxin, with only a few molecules binding to each cholinergic nerve terminal being lethal! Anticholinesterases Anticholinesterases are drugs that work by inhibiting the enzyme acetylcholinesterase, which is normally responsible for hydrolysing ACh to choline and acetate. Therefore, these drugs prolong the presence of ACh in cholinergic synaptic clefts, so increasing ACh’s effect. Anticholinesterase drugs can be divided into three classes: • Short acting e.g., edrophonium. • Medium duration acting e.g., physostigmine. • Irreversible e.g., dyflos. Edrophonium is a quaternary ammonium compound that forms reversible ionic bonds with acetylcholinesterase. Its duration of action is however too short to be of any therapeutic use, but it can be used in the diagnosis of myasthenia gravis. Physostigmine, sometimes called eserine, is a naturally occurring tertiary amine that can be extracted from Calabar beans. Its mechanism of action is to form carbamyl bonds to the anionic site of acetylcholinesterase instead of acetyl esters. These carbamyl bonds take minutes to hydrolyse, which therefore diminishes the enzyme’s rate of catalysis. Physostigmine can be formulated into eye drops and used to treat glaucoma. Dyflos forms an irreversible bond to the esteric site of acetylcholinesterase and is potentially a very dangerous compound. Dyflos has some agricultural uses and can be found in organophosphate pesticides, but it also has military applications too, since it can be used in chemical war gases. As a chemical weapon, its routes of administration are by inhalation, skin absorption and oral/enteric absorption. Effects of anticholinesterases Anticholinesterases can affect three main areas of the body: • Autonomic cholinergic synapses. • Neuromuscular junctions (NMJs). • The central nervous system (CNS). Since most postganglionic ACh is released from parasympathetic nerve terminals (remember that sweat glands are stimulated by ACh released from the sympathetic nervous system), anticholinesterase drugs will enhance parasympathetic actions. Since the parasympathetic nervous system innervates many tissues of the body, anticholinesterases will produce a wide range of effects e.g., bradycardia, hypotension, pupillary constriction, increased gut secretions etc. As you already know, ACh acts on nicotinic receptors in NMJs to stimulate muscle fibres to contract. So, when anticholinesterase drugs affect the NMJ, this will result in increasing the duration of single endplate potentials to produce short trains of action potentials, thus leading to an increase in muscle tension. If large doses of anticholinesterases are used, then this will lead to a paralysis caused by a depolarization block. 26 4PY019 Drug Targets TBL Pack The blood brain barrier normally stops large polar molecules from entering the CNS; therefore drugs such as edrophonium find it difficult to enter. Tertiary ammonium compounds, such as physostigmine however, can readily cross the blood brain barrier. Once inside the CNS, anticholinesterase drugs can initially produce excitatory effects that can result in convulsions as well as depressive effects, which can cause unconsciousness and respiratory failure. Myasthenia Gravis A rare autoimmune disease affecting around 1 in 200 individuals. It is characterised by muscle weakness so that muscles are unable to produce a sustained contraction. The initial symptoms are diplopia (drooping eyelids) which then progresses to other muscle groups. The disease is due to destruction of nicotinic receptors at the neuromuscular junction. Treatment - use of anticholinesterases. Monoamine oxidase inhibition Monoamine oxidase (MAO) enzymes are found in most tissues. These enzymes catalyse the conversion of catecholamines to aldehydes and so they are important for the degradation of neurotransmitters. MAOs can oxidize 5­HT, dopamine and NA. The enzyme has two isoforms: MAO­A and MAO­B. Dopamine and NA can be metabolized by both isoforms, whereas 5­HT is preferentially metabolized by MAO­A. MAO­B has a preference to metabolize phenylethylamine. Although MAO inhibitors do not work by causing an increase in the concentration of neurotransmitter that is stored in neuronal vesicles, they instead work by stopping the enzymes from catalyzing the oxidation of neurotransmitters in the cytoplasm. This therefore causes an overall rapid and sustained increase in monoamine neurotransmitter concentration in neurone terminals. This increased amount of cytoplasmic neurotransmitter is then free to spontaneously leak out of terminals into the synaptic clefts, so causing an effect. Examples of MAO inhibitors include phenelzine. These drugs can be clinically used to treat depression. One of the disadvantages of using MAO inhibitors is the fact that they enhance the effects of indirectly acting sympathomimetics, such as tyramine. Tyramine is a naturally occurring substance that is found in a wide variety of foods. Normally tyramine is metabolized by MAOs in the wall of the gut and in the liver, so that very little consumed tyramine enters into the circulation. Under conditions of MAO inhibition, however, dietary tyramine can be absorbed from the gut and be distributed around the body. It will then be taken up into neurone terminals, where it will displace NA from its vesicles. This NA cannot be metabolized by cytoplasmic MAOs, and so there is much more NA available to leak out of neurones and into synapses. If severe enough, this can result in a hypertensive crisis and an intracranial haemorrhage. References and Resources Rang & Dale’s Pharmacology 9th edition 2018 Rang, Dale, Ritter, Flower, Henderson. Chs 1-5 ebook (could be accessed through the library website) Rang and Dale's Pharmacology E-Book - University of Wolverhampton (wlv.ac.uk) 27