IDM Drug Targets TBL

what is a drug natural or synthetic substance that alters the physiological state of a living organism what targets proteins do drugs work on - receptors - enzymes - carrier proteins - ion channels what are receptors - relay external signals to the inside of cells to produce physiological effects - specific protein molecules expressed by cells - located on cell membrane or within cells - agonists bind to them causing physiological effect - ligand gated ion channels (ionotropic) receptors - G-protein coupled (metabotropic) rewhat are the four main types of receptors ceptors - kinase-linked receptors - nuclear/intracellular receptors what are ligand gated ion channels - plasma membrane protein ion channels containing a ligand binging site therefore the receptor is directly coupled to the ion channel - also known as ionotropic receptors examples of ligand gated ion channels - nicotinic - GABAa - GABAc - glutamate - plasma membranes of cells - one of the most numerous receptors in where are nicotinic acetylcholine recepthe body tors - in neuromuscular junction in autonomic ganglia and central nervous system 1 / 16 - 5 subunits: 2 alpha, 1 beta, 1 sigma, 1 pi - each subunit contains membrane spanning segment - pore: when the subunits cluster together Structure of Nicotinic Acetylcholine Re- - domains: proteins forming subunits ceptor have domains that line the channel of the pore which are sharply kinked inward. these kinks constrict the pore stopping any ions from crossing the membrane - each receptor has two alpha subunits and the junctions between them and neighbours form ACh binding sites - are not selective between nicotinic recan agonists tell the difference between ceptors that are in different locations nicotinic receptors in different parts of - the receptors can be distinguished from the body each other pharmacologically - all drugs apart from decamethonium can all drugs distinguish nicotinic recep- - this drug is a potent agonist at the NMJ tors in different locations but is a weak agonist on the autonomic ganglia what is a antagonist - substance that interferes with or inhibits the physiological action of another are antagonist selective to receptors - antagonists show strong selectivity to receptors - evidence: low conc of beta-bungarotoxin blocks NMJ nicotinic receptors but has no effect on autonomic ganglia how do nicotinic receptors work - ACh binds to binding site of alpha subunit - causes ion channels to open (the kinks are turned out of the way, so opening the channel) - Na+ and K+ flow down their conc gradient through the channel 2 / 16 - the inward rush of Na+ is what is most responsible for depolarisation - the intracellular side is negatively charged compared to extracellular so an why is the inward rush of Na+ ions more inward rush of positively charged Na+ responsible for depolarization ions will reduce the negative membrane potential, depolarizing the cell what is the neuromuscular junction - function: transmit action potentials conducted along motor (somatic) nerves across the synaptic clefts that occur in motor endplates - unlike other receptors the postsynaptic membrane is not part of another neuron, instead is is the sarcolemma (plasma membrane of a muscle cell)of a skeletal muscle fibre - the postsynaptic region contains many invaginations which increases total surface area of the membrane therefore increasing its exposure to ACh where is ACh made - in the motor neurone terminals - synthesis is catalysed by the cytosolic enzyme choline acetyltransferase (C.A.T) - acetylcoenzyme A donates acetyl groups which are then covalently bonded to choline forming ACh - ACh is stored in vesicles, ready for release how is ACh released - when action potential conducted along the motor nerves reaches their terminals, depolarization causes an influx of Ca2+ ions which triggers ACh to be released into the synaptic cleft 3 / 16 - if the depolarisation is large enough it will stimulate an action potential in the muscle fibre, causing it to contract - ACh in the synaptic cleft interacts with enzyme acetylcholinesterase which hydrolyses into choline and acetic acid (this what happens when ACh causes depo- happens the whole time ACh is present larisation and how is this response ter- in the synaptic cleft) minated - when no more ACh is released form terminal, acetylcholinesterase reduces the synaptic conc of ACh, terminating the response - 50% of choline produced is then recaptured by nerve terminal, which is then synthesized into ACh again What is myasthenia gravis? - autoimmune disease affecting neuromuscular transmission - chronic causing abnormal fatigability and weakness of skeletal muscles - affects adolescents and young adults under 40 - symptoms: ptosis (drooping of upper eyelid), double vision, dysarthria (speech disorder) - immunoglobin G (IgG) antibodies cause loss of nicotinic receptors in the NMJ - fatigue is so extreme because the muscles become temporarily paralysed - treatment: administering anticholinesterases, using prednisolone or azathioprine, performing plasmapheresis, surgically removing the thymus what are G-protein coupled receptors - also called metabotropic, 7-transmembrane spanning receptors - are not directly coupled to the processes they govern therefore are slower acting 4 / 16 - receptors use g-proteins to form link between receptor and the target. - muscarinic ACh - adrenoceptors - dopamine - purine (adenosine ATP) - opiate - peptide receptors Examples of G-protein coupled receptors - constructed from a single protein (1,100 residues) - consists of 7 transmembrane spanning region (goes through the membrane 7 times) - connected by extracellular and intracellular loops G-protein coupled receptors structure - extracellular loops are arranged into pockets which can accept ligands - extracellular domains very varied therefore large no. of diverse receptors exist, all with different affinities for ligands - 3D structure is due to many disulphide bonds - found in the plasma membrane G-protein coupled receptors structure more - named G-proteins due to their ability to bind GDP and GTP - made up of alpha, beta, gamme subunits - beta and gamma form a complex as they are both veyr hydrophobic - complex resides in the intracellular surface of plasma membranes where they are able to freely move - alpha subunits form a loose association witht he complex - alpha subunit binds GDP in its resting state - form a loose association with the beta gamma complex 5 / 16 - can bind to guanine nucleotides what are the properties of an alpha sub- has ability of catalysing hydrolysis of unit iN a G-protein coupled receptors GTP to GDP - agonist bind, causing conformational change - allows alpha subunit to bind to the receptor how does a G-protein coupled receptors - alpha subunit releases its GDP and work binds to GTP - activated alpha subunit dissociates from receptor and complex and diffuses across membrane to interact with the target protein, activating it - the alpha subunit associating with the target causes its GTPase activity to inhow is the alpha subunits activation tercrease minated in G-protein coupled receptors - therefore, bound GTP is hydrolysed to GDP and an inorganic phosphate group what are the advantages of G-protein coupled receptors - they can amplify a signal: one agonist/receptor complex can activate several alpha-subunits which then go on to stimulate several targets what are the different categories of G protiens - stimulation of GPCRs can produce either stimulatory or inhibitory effects, based on what receptor is controlling what type of target - Gs: stimulatory - Gi: inhibitory - Gq: activate phospholipase C what are the targets of G proteins - ion channels - adenylate cyclase/cyclic AMP system - the phospholipase C/inositol phosphate system What are muscarinic receptors? G-protein coupled receptors - named after drug muscarine - found on cells and tissues stimulated by 6 / 16 the parasympathetic nervous system - binds to ACh how do muscarinic receptors work - when muscarinic receptors of the heart are stimulated, it reduces heart rate and forces of contraction - muscarinic receptors on the cardiac muscle increase membrane permeability to K+ ions because when ACh binds with target, activated G-proteins open K+ channels - K+ then diffuses out of cell, down conc gradient therefore intracellular compartment loses positive charge therefore becomes more negatively charged - this hyperpolarization makes it harder for cells to depolarize and contract therefore heart rate and force of contraction decreases drug agonists which act on muscarinic receptors - ACh - Carbachol - Pilocarpine drug antagonists which act on muscarinic receptors - Atropine - Hyoscine - Ipratropium what are adrenergic receptors - name from the fact that neurotransmitter noradrenaline and hormone adrenaline can bind to them - also called adrenoceptors - are divided into alpha, beta subtypes - which are then further divided into a1, a2, b1, b2 subtypes - all these types can be distinguished from each other by the different levels of responses obtained by different drugs selectively binding to them 7 / 16 how do adrenergic receptors work - are involved with the sympathetic nervous system (fight or flight response) - send signals to parts to cells to induce a response - they use 'second messengers' : agents whose intracellular concs vary, depending on level of receptor stimulation. - agents diffuse away from cell membrane to alter rate at which cellular responses occur - when these receptors in the heart are stimulated, heart rate and force of contraction increases what are the common second messengers that adrenergic receptors use - Ca2+ ions - cyclic adenosine monophosphate (cAMP) - inositol-1,4,5-triphosphate (InsP3) - diacylglycerol (DG) how do adrenergic receptors differ around the body - the distribution and function of the subtypes is different around the body - a1 are generally stimulatory and in blood vessels will cause vasoconstriction - a2 are generally found on presynaptic nerve terminals and negatively feedback on neurotransmitter release - b1 are mostly concerned in cardiac tissue and stimulate the heart ot beat faster and with more force - b2 generally induce tissues to relax e.g bronchodilation in lung airways and vasodilation in blood vessels that supply muscles 8 / 16 what is adenylate cyclase/cyclic AMP - g-protein target - enzyme - cyclic AMP = nucleotide second messenger - adenylate cyclase (membrane bound) synthesizes cAMP from ATP - cAMP = second messenger whose conc varies in response to receptor activation - receptors linked to Gs-proteins stimulate adenylate cyclase, so increase intracellular cAMP levels - receptors linked to Gi-proteins inhibit adenylate cyclase, so reduce cAMP levels - therefore cAMPi can be tightly controlled, which therefore tightly regulates the function of cellular components more on cAMP target - it regulates many cellular function by activating various protein kinases - protein kinases = enzyme that catalyses phosphorylation reactions - ATP used to power the reactions - the phosphorylation of cellular inactive components regulated their function how is cAMP inactivated - by hydrolysis to 5'-AMP by phosphodiesterase enzymes - this enzyme is inhibited by methylxanthines such as caffeine and theophylline what is the phospholipase C/inositol phosphate system - g-protein target - enzyme - when it becomes activated it acts on PIP2 and breaks InsP3 (second messenger) down to give DAG (second messenger) - DAG and InsP3 are recycled back to PIP2 which takes time - InsP3 binds to receptors on the en9 / 16 doplasmic reticulum which opens Ca2+ ions channels therefore increasing they cytoplasmic conc of free Ca2+ - DAG binds to protein kinases C, which controls the phosphorylation of proteins such as enzymes what are kinase linked reactions - receptors linked to tyrosine kinase - most well known ligand = insulin - found bound to membranes - kinase enzymes responsible for catalysing the phosphorylation to various cellular components - they need ATP to work kinase linked receptors structure - have extracellular and intracellular domains - each domain has 400-700 amino acid residues - domains are connected together by a single alpha helix spanning the thickness of the membrane - not al kinase receptors are made the same - extracellular domain contains ligand binding site - amino acid sequences in this domain ^ are very variable giving ligand selectivity - intracellular domains more conserved. has bingind site for ATP - as only a simple alpha helix connects the domains, ligand binding in the extracellular domain doesn't lead to a conformational change in the intracellular dohow does ligand binding work in kinase main linked receptors - ligand binding results in dimerization of receptors (when two receptors pair up together forming a dimer) - this causes autophosphorylation: the tyrosine residues of the intracellular do10 / 16 main become automatically phosphorylated with the phosphate grps donated by the bound ATP - the phosphorylated tyrosines, now become high affinity binding sites for various intracellular proteins. what are citokines - a ligand - small proteins/ biological factors released by cells - have specific effects on cell-to-cell communication and behaviour - act on receptors with a fairly similar structure to kinase linked receptors but lack the intracellular tyrosine kinase domain How do cytokines work? - when cytokines ligands bind to receptors, receptor dimerization occurs - after this is when cytosolic tyrosine kinase units are able to bind - then SH2 containing proteins bind and become activated producing the cellular response - a second messenger - nitric oxide ( an enzyme) is the most famous activator of guanylate cyclase - the membrane bound form of nitric acid what is guanylate cyclase/cyclic guano- has a similar structure to those in the sine monophosphate system tyrosine kinase family - therefore when a peptide agonist binds to its receptor, the production of cGMP occurs via a tyrosine phosphorylation process what is insulin polypeptide hormone (6,000MW) produced by beta cells of the islets of langerhans in the pancreas - insulin's job: important in regulating blood glucose conc, with high blood sugar levels stimulating it's secretion 11 / 16 - 4 glycoprotein molecules made of 2 alpha and 2 beta subunits - total molecular weight 350000 - subunits contain cyteine residues enabling the formation os disulphide what is the structure of an insulin recep- bridges tor - these bridges hold the complex together - extracellular alpha subunits is where insulin binds - beta subunits are transmembrane proteins how do insulin receptors work - intracellular portion of beta subunits posses tyrosine kinase activity - when insulin binds, the beta subunits autophosphorylate <-- beta subunits act on tyrosine residues, phosphorylate them causing them to bind to the target proteins - autophosphorylation enhanced kinase activity of other target proteins how do insulin receptors work more - 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 - insulin receptors are very varied involving diverse pathways therefore cellular events triggered by activated insulin receptors have both short and long term actions - the short term actions are the immediate metabolic effects 12 / 16 - these effects are generally governed by regulating kinase and phospholipase enzymes via the tyrosine kinase activity of the receptor are the cellular events triggered by acti- insulins long term actions involve regvated insulin receptors long or short term ulating the synthesis of specific mRNA molecules, thus controlling the synthesis of structures such as enzymes, which in turn regulates cellular responses what are nuclear receptors - receptors found in cytoplasm (intracellular) - involved with regulating DNA transcription (synthesis of RNA a slow process) - ligands: steroid hormones, thyroid hormones, agents like vitamin D and retinoic acid - nuclear receptors are intracellular therefore ligands have to be lipid soluble to pass through plasma membranes how are the nuclear receptor ligands made - hormones are produced by endocrine glands, which are then passed into blood and distributed around the body, eventually binding to their receptors - large monomeric (single part) protein - containing 400-1000 amino acid residues - middle portion: highly conserved region of 60 resides what is the basic nuclear receptor struc- contains two finger shaped loops of ture peptide chain each being 15 residues long - "zinc fingers" as each contains a zn2+ ion. ion held in place by 4 cysteine residues - carboxyl end contains steroid binding domain - zinc finger region forms DNA binding region as the fingers can wrap around 13 / 16 DNA helixes - amino acid end = receptors regulatory basic nuclear receptor structure more domain that can activate the transcription of specific genes - steroid hormones are highly lipid soluble so easily gain access to the intracellular compartment where they can bind to their receptors in the nucleus - when a steroid binds, it induces a conformational change in which the receptor unfolds to expose the zinc finger domain - ligand/receptor complex can now bind to specific portions of NDA - these binding positions are well defined how does ligand binding work in nuclear regions of the DNA that contain 'horrecptors mone responsive elements' - these elements are 200 DNA base pairs upstream from the genes that are regulated - within minutes of binding to DNA, there is an increase in RNA polymerase activity - results in production of mRNA - the mRNA leaves the nucleus to form a complex with ribosomes - this enables synthesis of new protein What are glucocorticoids? - agent that binds to nuclear receptors - binds to their receptor with very high affinity What are mineralocorticoids? - agent that binds to nuclear receptors - stimulate the production of various transport proteins that are involved in renal tubular function no - some when bound to DNA repress the do all steroid/receptor complexes bind to transcription of particular genes. thus inDNA to initiate mRNA transcription hibiting the synthesis of certain proteins, so producing the physiological effect 14 / 16 what are carrier proteins - plasma membrane made of lipid bilayer - ions and naturally occuring drugs too polar to cross on their own - so need membrane carrie or transporter molecules to gain access to intracellular compartments examples of carrier proteins - ion - glucose - amino acid transporters - neurotransmitter reuptake transporter molecules inhibitors of acetylcholine synthesis - hemicholinium is a competitive inhibitor of choline uptake into the neuron terminal - triethylcholine inhibits uptake but is also transported into the neuron, where it is acetylated to form acetyl triethylcholine. - this is then stored in vesicles in place of ACh and is released as a 'false transmitter' meaning it has no depolarising effect on the postsynaptic membrane - ACh is packed into empty vesicles via active process using ACh carriers - drug vesamicol inhibits this process resulting in neurotransmitter blockades what are anticholinesterases - drugs which work by inhibiting enzyme acetylcholinesterase which is responsible for hydrolysing ACh to choline and acetate - therefore these drugs prolong the presence of ACh in cholinergic synaptic clefts therefore increasing ACh's effect - example: edrophonium - is a quaternary ammonium compound what are short acting anticholinesterasthat forms reversible ionic bonds with es acetylcholinesterases - the duration is too short to have thera15 / 16 peutic use - however, can be used in the diagnosis of Myasthenia gravis what are medium duration anticholinesterases - example: physostigmine (also called eserine) - naturally occuring tertiary amine extracted from calabar beans - it forms carbonyl bonds to the anionic site of acetylcholinesterases instead of acetyl esters - takes minutes to hydrolyse therefore diminishes the enzymes rate of catalysis - formulated into eye drops and used to treat glaucoma - example: dyflos - forms an irreversible bond to the esteric what are irreversible anticholinesterases site of acetylcholinesterases - found in organophosphate pesticides - used in chemical war gases - most postganglionic ACh is released from parasympathetic nerve terminals, and anticholinesterase drugs will enhow do anticholinesterases effect the auhance parasympathetic actions tonomic cholinergic synapses - since the parasympathetic system innervates tissues, anticholinesterases will produce a wide range of effects how do anticholinesterases effect the NMJ - when anticholinesterase drugs affect the NMJ it results in increasing the duration of single endplate potentials to produce short trains of action potentials causing an increase in muscle tension how do anticholinesterases effect the CNS - in the CNS, anticholinesterases initially produce excitatory effects resulting in convulsions and depressive effects which can cause unconsciousness and respiratory failure 16 / 16