Molecular Pharmacology 2024 Past Paper PDF

Molecular Pharmacology 2024 (4 Lectures) Aims 1.Introduce the idea of receptor families. 2. Describe how gene cloning and most recently genome sequencing have expanded view of receptor families. 3. Describe some of the better characterized receptor families and list aspects of their structure func...

Molecular Pharmacology 2024 (4 Lectures) Aims 1.Introduce the idea of receptor families. 2. Describe how gene cloning and most recently genome sequencing have expanded view of receptor families. 3. Describe some of the better characterized receptor families and list aspects of their structure function activity. 4. Use GPCR adrenergic receptor and nicotinic acetylcholine receptors to detail how current and emerging structures inform on protein conformations that allow receptor function. 5. Describe principles of tyrosine kinases and steroid receptors. 6. Introduce the key principles Pharmacogenomics. 7. Use examples of how genetic determinants effect drugs by changing their Pharmacokinetics and Pharmacodynamics. References Ritter, J.M., Flower, R.J., Henderson, G., Kong Loke, Y, MacEwan, D., Rang, H.P., Rang and Dale’s Pharmacology. Chapter 3 How drugs Act. Molecular Targets. Churchill Livingstone. Ninth Edition. Corringer PJ, Baaden M, Bocquet N, Delarue M, Dufresne V, Nury H, Prevost M, Van Renterghem C (2010) Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues. J Physiol. 588:565-72. Ritter, J.M., Flower, R.J., Henderson, G., Kong Loke, Y, MacEwan, D., Rang, H.P., Rang and Dale’s Pharmacology. Chapter 12 Individual variation, pharmacogenomics and personalized medicine. Churchill Livingstone. Ninth Edition. Hockings, J., Pasternak, A., Erwin, A., Mason, N., Eng, C., Hicks, J.K. (2020) Pharmacogenomics: An evolving clinical tool for precision medicine. Cleveland Clinic Journal of Medicine. 87. 91-99.  The international union of basic and clinical pharmacology committee: IUPHAR https://www.guidetopharmacology.org/ Listing of major receptor families. 1. Molecular characteristic. 2. Major Drugs-indicating affinities, efficacies and relative specificities. 3. Good for revising and drug related write ups.  Classics define idea of receptor families Adrenalin/Noradrenalin shown to have a wide range of effects. Anatomical classification Different vessels gave distinct response constrict or dilate. Pharmacological classification Ahlquist noted a rank order of potency for adrenergic agonists depending on the nature of the response. Constricting responses Noradrenalin>adrenalin>isoprenaline ALPHA RECEPTOR Dilating response Isoprenaline>adrenalin> noradrenalin BETA RECEPTOR  Differential Pharmacology (structure function) and receptor associated signalling Further sub-classification based on relative sensitivities a-1 a-2 b-1 b-2 Agonist Adrenalin +++ +++ ++ + Noradrenalin ++ ++ ++ + Antagonist Yohimbine + +++ - - Propanolol - - +++ +++ Pindolol - - +++ + Yohimbine Propranolol Pindolol Functional +IP3 -cAMP +cAMP +cAMP Classification Molecular classification and receptor family expansion through gene cloning. Identify and sequence the cDNA for the receptor. Predicts the amino acid sequence of the receptor. Repeat for all receptor sub-types compare amino acid to give a molecular classification. Often leads to identification of distinct sub-types not realized by pharmacological classification. b1 b2 b3 a1 a2 Low homology High homology Methods that reveal molecular basis of receptor sub-types Method Era Approach Receptor diversity based (e.g. adrenorecptors) Protein purification and 1980 Purify protein 1 or 2 cDNA cloning. sequence and screen cDNA libraries Homology cloning 1980-1990 Low stringency 10 or 20 Expressed sequence tags 1990 Assembling random 100 (EST) sequences into contiguous Genome data mining 1990- Extraction of 1000’s present homologies and day shared relationships from sequenced genomes. Sequencing technologies Bioinformatics  Molecular methods that open up diversity in receptor families cDNA screening (1980’s) Purify Receptor Full receptor sequence Partial Amino Acid MetGluArg……….. atg………taa Oligonucleotide ATG GAA CGX To mimic DNA G AGA cDNA sequence predicted G by protein Homology Screening (1980/90s) High Identical Stringency Low cDNA Stringency Homologous (novel receptor)  Molecular methods that open up diversity in receptor families Expressed sequence tags (ESTs) and data mining (1990’s) EST small piece of DNA sequence Identical cDNA Partial Sequences Derived from tissue mRNA predicting ESTs in data protein bases Homologous (novel receptor sub-type) Genome sequencing (1998 +): C.elegans, Yeast , human, Drosophila, Mouse, Rat plus close to hundred other genomes. Sequenced genome Computer Splicing annotated prediction Compare new genes with known receptors  Name that Genra from the title HARRYMETSALLY HARRYANDSALLY HARRY-ISSALLY HAIRYANDSALTY  Building Post-genomic Receptor Families cDNA sequences Predict protein sequences Sequence comparison, established function and protein prediction leads to diverse receptor being identified as relatives of one family. Exemplified by the G-protein receptor family (includes the humble adrenoceptors) Activate 1 23 4 5 6 7 G G-protein Protein Prediction Sequence Comparison Function (7 transmembrane domains)  Nomenclatures in receptors types and sub-types. “For example” Superfamily G-protein coupled receptors Rhodopsin family Family (Family 1) Sub-family Adreno like receptors (Class) Sub-type Alpha and beta receptors (sub-class)  G-protein receptor super-family Human genome 3-5% genes encode G-protein coupled receptors Estimate 30 000 genes in the genome >1000 G-protein receptors. Family 2 Family 3 Family 4 Family 5 Family 1 Pharmacological and clinical consequence of receptor subtypes. Differential signalling Differential expression Differential Pharmacology Tissue Receptor Drug Clinical use Heart Beta-1 Propranolol (Ant) Tachycardia Adipose Beta-1 Glycogenolysis Vascular Beta-2 Terbetuline (Ag) Premature labour Smooth muscle Airways smooth muscle Beta-2 Salbutamol (Ag) Asthma Smooth muscle contraction Alpha-1 Prazosin (Ant) Hypertension Inhibit transmitter release. Alpha-2 Yohimbine (Ant) Memory retrieval Vasoconstriction Blood pressure boosting  Receptor signalling: skinning a cat in different ways. Simple concept Stimulate Receptor R Transduce Signal G-protein coupled receptors 4 Specific Examples G Steroid Ligand gated ion channels Y Receptor tyrosine kinases Structure Function Studies: understanding receptor transduction Express cDNA for receptor Express mutated receptor cDNA Measure activity Resulting receptor is different Normal structure function If different activity you have modified structure function Useful structural information for structure function studies Primary Secondary Tertiary Quaternary MetGluTrpTyr…. AspGlyLeuMetVal SerTrpLeuIleVal,,, stop Hydropathy, 3-D Subunit predicted arrangement interactions structures  G-protein coupled receptors OFF ON Activate a GDP GTP exchange b b a g g GTP GTP hydrolysis Signal Signal Primary structure: Multifaceted family made up related sub-families (see previous lecture) Secondary structure: all receptors predicted to be seven transmembrane spanning Tertiary structure: Rhodopsin and many structures support the 7 transmembrane model. Quaternary structure: growing evidence that some GPCR may operate as dimers of individual subunits (e.g. Family 3). Transduction Bind agonist supplied from outside Alter receptor conformation Make contact to activate G-protein  G-Protein Coupled Receptors Ligand Binding Conformational change G-protein contact Family 1a Family 1b Family 1c e.g. Biogenic amines e.g. peptide Glycoprotein hormones hormones e.g. Luteinizing Hormone N-terminal domain cleaved. Held together by intramolecular forces Family 2 e.g. Secretin Family Bi-lobed STRUCTURE in extracellular domain G-protein contact loop-2 Family 3 Family 3 e.g. glutamate receptor ARE DIMERS  Cartoon of secondary structure of a Adrenergic GPCR 3D structure of a TurkeyBeta-1 adrenergic receptor showing tertiary structure. Warne et al., (2008) Nature 454. 486-491: Drug binding site (antagonist bound). Structure of beta-1 adrenergic receptor. Organization of TM domains. Adrenergic agonist binding sites in transmembrane domains. Show key amino acids with major hydrogen bonds in (red) and van der Waal forces in blue. Broken lines indicate weak bonding partners. Detailing important aspects of the agonist binding site and the conformational changes to fully activate. Agonist R R* Agonist R G-protein (GTP) R* Agonist binds moves helices (5 and 6) creates a G-protein binding site. Poor activation of the receptor if G-protein does not bind. Agonist bind moves helices (5 and 6) G-protein binds stabilizes change in helices conformation. Suggests interdependence of the agonist, binding transmembrane conformation and G-protein binding.  Ligand-gated ion channels Activate Ion flux Open or Gate Membrane potential Cellular Biochemistry Nicotinic Acetyl Receptor super-family as an example. Primary structures. A number of related genes encoding sub-units that make up distinct members of this family. Acetylcholine (Nicotinic) and 5-HT (5-HT3) excite allowing Na+ to flow in Glycine and GABA inhibit by allowing Cl- to flow in Secondary structure. Each subunits exhibit a common and distinct shared trans-membrane topology. Tertiary structure good models of tertiary structure are now supported by structural information for whole receptor or domains of the receptor. Quaternary structure exist as oligomeric receptors made up of a mix and match of individual subunits. 5 subunits associate to generate a PENTAMERIC functional receptor. Fast and self-contained ligand gated ion channels Transduction Mechanism Bind agonist from the outside Conformational change or gate of ion channel Select specific ions to flux through ion channel pass through membrane. Operate on a millisecond time scale. Key Features of the Nicotinic acetylcholine ligand gated ion channel superfamily Cartoon of 1 receptor Cartoon of 1 subunits Showing quaternary secondary structure. structure. Structural model of the Functional Receptor. Acetylcholine binding site is at the alpha subunit and on neighbouring non-alpha subunit. Minor contact Non alpha subunit Alpha subunit Major contact  Similar organization and key features of the M2 ion channel domain Other 3 M domains Organization of 5 M2 regions for one subunit Hydrated ion Hydrophobic amino acids Hydroxylated amino acids Partially Hydrated ion Charged amino acids Twisting the hydrophobic residues in M2 helix could open the ion channel but would twist the aligned hydroxyl residues (so nice idea that probably wrong)!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Amino acid alignment that is generally conserved in M2 of this receptor family. Charged residues (-2), hydroxyl residues 2 and 4 and hydrophobic residues (9 13 16) co-ordination, selectivity and flux.  Twisting the agonist binding site and levering the underlying ion channel moves the M2 ion channel lining helix so it opens receptor Twisting (or rotating) Agonist binding site Levering the helixes Important helical domains that make ion flux pathway Rotation in an individual subunit co-ordinated across subunits of oligomeric protein increases the size of the ion channel thus it opens. R BASAL(CLOSED) R* Active ( Open) Note only 3 of the 5 subunits that make up the receptor are shown Usually these receptors have 5 subunits and 2 that bind agonist.  Tyrosine-kinase linked receptors Activate Y Y Y Y Y P P P P Signal Primary structure A number of different growth factor receptor gene families (e.g. NGF, cytokines and Insulin receptors same basic transduction pathway) Secondary structure Single transmembrane spanning or membrane associated domain. Tertiary structure. Isolated ligand binding domain or kinase domains confirm likely structures. Quaternary Clear dimerization required during signal transduction and some exist as oligomers before ligand. Transduction mechanism Activate by extracellular ligand Conformational change drives receptor dimerization Dimerization activates intrinsic tyrosine kinase or recruits non membrane associated kinase Creates site for phosphotyrosine or SH2 binding sites  Steroid receptors Cholesterol starting point for synthesis of mineralcorticoids (aldosterone), glucocorticoids (corticosterone) and sex steroids (e.g. oestrogens). Lipid soluble, diffuse into the cell and act on intracellular receptors. S Inactive receptor S CELL Heat shock protein Dissociation (HSP-90) Nucleus S Dimerization Activation DNA Binding Gene Transcription S DNA  Steroid Receptors Transduction mechanism Diffuse into cell and nucleus and bind Hsp-90 inactivated receptor. Dissociate HSP-90 and dimerize to give active form of the receptor. Activate transcription Schematic of steroid receptor: 5 Domain structure 1 3 4 5 2 N S C Zn2+ Zn2+ Zinc finger DNA Hinge Steroid binding Nuclear Localization Gene transcription binding (Dimer Dimer formation Facilitation) Hsp-90  Pharmacogenomics. How Genetic determinants and variability effect the way in which a drug works. Control for this to ensure efficacy and reduce side effects.  Principles of drug action the outcome of Pharmacokinetics and Pharmacodynamics Pharmacokinetics Pharmacodynamics Genetic determinants of biology: as they pertain to drug action. Several drug determinants express as Mendelian traits e.g. Succinylcholine (Suxamethonium) neuromuscular junction block Short lived drug used as muscle relaxant to immobilize in surgery. Short lived (thus controlled by its breakdown by serum cholinesterase). 1:3000 have a genetically altered enzyme that has reduced activity. The homozygous and heterozygous unaffected copy of gene enzyme activity. The individuals carrying two copies of the modified gene have adverse reaction. Sustained neuromuscular junction muscle block.  High throughput genome sequencing reveals a range of genetic variation. Human genome varies between individuals every 500 or so nucleotides. e.g. 14 million Single Nucleotide Polymorphisms (SNP) across the 3.2 billion nucleotides (3.2 Gigabases). Not all variations are functionally relevant but enough are to contribute variability. Functional consequences of some genome variation.  Genomic modulation of Pharmacokinetic parameters. Cytochrome P450 (Cyp family). Major route for drug metabolism Essentially detoxifying oxidation reactions (other physiological functions). Transform drug structure to increase elimination. Transform drug structure to inactivate or reduce potency. Transform drug structure to increase potency relative to the parent (pro-drug activation). Transform drug structure to generate toxic intermediate. Genomic changes that exist for example P450 Spectrum of pharmacogenomic consequences. Cytochrome P450 2D6 (CYP2D6) Chromosome 22 position q13.1 Nine exons and eight introns >100 genetic variants across the gene. Important substrates tricyclic and selective serotonin reuptake blockers (antidepressants), Beta adrenergic receptor blockers, antiarrhythmic drugs. >10 fold variability in plasma concentrations. Inactive Active UM have very reduced levels of active drug- problem if there is a narrow therapeutic window. PM excess P450 2D6 Increases pro-drug metabolism e.g. codeine to morphine: latter more potent and CYP2D6 effects analgesia PM don’t use/UM overdose. Genetic variation in the drug targets. Clinically relevant pharmacodynamics determinant.  Modified signalling in the Arg389Gly Beta 1 receptor Easier to activate the down stream signalling  Pharmacogenomic significance but it is complicated. After heart attack Beta blockers are used and helpful. Compare Arg389 and Gly389 cohorts there is a benefit to Arg 389. This outcome only seen if the comparison is made between treated and untreated Beta blockers are used in hypertension. Compare Arg 389 and Gly 389 cohorts there is a benefit to Arg 389 Again most striking (clinical relevant) if compare treated and untreated. Supports drug induced mitigation against underlying susceptibility. Genetic profiling might highlight cohorts that best served by given drug treatment.