Pharmacology of Peptides and Proteins

Pharmacology of Peptides and Proteins

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  Pharmacology of Proteins and Peptides Dr. RohanKolla Oxytocin
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  2 Contents         Historical perspective Introduction Comparison ofneuropeptides and conventional neurotransmitters Bisosynthesis Proteins and peptides as drugs Peptide agonists and antagonists Identification, Isolation And charachterization Future Dr. Rohan Kolla
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  3 Historical perspective Dr. RohanKolla
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  4 Low molecular weight andnonpeptide signaling molecules. ACh Adrenaline the 1970s  peptides and proteins Since Dr. Rohan Kolla
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  5 Bias  Substance P Most drugs  natural (mainly plant) products.  Very few  peptides or acted through peptide signaling systems.  Methodology required to study peptides  1930    HPLC, HPTLC, Solid-phase peptide synthesis, and Radioimmunoassay and immunocytochemistry Dr. Rohan Kolla
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  6 The Beginnings Dr. Vincentdu Vigneaud • • • • Pioneer in peptide pharmacology. Nobel prize in Chemistry for elucidating the structure of and later synthesizing OXYTOCIN - 1955. Vasopressin. Disulphide bonds in insulin structure. Dr. Rohan Kolla
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  7 Progress  Bradykinin, SubstanceP and Angiotensin  Angiotensin (octapeptide)  1957  Bradykinin (nonapeptide)  1960  Substance P (undecapeptide)  1970  1930s (21 aminoacids)  fully characterised, synthesised and cloned in 1988  Endothelin Dr. Rohan Kolla
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  8 Dr. Rohan Kolla
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  9  Protein mediators (cytokinesand growth factors) containing 50 or more residues are still difficult to synthesize chemically.  Molecular biology in the form of Recombinant DNA technology – an harbinger of peptide revolution. Peptide and protein molecules Small molecule mediators Dr. Rohan Kolla
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  10 Laurels 7 Nobels in Chemistry 5 Nobels in Physiology or medicine Dr. Rohan Kolla
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  11 Introduction Dr. Rohan Kolla
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  12 Terminology (from Gr. "digested") short chains of amino acid monomers linked by peptide (amide) bonds, the covalent chemical bonds formed when the carboxyl group of one amino acid reacts with the amino group of another.  Peptides  long, unbranched peptide chain  Polypeptide continuous, and Dr. Rohan Kolla
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  13 and peptide mediators 3 to 200 residues  Protein  Difference between peptides and proteins  arbitrary dividing line of 50 amino acid residues Dr. Rohan Kolla
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  14 Dr. Rohan Kolla
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  15 Classification 1. Ribosomal peptides    2. synthesized bytranslation of mRNA subjected to proteolysis to generate the mature form posttranslational modifications Non – ribosomal peptides  assembled by enzymes that are specific to each peptide e.g.: glutathione, cyclosporine Dr. Rohan Kolla
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  16 Peptide mediators : Neurotransmittersand neuroendocrine mediators 2. Hormones from non-neural sources: The a) Plasma-derived peptides, notably angiotensin peripherally distinction between neuropeptides and and bradykinin, acting hormones is useful but not absolute. substances such and insulin, angiotensin, atrial Thus b) the incretins as insulin, endothelin, atrial natriuretic peptide and leptin natriuretic peptide and oxytocin are best known as 3. Growth factors: produced by many different hormones that are formed, released and act in the cells periphery. and tissues that control cell growth and differentiation They are, however, also found in the brain, although their role there is uncertain. immune system (cytokines 4. Mediators of the Similarly, endothelin was first discovered in blood vessels and chemokines) Dr. Rohan Kolla but is now known to occur extensively in the brain as well. 1. • • • •
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  17 The neuropeptide concept Peptides produced in brain and gut have direct effect on central and peripheral neurons.  90 genes have been identified which code >100 neuropeptides  Many of them coexist with the classical neurotransmitters ( Adr, Ach, GABA). Dr. Rohan Kolla
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  18 Functions of Neuropeptides Reproduction Nerve development& regeneration Autonomic response Salt & Water Growth Neuropeptide CVS & Resp. fn Temperat ure GI fn Affect Food & water Dr. Rohan Kolla
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  19 Enteric nervous system Dr.Rohan Kolla
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  20 Neuropeptide receptors andSecond Messenger Systems 1. 2. GPCRs  >80% of neuropeptides are coupled to G-proteins and stimulate cAMP formation. PIP – IP3 pathway. 1. 2. 3. 4. TSH Bombesin Vasopressin GnRH Dr. Rohan Kolla
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  21 3. cGMP receptors 1. 4. Tyrosine kinasecoupled receptors 1. 2. 5. Atrial natriuretic peptide Insulin IGF Cytokine receptors 1. 2. 3. 4. GH PRL Interleukins Erythropoetin Dr. Rohan Kolla
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  22 Comparison of neuropeptides andconventional transmitters Dr. Rohan Kolla
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  23  Vesicles are loadedwith peptide precursors in the cell body, the active peptides being generated within the vesicles as they move to the nerve terminals.  Vesicles for neuropeptides are called LDCVs  Following exocytosis, the vesicles cannot be reloaded in situ.  Transmitter turnover is therefore less rapid and recapture of the released transmitter does not occur Dr. Rohan Kolla
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  24 – excitatory/inhibitory and presynaptic/postsynaptic.  Endogenous peptidesrarely activate ligandgated ion channels.  Effects [Some spider venom peptides, for example, produce pain by activating the ion-channel linked capsaicin receptor TRPV1]   Peptides are much more susceptible to evolutionary change than are the structures of non-peptide mediators. But conversely there is high degree of conservation evolutionarily across taxa. e.g.: GnRH, Insulin in mammals Dr. Rohan Kolla
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  25 Co-transmitters  Two well-documented examples: The parasympathetic nerves innervating the salivary glands (where the secretory response is produced by acetylcholine and the vasodilatation partly by vasoactive intestinal peptide) and  The sympathetic innervation to many tissues, which releases the vasoconstrictor neuropeptide Y in addition to noradrenaline Dr. Rohan Kolla (norepinephrine).
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  26 Biosynthesis and Regulation ofProteins Dr. Rohan Kolla
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  27 Peptide precursors  Peptide synthesisbegins with the manufacture of a precursor protein in which the peptide sequence is embedded, along with specific proteolytic enzymes that excise the active peptide.  Preprohormone:  Signal peptide  Prohormone Dr. Rohan Kolla
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  28 Trypsin like proteases– ‘Prohormone convertases’ Dr. Rohan Kolla
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  29 Dr. Rohan Kolla
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  30 Dr. Rohan Kolla
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  31 Diversity within peptidefamilies  Peptides commonly occur in families with similar or related sequences and actions.  Opioid peptides, defined as peptides with opiate-like pharmacological effects, are coded by three distinct genes whose products are, respectively,    prepro-opiomelanocortin (POMC), preproenkephalin and preprodynorphin.  Each of these precursors contains the Dr. sequences of a number of opioid peptides Rohan Kolla
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  32 Dr. Rohan Kolla
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  33 Family Peptides POMC family ACTH, MSH,Opiates, βlipotropin, β-endorphin Bombesin like peptides Bombesin, Gastrin-releasing peptide, Meuromedin B, Rantensin Calcitonin gene related peptides Calcitonin, CGRP CCK like peptides Gastrin, CCK Enkephalins Met-enkephalins, Leuenkephalins, Dynorphin Glucagon, Secretin family Glucagon, secretin, VIP, GIP, GHRH, PHI, PACAP Glycoprotein hormones TSH, FSH, LH, HCG Dr. Rohan Kolla
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  34 Family Peptides Oxytocin, Vasopressin Oxytocin, Vasopressin, Vasotocin Pancreaticpolypeptides Pancreatic polypeptide, Neuropeptide Y, Peptide YY Somatotropin Growth hormone, prolactin Tachykinins Substance P, Neurokinin A, Neurokinin B Insulin-like Growth Factors Insulin, IGF-I & IGF-II, Relaxin Neurotensin family Neurotensin, Neuromedin, Angiotensin II Dr. Rohan Kolla
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  35 Gene Splicing asa source for peptide diversity  When the gene is transcribed, RNA (heterologous nuclear RNA; hnRNA) is spliced to remove the introns and some of the exons, forming the final mRNA that is translated.  Control of the splicing process allows a measure of cellular control over the peptides that are produced.  Good examples of this are calcitonin/CGRP and substance P/neurokinin A. Dr. Rohan Kolla
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  36 Dr. Rohan Kolla
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  37 Preprotachykinin - A Dr.Rohan Kolla
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  38   Tissues may alsogenerate peptides of varying length from the same primary sequence by the action of specific peptidases that cut the chain at different points. E.g.:   procholecystokinin (pro-CCK) contains the sequences of at least five CCK-like peptides ranging in length from 4 to 58 amino acid residues, all with the same Cterminal sequence. CCK itself (33 residues) is the main peptide produced by the intestine, whereas the brain produces mainly CCK-8. Dr. Rohan Kolla
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  39 Proteins and peptidesas drugs Dr. Rohan Kolla
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  40  1. 2. 3. 4. 5. Many of theproteins currently in therapeutic use  functional human proteins prepared by recombinant technology, which are used to supplement the action of endogenous mediators. Insulin Growth hormone ACTH Erythopoetin GM-CSF Dr. Rohan Kolla
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  41   Despite the largenumber of known peptide mediators, only a few peptides, mostly close analogues of endogenous mediators, are currently useful as drugs. In most cases, peptides make poor drugs, because:       - they are poorly absorbed when given orally - they have a short duration of action because of rapid degradation in vivo - they do not predictably cross the blood-brain barrier - they are expensive and difficult to manufacture - they may be immunogenic. Smaller peptides are used therapeutically mainly when there is simply no viable alternative Dr. Rohan Kolla
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  42 Peptide agonists and antagonists Dr.Rohan Kolla
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  43 Peptide antagonists     They canpeptide or non-peptide molecules. Substitution into endogenous peptides of unnatural amino acids, such as D-amino acids. 'peptoids' have been produced by modifying the peptide backbone, while retaining as far as possible the disposition of the side-chain groups that are responsible for binding to the receptor. random screening of large compound libraries Dr. Rohan Kolla
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  44 The most importantpeptide receptor antagonists in clinical use : Naloxone, Naltrexone (μ-opioid receptors): used to antagonise opiate effects  Losartan, Valsartan, etc. (angiotensin AT1 receptors)  Bosentan (endothelin ET1/ET2 receptors)  Atosiban (Oxytocin antagonist)  Aprepitant (substance P antagonist)  Ganirelix, Cetrorelix etc (GnRH antagonists) Dr. Rohan Kolla
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  45 Peptide agonists –‘Peptidomimetics’  Octreotide (Somatostatin analogue)  Desmopressin, Terlipressin (AVP analogues)  Buserelin, Goserelin, Leuprolide ( GnRH Analogues)  Opioid agonists Dr. Rohan Kolla
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  46 Identification, Isolation and Characterizationof Peptides Dr. Rohan Kolla
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  47 Techniques for Identification 1. Bioassay   2. Insulin Endogenousopioids Cytochemical assay  Coloured precipitate formed d/t hormone dependant intracellular reaction detedted by microspectrometry & microdensitometry Dr. Rohan Kolla
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  48 3. 4. 5. 6. 7. Radioimmunoassay (RIA) Immunocytochemistry Immediate earlygenes Autoradiography InSitu Hybridization and Histochemistry - mRNA concentrations for that particular peptide Dr. Rohan Kolla
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  49 Immunocytochemistry  Anatomical localization ofneuropeptides through their immunoreactivity as detected by specific antisera.  IPSCON -2012 conference in Nagpur – workshop on Immunocytochemistry Dr. Rohan Kolla
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  50 Dr. Rohan Kolla
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  51 Dr. Rohan Kolla
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  52 Tools for isolationand characterization 1. 2. 3. 4. 5. Capillary electrophoresis Immunofluorescence Fast atom bombardment spectrometry LC-MS MALDI-TOF MS Dr. Rohan Kolla
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  53 Peptidomics  Refers to thetechniques that permit quantitative determination of the peptide content of whole cells.  This novel concept aims at the comprehensive visualization and analysis of small polypeptides. Dr. Rohan Kolla
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  54 Peptidomics Dr. Rohan Kolla
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  55 Genetic manipulations 1. 2. 3. 4. Transgenic animals Genetargeting and knockout mice Genomics Site-directed mutagenesis Dr. Rohan Kolla
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  56 Future Dr. Rohan Kolla
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  57 Designer Proteins – Dawnof new era therapeutics  'Designer proteins'-genetically engineered variants of natural proteins-for specific purposes are already a reality .  E.g.: 'humanised antibodies' and fusion proteins consisting of an antibody (targeted, for example, at a tumour antigen) or a peptide (e.g. bombesin or somatostatin, which bind to receptors on tumour cells) linked to a toxin (such as ricin or diphtheria toxin) to kill the target cells Dr. Rohan Kolla
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  58 References      Rang & Dale’sPharmacology 7th ed, Basic & Clinical Pharmacology, Katzung’s, 12th ed. Neuropeptides, Contemporary Neuropharmacogy, Wiley Press, London. 2007 Holmgren S, Jörgen J. Evolution of vertebrate neuropeptides, Brain Research Bulletin, Volume 55, Issue 6, August 2001, Pages 723-735 Alexander, S.P., Mathie, A., Peters, J.A. (Eds.), 2006. Guide to receptors and channels, 2nd ed. Br. J. Pharmacol. 147 (Suppl. 3), S1-S168 Dr. Rohan Kolla
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  59 References (Contd.)    Banks, W.A.,2006. The CNS as a target for peptides and peptide-based drugs. Expert Opin. Drug. Deliv. 3, 707-712 Meunier, J.-C., Mollereau, C., Toll, L., et al., 1995. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor Yanagisawa, M., Kurihara, H., Kimura, S., et al., 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-415 Dr. Rohan Kolla
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  60 Thank you Dr. RohanKolla