Pharmacology of Proteins and Peptides

Pharmacology of Proteins and Peptides

Dr. Rohan Kolla

Oxytocin

ContentsHistorical perspectiveIntroductionComparison of neuropeptides and conventional neurotransmittersBisosynthesisProteins and peptides as drugsPeptide agonists and antagonistsIdentification, Isolation And charachterizationFuture2Dr. Rohan Kolla

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Historical perspective

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Low molecular weight and non-peptide signaling molecules.

Since the 1970s peptides and proteins4

AChAdrenalineDr. Rohan Kolla

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Bias1930 Substance PMost drugs natural (mainly plant) products.Very few peptides or acted through peptide signaling systems. Methodology required to study peptides - HPLC, HPTLC,Solid-phase peptide synthesis, and Radioimmunoassay and immunocytochemistry5Dr. Rohan Kolla

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The Beginnings

Dr. Vincent du 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.6Dr. Rohan Kolla

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Progress1930s Bradykinin, Substance P and AngiotensinAngiotensin (octapeptide) 1957Bradykinin (nonapeptide) 1960Substance P (undecapeptide) 1970

Endothelin (21 aminoacids) fully characterised, synthesised and cloned in 19887Dr. Rohan Kolla

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Protein mediators (cytokines and 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.

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Small molecule mediatorsPeptide and protein moleculesDr. Rohan Kolla

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Laurels7 Nobels in Chemistry5 Nobels in Physiology or medicine10

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Introduction

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TerminologyPeptides (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.

Polypeptide long, continuous, and unbranched peptide chain12Dr. Rohan Kolla

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Protein and peptide mediators 3 to 200 residues

Difference between peptides and proteins arbitrary dividing line of 50 amino acid residues13Dr. Rohan Kolla

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ClassificationRibosomal peptidessynthesized by translation of mRNAsubjected to proteolysis to generate the mature formposttranslational modifications

Non ribosomal peptidesassembled by enzymes that are specific to each peptidee.g.: glutathione, cyclosporine15Dr. Rohan Kolla

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Peptide mediators :Neurotransmitters and neuroendocrine mediators Hormones from non-neural sources:Plasma-derived peptides, notably angiotensin and bradykinin, substances such as insulin, endothelin, atrial natriuretic peptide and leptinGrowth factors: produced by many different cells and tissues that control cell growth and differentiationMediators of the immune system (cytokines and chemokines)16The distinction between neuropeptides and peripherally acting hormones is useful but not absolute. Thus the incretins and insulin, angiotensin, atrial natriuretic peptide and oxytocin are best known as hormones that are formed, released and act in the periphery. They are, however, also found in the brain, although their role there is uncertain. Similarly, endothelin was first discovered in blood vessels but is now known to occur extensively in the brain as well.Dr. Rohan Kolla

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The neuropeptide conceptPeptides produced in brain and gut have direct effect on central and peripheral neurons.90 genes have been identified which code >100 neuropeptidesMany of them coexist with the classical neurotransmitters ( Adr, Ach, GABA).

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18Functions of NeuropeptidesDr. Rohan Kolla

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Enteric nervous systemDr. Rohan Kolla

Neuropeptide receptors and Second Messenger SystemsGPCRs >80% of neuropeptides are coupled to G-proteins and stimulate cAMP formation.PIP IP3 pathway.TSHBombesinVasopressinGnRH20Dr. Rohan Kolla

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cGMP receptorsAtrial natriuretic peptideTyrosine kinase coupled receptorsInsulinIGFCytokine receptorsGHPRLInterleukinsErythropoetin21Dr. Rohan Kolla

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Comparison of neuropeptides and conventional transmitters

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Vesicles are loaded with 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 LDCVsFollowing exocytosis, the vesicles cannot be reloaded in situ.Transmitter turnover is therefore less rapid and recapture of the released transmitter does not occur23Dr. Rohan Kolla

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Effects excitatory/inhibitory and presynaptic/postsynaptic.Endogenous peptides rarely activate ligand-gated ion channels. [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 mammals24

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Co-transmittersTwo 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 (norepinephrine). 25Dr. Rohan Kolla

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Biosynthesis and Regulation of Proteins

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Peptide precursorsPeptide synthesis begins 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 peptideProhormone 27Dr. Rohan Kolla

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Trypsin like proteases Prohormone convertasesDr. Rohan Kolla

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Diversity within peptide familiesPeptides 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 sequences of a number of opioid peptides31Dr. Rohan Kolla

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FamilyPeptidesPOMC familyACTH, MSH, Opiates, -lipotropin, -endorphinBombesin like peptidesBombesin, Gastrin-releasing peptide, Meuromedin B, RantensinCalcitonin gene related peptidesCalcitonin, CGRPCCK like peptidesGastrin, CCKEnkephalinsMet-enkephalins, Leu-enkephalins, DynorphinGlucagon, Secretin familyGlucagon, secretin, VIP, GIP, GHRH, PHI, PACAPGlycoprotein hormonesTSH, FSH, LH, HCG

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FamilyPeptidesOxytocin, VasopressinOxytocin, Vasopressin, VasotocinPancreatic polypeptidesPancreatic polypeptide, Neuropeptide Y, Peptide YYSomatotropinGrowth hormone, prolactinTachykininsSubstance P, Neurokinin A, Neurokinin BInsulin-like Growth FactorsInsulin, IGF-I & IGF-II, RelaxinNeurotensin familyNeurotensin, Neuromedin, Angiotensin II

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Gene Splicing as a source for peptide diversityWhen 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. 35Dr. Rohan Kolla

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Preprotachykinin - A37

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Tissues may also generate 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 C-terminal sequence. CCK itself (33 residues) is the main peptide produced by the intestine, whereas the brain produces mainly CCK-8.38Dr. Rohan Kolla

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Proteins and peptides as drugs

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Many of the proteins currently in therapeutic use functional human proteins prepared by recombinant technology, which are used to supplement the action of endogenous mediators.InsulinGrowth hormoneACTHErythopoetinGM-CSF40Dr. Rohan Kolla

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Despite the large number 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 41Dr. Rohan Kolla

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Peptide agonists and antagonists

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Peptide antagonistsThey can peptide 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 libraries43Dr. Rohan Kolla

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The most important peptide receptor antagonists in clinical use :-Naloxone, Naltrexone (-opioid receptors): used to antagonise opiate effectsLosartan, Valsartan, etc. (angiotensin AT1 receptors)Bosentan (endothelin ET1/ET2 receptors)Atosiban (Oxytocin antagonist)Aprepitant (substance P antagonist)Ganirelix, Cetrorelix etc (GnRH antagonists)

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Peptide agonists PeptidomimeticsOctreotide (Somatostatin analogue)Desmopressin, Terlipressin (AVP analogues)Buserelin, Goserelin, Leuprolide ( GnRH Analogues)Opioid agonists

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Identification, Isolation and Characterization of Peptides

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Techniques for IdentificationBioassayInsulinEndogenous opioids

Cytochemical assayColoured precipitate formed d/t hormone dependant intracellular reaction detedted by microspectrometry & microdensitometry47Dr. Rohan Kolla

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Radioimmunoassay (RIA)ImmunocytochemistryImmediate early genesAutoradiographyInSitu Hybridization and Histochemistry- mRNA concentrations for that particular peptide

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ImmunocytochemistryAnatomical localization of neuropeptides through their immunoreactivity as detected by specific antisera.IPSCON -2012 conference in Nagpur workshop on Immunocytochemistry

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Tools for isolation and characterization Capillary electrophoresisImmunofluorescenceFast atom bombardment spectrometryLC-MSMALDI-TOF MS52Dr. Rohan Kolla

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PeptidomicsRefers to the techniques that permit quantitative determination of the peptide content of whole cells.This novel concept aims at the comprehensive visualization and analysis of small polypeptides.53Dr. Rohan Kolla

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PeptidomicsDr. Rohan Kolla

Genetic manipulationsTransgenic animalsGene targeting and knockout miceGenomicsSite-directed mutagenesis55Dr. Rohan Kolla

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Future

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Designer Proteins Dawn of 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 57Dr. Rohan Kolla

ReferencesRang & Dales Pharmacology 7th ed,Basic & Clinical Pharmacology, Katzungs, 12th ed.Neuropeptides, Contemporary Neuropharmacogy, Wiley Press, London. 2007Holmgren S, Jrgen J. Evolution of vertebrate neuropeptides, Brain Research Bulletin, Volume 55, Issue 6, August 2001, Pages 723-735Alexander, S.P., Mathie, A., Peters, J.A. (Eds.), 2006. Guide to receptors and channels, 2nd ed. Br. J. Pharmacol. 147 (Suppl. 3), S1-S168

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References (Contd.)Banks, W.A., 2006. The CNS as a target for peptides and peptide-based drugs. Expert Opin. Drug. Deliv. 3, 707-712Meunier, J.-C., Mollereau, C., Toll, L., et al., 1995. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptorYanagisawa, M., Kurihara, H., Kimura, S., et al., 1988. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-41559Dr. Rohan Kolla

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Thank you

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