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Pharmacology
of
Proteins and
Peptides
Dr. Rohan Kolla

Oxytocin
2

Contents










Historical perspective
Introduction
Comparison of neuropeptides and conventional
neurotransm...
3

Historical perspective

Dr. Rohan Kolla
4

Low

molecular weight and nonpeptide signaling molecules.
ACh

Adrenaline

the 1970s  peptides and
proteins

Since

...
5

Bias
 Substance P
 Most drugs  natural (mainly plant) products.
 Very few  peptides or acted through peptide
signa...
6

The Beginnings
Dr. Vincent du Vigneaud
•
•

•
•

Pioneer in peptide
pharmacology.
Nobel prize in Chemistry
for elucidat...
7

Progress
 Bradykinin, Substance P and
Angiotensin
 Angiotensin (octapeptide)  1957
 Bradykinin (nonapeptide)  1960...
8

Dr. Rohan Kolla
9

 Protein

mediators (cytokines and growth
factors) containing 50 or more residues are still
difficult to synthesize ch...
10

Laurels
7

Nobels in Chemistry
 5 Nobels in Physiology or medicine

Dr. Rohan Kolla
11

Introduction

Dr. Rohan Kolla
12

Terminology
(from Gr. "digested")  short chains
of amino acid monomers linked by peptide
(amide) bonds, the covalent ...
13

and peptide mediators 
3 to 200 residues

 Protein

 Difference

between
peptides
and
proteins  arbitrary dividing...
14

Dr. Rohan Kolla
15

Classification
1.

Ribosomal peptides




2.

synthesized by translation of mRNA
subjected to proteolysis to genera...
16

Peptide mediators :
Neurotransmitters and neuroendocrine
mediators
2. Hormones from non-neural sources:
The a) Plasma-...
17

The neuropeptide concept
 Peptides

produced in brain and gut have direct
effect on central and peripheral neurons.
...
18

Functions of Neuropeptides
Reproduction
Nerve
development &
regeneration

Autonomic
response

Salt &
Water

Growth
Neu...
19

Enteric nervous system

Dr. Rohan Kolla
20

Neuropeptide receptors and Second
Messenger Systems
1.

2.

GPCRs  >80% of neuropeptides are
coupled to G-proteins an...
21

3.

cGMP receptors
1.

4.

Tyrosine kinase coupled receptors
1.
2.

5.

Atrial natriuretic peptide
Insulin
IGF

Cytoki...
22

Comparison of neuropeptides
and conventional transmitters

Dr. Rohan Kolla
23

 Vesicles

are loaded with peptide precursors in
the cell body, the active peptides being
generated within the vesicl...
24

–
excitatory/inhibitory
and
presynaptic/postsynaptic.
 Endogenous peptides rarely activate ligandgated ion channels.
...
25

Co-transmitters
 Two

well-documented examples : The parasympathetic nerves innervating the
salivary glands (where t...
26

Biosynthesis and Regulation
of Proteins

Dr. Rohan Kolla
27

Peptide precursors
 Peptide

synthesis begins with the manufacture
of a precursor protein in which the peptide
sequen...
28

Trypsin like proteases – ‘Prohormone convertases’

Dr. Rohan Kolla
29

Dr. Rohan Kolla
30

Dr. Rohan Kolla
31

Diversity within peptide families
 Peptides

commonly occur in families with
similar or related sequences and actions...
32

Dr. Rohan Kolla
33

Family

Peptides

POMC family

ACTH, MSH, Opiates, βlipotropin, β-endorphin

Bombesin like peptides

Bombesin, Gastrin...
34

Family

Peptides

Oxytocin, Vasopressin

Oxytocin, Vasopressin,
Vasotocin

Pancreatic polypeptides

Pancreatic polypep...
35

Gene Splicing as a source for
peptide diversity
 When

the gene is transcribed, RNA
(heterologous nuclear RNA; hnRNA)...
36

Dr. Rohan Kolla
37

Preprotachykinin - A

Dr. Rohan Kolla
38





Tissues may also generate peptides of varying
length from the same primary sequence by the
action of specific pe...
39

Proteins and peptides as
drugs

Dr. Rohan Kolla
40



1.
2.

3.
4.
5.

Many of the proteins currently in therapeutic use 
functional human proteins prepared by recombin...
41





Despite the large number of known peptide mediators,
only a few peptides, mostly close analogues of
endogenous m...
42

Peptide agonists and
antagonists

Dr. Rohan Kolla
43

Peptide antagonists






They can peptide or non-peptide molecules.
Substitution into endogenous peptides of unna...
44

The most important peptide receptor
antagonists in clinical use : Naloxone,

Naltrexone (μ-opioid receptors):
used to...
45

Peptide agonists – ‘Peptidomimetics’
 Octreotide

(Somatostatin analogue)
 Desmopressin, Terlipressin (AVP analogues...
46

Identification, Isolation and
Characterization of Peptides

Dr. Rohan Kolla
47

Techniques for Identification
1.

Bioassay



2.

Insulin
Endogenous opioids

Cytochemical assay


Coloured precipi...
48

3.
4.
5.
6.
7.

Radioimmunoassay (RIA)
Immunocytochemistry
Immediate early genes
Autoradiography
InSitu Hybridization ...
49

Immunocytochemistry
 Anatomical

localization of neuropeptides
through their immunoreactivity as detected by
specific...
50

Dr. Rohan Kolla
51

Dr. Rohan Kolla
52

Tools for isolation and
characterization
1.
2.
3.
4.
5.

Capillary electrophoresis
Immunofluorescence
Fast atom bombar...
53

Peptidomics
 Refers

to the techniques that permit quantitative
determination of the peptide content of whole
cells.
...
54

Peptidomics

Dr. Rohan Kolla
55

Genetic manipulations
1.
2.
3.
4.

Transgenic animals
Gene targeting and knockout mice
Genomics
Site-directed mutagene...
56

Future

Dr. Rohan Kolla
57

Designer Proteins –
Dawn of new era therapeutics
 'Designer

proteins'-genetically engineered
variants of natural pro...
58

References








Rang & Dale’s Pharmacology 7th ed,
Basic & Clinical Pharmacology, Katzung’s, 12th ed.
Neuropep...
59

References (Contd.)






Banks, W.A., 2006. The CNS as a target for
peptides and peptide-based drugs. Expert Opin....
60

Thank you
Dr. Rohan Kolla
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Pharmacology of Peptides and Proteins

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Pharmacology of Peptides and Proteins

  1. 1. Pharmacology of Proteins and Peptides Dr. Rohan Kolla Oxytocin
  2. 2. 2 Contents         Historical perspective Introduction Comparison of neuropeptides and conventional neurotransmitters Bisosynthesis Proteins and peptides as drugs Peptide agonists and antagonists Identification, Isolation And charachterization Future Dr. Rohan Kolla
  3. 3. 3 Historical perspective Dr. Rohan Kolla
  4. 4. 4 Low molecular weight and nonpeptide signaling molecules. ACh Adrenaline the 1970s  peptides and proteins Since Dr. Rohan Kolla
  5. 5. 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
  6. 6. 6 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. Dr. Rohan Kolla
  7. 7. 7 Progress  Bradykinin, Substance P 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
  8. 8. 8 Dr. Rohan Kolla
  9. 9. 9  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. Peptide and protein molecules Small molecule mediators Dr. Rohan Kolla
  10. 10. 10 Laurels 7 Nobels in Chemistry  5 Nobels in Physiology or medicine Dr. Rohan Kolla
  11. 11. 11 Introduction Dr. Rohan Kolla
  12. 12. 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
  13. 13. 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
  14. 14. 14 Dr. Rohan Kolla
  15. 15. 15 Classification 1. Ribosomal peptides    2. synthesized by translation 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
  16. 16. 16 Peptide mediators : Neurotransmitters and 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. • • • •
  17. 17. 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
  18. 18. 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
  19. 19. 19 Enteric nervous system Dr. Rohan Kolla
  20. 20. 20 Neuropeptide receptors and Second 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
  21. 21. 21 3. cGMP receptors 1. 4. Tyrosine kinase coupled receptors 1. 2. 5. Atrial natriuretic peptide Insulin IGF Cytokine receptors 1. 2. 3. 4. GH PRL Interleukins Erythropoetin Dr. Rohan Kolla
  22. 22. 22 Comparison of neuropeptides and conventional transmitters Dr. Rohan Kolla
  23. 23. 23  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 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
  24. 24. 24 – excitatory/inhibitory and presynaptic/postsynaptic.  Endogenous peptides rarely 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
  25. 25. 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).
  26. 26. 26 Biosynthesis and Regulation of Proteins Dr. Rohan Kolla
  27. 27. 27 Peptide precursors  Peptide 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 peptide  Prohormone Dr. Rohan Kolla
  28. 28. 28 Trypsin like proteases – ‘Prohormone convertases’ Dr. Rohan Kolla
  29. 29. 29 Dr. Rohan Kolla
  30. 30. 30 Dr. Rohan Kolla
  31. 31. 31 Diversity within peptide families  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
  32. 32. 32 Dr. Rohan Kolla
  33. 33. 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
  34. 34. 34 Family Peptides Oxytocin, Vasopressin Oxytocin, Vasopressin, Vasotocin Pancreatic polypeptides 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
  35. 35. 35 Gene Splicing as a 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
  36. 36. 36 Dr. Rohan Kolla
  37. 37. 37 Preprotachykinin - A Dr. Rohan Kolla
  38. 38. 38   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 Cterminal sequence. CCK itself (33 residues) is the main peptide produced by the intestine, whereas the brain produces mainly CCK-8. Dr. Rohan Kolla
  39. 39. 39 Proteins and peptides as drugs Dr. Rohan Kolla
  40. 40. 40  1. 2. 3. 4. 5. 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. Insulin Growth hormone ACTH Erythopoetin GM-CSF Dr. Rohan Kolla
  41. 41. 41   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 Dr. Rohan Kolla
  42. 42. 42 Peptide agonists and antagonists Dr. Rohan Kolla
  43. 43. 43 Peptide antagonists     They 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 libraries Dr. Rohan Kolla
  44. 44. 44 The most important peptide 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
  45. 45. 45 Peptide agonists – ‘Peptidomimetics’  Octreotide (Somatostatin analogue)  Desmopressin, Terlipressin (AVP analogues)  Buserelin, Goserelin, Leuprolide ( GnRH Analogues)  Opioid agonists Dr. Rohan Kolla
  46. 46. 46 Identification, Isolation and Characterization of Peptides Dr. Rohan Kolla
  47. 47. 47 Techniques for Identification 1. Bioassay   2. Insulin Endogenous opioids Cytochemical assay  Coloured precipitate formed d/t hormone dependant intracellular reaction detedted by microspectrometry & microdensitometry Dr. Rohan Kolla
  48. 48. 48 3. 4. 5. 6. 7. Radioimmunoassay (RIA) Immunocytochemistry Immediate early genes Autoradiography InSitu Hybridization and Histochemistry - mRNA concentrations for that particular peptide Dr. Rohan Kolla
  49. 49. 49 Immunocytochemistry  Anatomical localization of neuropeptides through their immunoreactivity as detected by specific antisera.  IPSCON -2012 conference in Nagpur – workshop on Immunocytochemistry Dr. Rohan Kolla
  50. 50. 50 Dr. Rohan Kolla
  51. 51. 51 Dr. Rohan Kolla
  52. 52. 52 Tools for isolation and characterization 1. 2. 3. 4. 5. Capillary electrophoresis Immunofluorescence Fast atom bombardment spectrometry LC-MS MALDI-TOF MS Dr. Rohan Kolla
  53. 53. 53 Peptidomics  Refers 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. Dr. Rohan Kolla
  54. 54. 54 Peptidomics Dr. Rohan Kolla
  55. 55. 55 Genetic manipulations 1. 2. 3. 4. Transgenic animals Gene targeting and knockout mice Genomics Site-directed mutagenesis Dr. Rohan Kolla
  56. 56. 56 Future Dr. Rohan Kolla
  57. 57. 57 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 Dr. Rohan Kolla
  58. 58. 58 References      Rang & Dale’s Pharmacology 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
  59. 59. 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
  60. 60. 60 Thank you Dr. Rohan Kolla

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