Peptide pharmacology

1. Pharmacology
of
Proteins & Peptides
RVS Chaitanya Koppala

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

3. Historical perspective

4. Low molecular weight and non-peptide signaling
molecules.
Since the 1970s  peptides and proteins
ACh
Adrenaline

5. 1930  Substance P
Most 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 immunocytochemistry

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.

7. Progress
1930s  Bradykinin, Substance P and Angiotensin
Angiotensin (octapeptide)  1957
Bradykinin (nonapeptide)  1960
Substance P (undecapeptide)  1970
Endothelin (21 aminoacids)  fully characterised, synthesised
and cloned in 1988

8. 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.

9. Introduction

10. Terminology
Peptides (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 chain

11. Protein and peptide mediators  3 to 200 residues
Difference between peptides and proteins  arbitrary dividing
line of 50 amino acid residues

13. Classification
1. Ribosomal peptides
 synthesized by translation of mRNA
 subjected to proteolysis to generate the mature form
 posttranslational modifications
2. Non – ribosomal peptides
 assembled by enzymes that are specific to each peptide
e.g.: glutathione, cyclosporine

14. Peptide mediators :
1. Neurotransmitters and neuroendocrine mediators
2. Hormones from non-neural sources:
a) Plasma-derived peptides, notably angiotensin and bradykinin,
b) substances such as insulin, endothelin, atrial natriuretic peptide and leptin
3. Growth factors: produced by many different cells and tissues that
control cell growth and differentiation
4. Mediators of the immune system (cytokines and chemokines)

15.  The 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.

16. 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).

17. Neuropeptide
Reproduction
Growth Salt &
Water
Temperat
ure
Food &
water
Affect
GI fn
CVS &
Resp. fn
Autonomic
response
Nerve
development &
regeneration
Functions of Neuropeptides

18. Neuropeptide receptors and Second Messenger Systems
1. GPCRs  >80% of neuropeptides are coupled to G-proteins
and stimulate cAMP formation.
2. PIP – IP3 pathway.
TSH
Bombesin
Vasopressin
GnRH

19. 3. cGMP receptors
Atrial natriuretic peptide
4. Tyrosine kinase coupled receptors
Insulin
IGF
5. Cytokine receptors
GH
PRL
Interleukins
Erythropoetin

20. Comparison of
neuropeptides and
conventional transmitters

21. 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

22. 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.
e.g.: GnRH, Insulin in mammals

23. 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 (norepinephrine).

24. 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

27. 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 sequences of a number of opioid peptides

28. Family Peptides
POMC family ACTH, MSH, Opiates, β-lipotropin, 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, Leu-enkephalins,
Dynorphin
Glucagon, Secretin family Glucagon, secretin, VIP, GIP, GHRH, PHI,
PACAP
Glycoprotein hormones TSH, FSH, LH, HCG

29. 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

30. Proteins
and
peptides as drugs

31. 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.
1. Insulin
2. Growth hormone
3. ACTH
4. Erythopoetin
5. GM-CSF

32.  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

33. Peptide agonists and
antagonists

34. 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

35. 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)

36. Peptide agonists – ‘Peptidomimetics’
 Octreotide (Somatostatin analogue)
 Desmopressin, Terlipressin (AVP analogues)
 Buserelin, Goserelin, Leuprolide ( GnRH Analogues)
 Opioid agonists

37. Identification, Isolation and
Characterization of Peptides

38. Techniques for Identification
1. Bioassay
 Insulin
 Endogenous opioids
2. Cytochemical assay
 Coloured precipitate formed d/t hormone dependant intracellular reaction
detedted by microspectrometry & microdensitometry

39. 3. Radioimmunoassay (RIA)
4. Immunocytochemistry
5. Immediate early genes
6. Autoradiography
7. InSitu Hybridization and Histochemistry- mRNA concentrations for that
particular peptide

40. Tools for isolation and characterization
1. Capillary electrophoresis
2. Immunofluorescence
3. Fast atom bombardment spectrometry
4. LC-MS
5. MALDI-TOF MS

41. 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.

42. Thank you