The document provides an overview of the pharmacology of proteins and peptides. It discusses the historical perspective of using peptides as therapeutic drugs, beginning in the 1970s. It compares neuropeptides to conventional neurotransmitters in their biosynthesis, storage, and mechanisms of action. The document outlines different classes of biologically active peptides and techniques used to identify, isolate, and characterize peptides. It also discusses using peptides and proteins as therapeutic drugs, including peptide agonists and antagonists that target endogenous receptors.

1. The Pharmacology of Proteins and
Peptides
Edit: Odette Heyneke (Full credit to Dr. Rohan Kolla for original presentation)

2. Contents
Historical perspective
Introduction
Comparison of neuropeptides and conventional neurotransmitters
Biosynthesis
Proteins and peptides as drugs
Peptide agonists and antagonists
Identification, Isolation and characterisation
Future
2

3. Historical Perspective
• Low molecular weight and nonpeptide signalling molecules were first use as
therapeutic drug interventions.
• Since the 1970s → peptides and proteins
3

4. Historical Perspective -Bias
1930 → Substance P
Most drugs → natural (mainly plant) products.
Very few → peptides or acted through peptide signalling systems.
Methodology required to study peptides –
• HPLC, HPTLC,
• Solid-phase peptide synthesis, and
• Radioimmunoassay and immunocytochemistry
4

5. 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.
5

6. 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 19886

7. 7

8. Progress (cont.)
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 – a forerunner of
peptide revolution.

9. 9
Laurels
- 7 Nobles in
chemistry
- 5 Nobles in
physiology or
medicine
Andrew Shally
Stanley Cohen
Günter Blobel
Paul Greengard
Frederick Sanger

10. Introduction
10

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

12. 12
Biologically Active
Peptides

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
13

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

15. Peptide mediators
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. 17
EntericNervousSystem

18. 18
Functions of neuropeptides
Neuropeptide
Reproduction
Autonomic
response
Salt and
Water
Temperature
Food and
Water
Affect
GI function
CVS and
RESP
function
Growth
Nerve
development
and
regeneration

19. Neuropeptide receptors and Second Messenger
Systems
5. Cytokine receptors
GH PRL Interleukins Erythropoetin
4. Tyrosine kinase coupled receptors
Insulin IGF
3. cGMP receptors
Atrial natriuretic peptide
2. PIP – IP3 pathway.
TSH Bombesin Vasopressin GnRH
1. GPCRs
>80% of neuropeptides are coupled to G-proteins and stimulate cAMP formation.
19

20. 20
Comparisonofneuropeptidesand
conventionaltransmitters
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
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
ionchannel 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

21. 21
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)
The sympathetic
innervation to
many tissues,
which releases
the
vasoconstrictor
neuropeptide Y in
addition to
noradrenaline
(norepinephrine).

22. 22
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

23. 23

24. 24

25. 25
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

26. 26

27. 27

28. Proteins
and
peptides as drugs
28

29. 29
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

30. 30
Despite the large number of
known peptide mediators, only
a few peptides, mostly close
analogues of endogenous
mediators, are currently useful
as drugs.
• 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.
In most cases, peptides make
poor drugs, because:
Smaller peptides are used
therapeutically mainly when
there is simply no viable
alternative

31. Peptide agonists and
antagonists
31

32. 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

33. 33
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)

34. 34
Peptide agonists – ‘Peptidomimetics’
Octreotide (Somatostatin analogue)
Desmopressin, Terlipressin (AVP
analogues)
Buserelin, Goserelin, Leuprolide ( GnRH
Analogues)
Opioid agonists

35. Identification, Isolation and
Characterization of Peptides

36. Techniques for Identification
• Insulin
• Endogenous opioidsBioassay
• Coloured precipitate formed d/t hormone
dependant intracellular reaction detedted
by microspectrometry &
microdensitometry
Cytochemical
assay

37. 37
3. Radioimmunoassay (RIA)
4. Immunocytochemistry
5. Immediate early genes
6. Autoradiography
7. InSitu Hybridization and Histochemistry- mRNA
concentrations for that particular peptide

38. 38

39. 39
Tools for isolation and
characterization
Capillary
electrophoresis
Immunofluorescence
Fast atom
bombardment
spectrometry
LC-MS MALDI-TOF MS

40. 40
This novel concept aims at
the comprehensive
Visualization and analysis of
small polypeptides.
Refers to the techniques that
permit quantitative
Determination of the peptide
content of whole cells.
Peptidomics

41. 41

42. 42
Genetic
manipulations
Transgenic
animals
Gene
targeting and
knockout mice
Genomics
Site-directed
mutagenesis

43. 43
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

44. 44
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
• 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

45. THE END
Thank you!
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