4. 4
Low
molecular weight and nonpeptide signaling molecules.
ACh
Adrenaline
the 1970s peptides and
proteins
Since
Dr. Rohan Kolla
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
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
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
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
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
and peptide mediators
3 to 200 residues
Protein
Difference
between
peptides
and
proteins arbitrary dividing line of 50
amino acid residues
Dr. Rohan Kolla
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
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
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
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
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
–
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
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).
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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