Pharmacology

of

Proteins & Peptides

RVS Chaitanya Koppala

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

Historical perspective

Low molecular weight and non-peptide signaling

molecules.

Since the 1970s peptides and proteins

ACh

Adrenaline

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

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.

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

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.

Introduction

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

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

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)

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.

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).

Neuropeptide

Reproduction

GrowthSalt & Water

Temperature

Food & water

Affect

GI fn

CVS & Resp. fn

Autonomic response

Nerve development & regeneration

Functions of Neuropeptides

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

3. cGMP receptors

Atrial natriuretic peptide

4. Tyrosine kinase coupled receptors

Insulin

IGF

5. Cytokine receptors

GH

PRL

Interleukins

Erythropoetin

Comparison of

neuropeptides and

conventional transmitters

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 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

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).

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

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

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

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

Proteins

and

peptides as drugs

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

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

Peptide agonists and

antagonists

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

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)

Peptide agonists – ‘Peptidomimetics’

Octreotide (Somatostatin analogue)

Desmopressin, Terlipressin (AVP analogues)

Buserelin, Goserelin, Leuprolide ( GnRH Analogues)

Opioid agonists

Identification, Isolation and

Characterization of Peptides

Techniques for Identification

1. Bioassay

Insulin

Endogenous opioids

2. Cytochemical assay

Coloured precipitate formed d/t hormone dependant intracellular reaction

detedted by microspectrometry & microdensitometry

3. Radioimmunoassay (RIA)

4. Immunocytochemistry

5. Immediate early genes

6. Autoradiography

7. InSitu Hybridization and Histochemistry- mRNA concentrations for that

particular peptide

Tools for isolation and characterization

1. Capillary electrophoresis

2. Immunofluorescence

3. Fast atom bombardment spectrometry

4. LC-MS

5. MALDI-TOF MS

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.

Thank you