Transcript Agonism at melanocortin receptor type 3 on murine

MELANOCORTIN PHARMACOLOGY
Dr Stephen J. Getting
Department of Biochemical Pharmacology
William Harvey Research Institute
Charterhouse Square
Barts & the London
Queen Mary, University of London
[email protected]
Melanocortin
Learning Objectives
 POMC Gene
 Melanocortin receptors
 Physiological/Pharmacologic
al actions
POMC gene and ACTH-derived fragments
POMC Product
ACTH1-39
g- MSH
b - lipotropin
g-lipotropin
ACTH1-13
( a-MSH)
CLIP
b- MSH
ACTH 4-10
(MEHFRWG)
ACTH 4-10
(MEHFRWG)
b -endorphin
Features of the POMC gene
209-240 amino acid sequence
Stimulated by CRH from the
Hypothalamus. Its then glycosolated
(sugars added) and then cleaved
Better drug design
Find agonists and
antagonists of its
Peptide products.
Wonder Drug?
Altering POMC levels
Will alter ACTH, MSH
Levels and endorphins
So side effects would
Be immense
POMC gene
Neuro-peptides
ACTH and LPH are derived from POMC
In the pars distalis of the anterior pituitary.
In the pars intermedia of the anterior pituitary,
ACTH is further cleaved to produce a-MSH.
Other peptides
Endorphins which make you feel good
Features
Protein that tans skin
Makes you feel good
Makes you thin
Peripheral synthesis
Synthesised in GI tract, immune cells,
Placenta and gonads.
Amino acid sequence for peptide fragments
Peptide
ACTH
ACTH4-10
aMSH
ßMSH
gMSH
Amino acid sequence (single letter code)
SYSMEHFRWGKPVKKRRPVKVYPNGAEDESAEAFPLEF
MEHFRWG
SYSMEHFRWGKPV
AEKKDEGPYRMEHFRWGSPPKD
YVMGHFRWDRFG
Melanocortin Receptors (MC-Rs)
 Seven transmembrane GPCRs
positively coupled to adenylate cyclase
melanocortins
extracellular
plasma
membrane
 Activation leads to elevation of cAMP
NH2
I
II III IV V
VI VII
 Diverse physiological role by
melanocortins are mediated via different
receptors.
intracellular
 To date five MC-Rs have been identified
and cloned and are termed as MC1-5R
HOOC
 MC-Rs share a sequence homology of
40 - 60%
MC1-R-MELANOCORTIN TYPE 1 RECEPTOR
Agonist profile: a-MSH > ACTH > g-MSH
a-MSH
. ..
out
plasma
membrane
 Identified in 1992 in Melanoma tumours
1996 in testis, pituitary
NH2
I
II III
IV V
VI VII
1995-1997 in cell lines of different
lineage (macrophages, monocytes, endothelial,
fibroblasts, Keratinocytes)
in
HOOC
 External receptor expressed on the cellular
cell-surface although reports of internalisation
upon exposure to peptides.
RECEPTOR INVOVLVED IN SKIN PIGMENTATION
MC1-R involved in skin pigmentation.
Recessive yellow e/e
Wild type
MC2-R-MELANOCORTIN TYPE 2 RECEPTOR
Agonist profile: ACTH
ACTH
. ..
out
plasma
membrane
Expressed on the adrenal cortex
(Zona fasiculata) and adipose tissue
NH2
I
II III
IV V
VI VII
in
HOOC
RECEPTOR INVOLVED IN STEROIDGENESIS
Circadian Variation in plasma cortisol
Endogenous corticosteroids
11b hydroxysteroid dehydrogenase
OH
Cortisol
1
Cortisone
0.5
Glucocorticoid activity
Synthetic corticosteroids
F
CH3
Methylprednisolone
6
Dexamethasone
30
Edward C Kendall 1886-1972
Philip S Hench 1896-1965
Identified, isolated, purified and synthesised
corticosteroids
Discovered the clinical anti-inflammatory effects of
steroids
Corticosteroids are widely used in the
treatment of inflammatory and
autoimmune diseases.
“…over 250 000 people in the
United Kingdom are taking
continuous oral steroids…”
(Walsh LJ et al. BMJ 1996:313,344-6)
Prednisone 20mg tabs
Glucocorticoids exert effects on multiple
organs and metabolic pathways
Immune system
Cardiovascular
Hypertensive heightened pressor
responsiveness
Carbohydrate metabolism
Hyperglycaemic due
to inhibition of insulin
and stimulation of
glucagon secretion
Lipid metabolism
Redistribution of
body fat - increased
lipolysis by
adipocytes
Immunosuppressive leukocyte
redistribution,
maintenance of
lymphoid tissues,
feedback regulation of
the HPA axis.
Glucocorticoids
CNS
Regulation of
neuronal excitability
Kidney
Permissive action on
tubular function and
glomerular filtration
Skeletal system
Maintenance of
muscular tone
Unwanted effects of systemic corticosteroids
Oedema
Weight gain
Myopathy
Glycosuria
Hypertension
Hyperlipidaemia
Thrombosis
GI bleeding
Peptic ulcers
Pancreatitis
Colonic perforation
Glaucoma
Cataracts
Insomnia
Depression / Psychosis
HPA axis suppression
Increased infection risk
Osteoporosis
Avascular / aseptic necrosis
Juvenile growth retardation
Corticosteroid-induced peptic ulceration
Steroid -associated peptic ulcers of the stomach and
oesophagus (left) are usually small, superficial and multiple.
Corticosteroid-induced glaucoma
Photo of normal optic nerve on left and optic nerve with
glaucoma damage on the right (note the central white
cupping or depression representing nerve fiber loss)
Normal & Osteoporotic Bone Architecture
Normal Bone
Osteoporotic Bone
Reproduced from J Bone Miner Res 1986;1:15-21 with permission of the American Society for Bone and
Mineral Research. © 1986 by Massachusetts Medical Society. All rights reserved.
response to
stress
stressor
physiological
'tone'
Glucocorticoid hormones prevent overzealous
rebound responses to stress.
Cushing’s Syndrome
Caused by too much ACTH which results in overproduction of cortisol
Caused by benign pituitary adenoma (70% of endogenous cases)
Lung tumours (15% of endogenous cases)
Benign and malignant adrenal tumours (15% of endogenous cases)
The most common cause is exogenous ingestion of glucorticoids.
Symptoms of Cushing's syndrome
Hypertension
Atherosclerosis
Congestive heart failure
Oedema
Menstrual irregularities
Psychological disturbances
Osteoporosis
Increased infections
The classical ‘moon face’ associated with
corticosteroid use
Buffalo hump in a 34yr old woman taking corticosteroids
MC3-R-MELANOCORTIN TYPE 3 RECEPTOR
Agonist profile:
g-MSH
plasma
membrane
Identified in 1993 in Brain, Gut and
Placenta
. ..
out
NH2
I
II III
in
HOOC
g-MSH = ACTH > a-MSH
IV V
1996 in Heart
VI VII
1999 in leucocytes (macrophages)
Found to have a wide distribution within
the brain, including the Hippocampus,
thalamus and midbrain.
It has been reported that it may function as
an auto-receptor, regulating the release of
MSH peptides from POMC neurones.
Receptor proposed to be involved in modulating inflammation
MC4-R-MELANOCORTIN TYPE 4 RECEPTOR
Agonist profile: a-MSH = ACTH > g-MSH
a-MSH
. ..
out
plasma
membrane
Expressed in multiple sites within the
brain (cotex, thalamus, hypothalamus,
brain stem)
NH2
I
II III
in
IV V
VI VII
Not detected in any other organ in
human, although one study has
proposed message on adipose tissue.
Distribution much wider than MC3-R
HOOC
RECEPTOR INVOLVED IN CONTROLLING FOOD INTAKE
MC5-R-MELANOCORTIN TYPE 5 RECEPTOR
Agonist profile: a-MSH > ACTH > g-MSH
a-MSH
. ..
out
plasma
membrane
MC5-R is ubiquitously expressed in many
peripheral tissues including adrenal glands,
fat cells, kidneys, lung , liver etc
NH2
I
II III
in
HOOC
IV V
VI VII
It has been proposed that the seborrhoea
observed in Parkinson’s
Disease has been attributed to an increased
activity of MSH and that receptor blocking
might lead to a treatment.
RECEPTOR INVOLVED IN SEBORRHOEA
Cardinal signs of inflammation
The accumulation and subsequent activation of leukocytes are central events in the pathogenesis of
virtually all forms of inflammation
Inflammation: Basic Principles
and Clinical Correlates
2nd Ed. Gallin JI, Goldstein IM
and Snyderman R, 1992
%
detachment
%
rolling
adhesion
emigration
Experimental Murine Colitis
Reduces Faecal Blood
Inhibits weight loss
Reduces TNF-a and Nitric Oxide
In lower colon
[Rajora, N. et al., Peptides 1997, 18, 381-385]
Endotoxin-induced intestinal inflammation
Control
ileitis
a-MSH-treated
reducing neutrophil migration
reduce severity of the lesions macroscopically and microscopically
in the distal ileum
[San, T. et al., Peptides 2001, 22(12), 2077-2082.]
Mesenteric I/R Injury
a-MSH reduced depressed intestinal transit
 reduced myeloperoxidase activity in ileal
cytoplasmic extracts
 reduced NF-kB activation in ileal nuclear
extracts following I/R
[Hassoun, H.T. et al., Am. J. Physiol. 2002, 282, G1059-1068]
Lung Inflammation
Control
a-MSH/OVA
 a-MSH inhibits eosinophil migration
 and mucus secretion
(Rapp et al., J. Immunol, 2003, 171(1): 353-359.)
OVA
“The Gout” by James Gilray, 1799. Gout
depicted as an evil demon attacking a toe.
“By Royal Authority” by George
Cruickshank. A gout sufferer helped
onto his horse.
2.2 million cases of gout in the USA equivalent to 1:60 American males
37 million working days are lost in the USA every year to gout.
GOUT-CLINICAL PICTURES
After years of gouty attacks, patients develop a chronic arthritis resulting
in bone and cartilage destruction and deformity. Uric acid crystals deposit
within and surrounding the joint causing a chronic destructive inflammatory
process. X-rays characteristically show “punched out” erosions.
X-ray showing soft tissue swelling and erosion
ACTH is effective in the clinical treatment of gout
with an efficacy over and above that obtainable with
conventional glucocorticoid therapy
[Ritter et al., J. Rheumatol, 1994; 696-699]
Must be another mechanism
Action besides reflex stimulation
Of the HPA axis
in vivo Inflammation
Time (h)
-0.5
0
2
6
Agonists
and Antagonists
PMN accumulation
MSU crystals
(3 mg in 0.5 ml PBS)
[Getting et al., 1999; J. Immunol, 162; 7446-7453]
Chemokine levels by ELISAs
MELANOCORTIN AGONISTS INHIBIT PMN
RECRUITMENT: EFFECT BLOCKED BY
MC3-R ANTAGONISTS
PMN (10 per mouse)
75
50
**
ACTH4-10
25
8
*
b-MSH
a-MSH
*
*
6
6
PMN Migration
(% of control)
#
10
100
4
*
2
0
0
0.1
1
10
100
Peptide (nmol s.c.)
1000
ACTH4-10
Antagonist
+
None
-
+
SHU9119
[3 mg]
Similar observation with respect to cytokine/chemokine levels
[Getting et al., 1999; J. Immunol, 162; 7446-7453]
-
+
SHU9119
[10 mg]
MC3-R mRNA AND FUNCTIONALITY
[Getting et al., 1999; J. Immunol, 162; 7446-7453]
Injection of
MSU crystals
(1 mg in 50 ml)
2-72 h knee joints were examined and inflammatory
exudates collected and analysed for:
1 ] Arthritic Score
2 ] Size of knee joint
3 ] PMN accumulation by light microscopy
4 ] Cytokine contents by specific ELISAs
[Getting et al., 2002; Arthritis & Rheumatism, 46; 2765-2775]
MC3-R AGONISTS INHIBIT JOINT INFLAMMATION
EFFECT BLOCKED BY ANTAGONIST
A
Neutrophil influx
(105per joint)
2.4
1.8
1.2
*
*
ACTH
+
g2-MSH
+
0.6
0
SHU
PBS
+
B
IL-1b
IL-6
Cytokine release
(pg per joint)
80
60
40
20
0
SHU
*
*
PBS ACTH
- + - +
PBS ACTH
- + - +
Neutrophil influx
(105per joint)
REPEATED ADMINISTRATION OF MSU CRYSTALS
A
4.5
3.0
1.5
*
*
0
0 6 16
72
96
Time (h)
B
*
60
*
40
#
20
0
-20
SHU
#
PBS
+
ACTH
+
g2-MSH
+
Lipton et al News physiol. Sci, 15,192-195.
Diverse Physiological Roles
Mediated By MC-Rs
AGONIST PROFILE
TISSUE mRNA
FUNCTION
MC1-R
a-MSH > ACTH > g-MSH
Melanocyte, Endothelial Cells,
Fibroblast, Monocytes
Pigmentation
MC2-R
ACTH
Adrenal Cortex, Adipocytes
Steroidgenesis
MC3-R
g-MSH = ACTH =a-MSH
MØ, Brain, Gut, Placenta
Feeding Control,
Cardiovascular Function,
Inflammation
MC4-R
a-MSH = ACTH > g-MSH
Brain
Obesity
MC5-R
a-MSH > ACTH > g-MSH
Brain, Several Peripheral Tissues
Control of the Sebaceous
Gland
Effects of melanocortins
Anti-Pyretic
Inhibit cytokine induced pyresis
Antiserum directed against a-MSH
Prolongs fever induced by LPS.
Cardiovascular
Prevent ischaemic
damage
Obesity and anorexia
Agonists cause increase
In food intake, whilst
Antagonists inhibit food
intake
Melanocortins
Immune system
Inhibit pro-inflammatory
cytokine production
Inhibit leukocyte migration
Induce anti-inflammatory
Cytokines.
Modulates disease pathologies
Arthritis, asthma, IBD, Dermatitis, NeuroInflammation, Brain and Kidney ischaemia
Endotoxemia
Plasma a-MSH increases
Plasma a-MSH increases after endotoxin injection.
In CDC III and IV patients
And is a marker for 6 month
Survival.
HIV infection