|
|
|
[D-Arg1 D-Phe5 D-Trp7 ,9 Leu11] - Substance P |
A Potent Ghrelin Inverse Agonist |
.gif)
  
Holst B, et al. Mol Pharmacol. 2006 Jun 23
[D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P functions as a low potency antagonist but a high potency inverse agonist on the ghrelin receptor. Through a systematic deletion and substitution analysis of this peptide the C-terminal, carboxyamidated pentapeptide wFwLX was identified as the core structure, which however in it self displayed relatively low inverse agonist potency. Mutational analysis at 17 selected positions in the main ligand-binding crevice of the ghrelin receptor demonstrated that ghrelin apparently interacts only with residues in the middle part of the pocket i.e. between TM-III, -VI and -VII. In contrast the inverse agonist peptides bind in a pocket, which extends all the way from the extracellular end of TM-II (AspII:20) across between TM-III and TM-VI / -VII over to TM-V and TM-IV. The potency of the main inverse agonist could be improved up to 20-fold by a number of space-generating mutants located relatively deep in the binding pocket at key positions in TM-III, -IV and -V. It is proposed that the inverse agonists prevent the spontaneous receptor activation by inserting relatively deep across the main ligand-binding pocket and sterically blocking the movement of TM-VI and -VII into their inward-bend, active conformation. The combined structure-functional analysis of both the ligand and the receptor allowed for the design of a novel, N-terminally Lys-extended analog of wFwLL, which rescued the high potency, selective inverse agonism being dependent upon both AspII:20 and GluIII:09. The identified pharmacophore can possibly serve as the basis for targeted discovery of also non-peptide inverse agonists for the ghrelin receptor.
Holst B, et al. Mol Pharmacol. 2006 Jun 23; [Epub ahead of print]
Ghrelin is a GH (GH) releasing peptide, which
also has an important role as an orexigenic hormone stimulating food
intake. By measuring inositol phosphate turnover or by using a
reporter assay for transcriptional activity controlled by cAMP
responsive elements, the ghrelin receptor showed strong,
ligand-independent signaling in transfected COS-7 or HEK293 cells.
Ghrelin and a number of the known non-peptide GH secretagogues acted
as agonists stimulating inositol phosphate turnover further. In
contrast, the low potency ghrelin antagonist,
[D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P was surprisingly
found to be a high potency (EC50 = 5.2 nM) full inverse agonist as
it decreased the constitutive signaling of the ghrelin receptor down
to that observed in un-transfected cells. The homologous motilin
receptor functioned as a negative control as it did not display any
sign of constitutive activity; however, upon agonist stimulation the
motilin receptor signaled as strongly as the un-stimulated ghrelin
receptor. It is concluded that the ghrelin receptor is highly
constitutively active and that this activity could be of
physiological importance in its role as a regulator of both GH
secretion and appetite control. It is suggested that inverse
agonists for the ghrelin receptor could be particularly interesting
for the treatment of obesity.
Holst B, et al. Mol Endocrinol. 2003 Aug 7
[Epub ahead of print].
 
Holst B, et al. Mol Pharmacol. 2006 Jun 23; [Epub ahead of
print]
Ghrelin is a 28-amino-acid peptide,
with an essential n-octanoyl modification at Ser3, that elicits
growth-hormone (GH) secretion in rats and humans. At present, the
mechanisms of ghrelin action and its interactions with other systems
controlling GH secretion remain poorly characterized. In this
context, the present study was undertaken to obtain information
about ontogeny and possible gender differences in the GH-releasing
activity of ghrelin, and to delineate its primary site(s) of action
at the hypothalamus and/or pituitary. In addition, the interactions
between ghrelin and other relevant signals in the control of GH
secretion, such as excitatory amino acids (EAAs), nitric oxide (NO)
and serotonin, were assessed. Experiments were carried out in
infantile-prepubertal animals, when GH pulsatility is not yet
established. Systemic administration of ghrelin (25 nmol/rat, i.p.)
to 5-, 10- and 23-day-old male and female rats increased plasma GH
levels from day 10 onwards. This action was NO dependent, since it
disappeared in 23-day-old males after pretreatment with an inhibitor
of NO synthase (NAME). Similarly, central infusion of ghrelin (3
nmol/rat, i.c.v.) elicited GH responses in 10- and 23-day-old
animals significantly higher than after systemic administration. By
contrast, in vitro challenge of pituitary tissue with increasing
doses of ghrelin (10(-9)-10(-7) M) failed to enhance GH release into
the incubation medium, whereas stimulation with GH-releasing hormone
(GHRH; 10(-7) M) or GHRP-6 (10(-7) M) was effective. Finally,
effects of ghrelin were blocked by pretreatment with MK-801 and NBQX
antagonists of EAA ionotropic receptors and after manipulation of
endogenous serotoninergic tone. In addition, the potent releasing
activity of EAA agonists NMDA and AMPA was blunted by pretreatment
with D-Lys3-GHRP-6, a selective antagonist of the cognate ghrelin
receptor, i.e. the GH-secretagogue receptor. In conclusion, our
results demonstrate that GH-releasing activity of ghrelin appears
early in the infantile period, is NO dependent and involves a
primary hypothalamic site of action. The data also demonstrate for
the first time the existence of a cross-talk between ghrelin and
other neurotransmitter systems, such as EAAs and serotonin, in
precise control of GH secretion.
Pinilla L, et al.
Neuroendocrinology 2003 Feb;77(2):83-90
BACKGROUND AND AIMS: Ghrelin, an endogenous ligand
for growth hormone secretagogue receptor (GHS-R), is an appetite
stimulatory signal from the stomach with structural resemblance to
motilin. We examined the effects of the gastric peptide ghrelin and
GHS-R antagonists on energy balance and glycaemic control in mice.
MATERIALS AND METHODS: Body weight, fat mass, glucose, insulin, and
gene expression of leptin, adiponectin, and resistin in white
adipose tissue (WAT) were measured after repeated administrations of
ghrelin under a high fat diet. Gastric ghrelin gene expression was
assessed by northern blot analysis. Energy intake and gastric
emptying were measured after administration of GHS-R antagonists.
Repeated administration of GHS-R antagonist was continued for six
days in ob/ob obese mice.
RESULTS: Ghrelin induced remarkable
adiposity and worsened glycaemic control under a high fat diet. Pair
feeding inhibited this effect. Ghrelin elevated leptin mRNA
expression and reduced resistin mRNA expression. Gastric ghrelin
mRNA expression during fasting was increased by a high fat diet.
GHS-R antagonists decreased energy intake in lean mice, in mice with
diet induced obesity, and in ob/ob obese mice; it also reduced the
rate of gastric emptying. Repeated administration of GHS-R
antagonist decreased body weight gain and improved glycaemic control
in ob/ob obese mice.
CONCLUSIONS: Ghrelin appears to be closely
related to excess weight gain, adiposity, and insulin resistance,
particularly under a high fat diet and in the dynamic stage. Gastric
peptide ghrelin and GHS-R may be promising therapeutic targets not
only for anorexia-cachexia but also for obesity and type 2 diabetes,
which are becoming increasingly prevalent worldwide.
Asakawa A, et al. Gut. 2003
Jul;52(7):947-52
Effects of intraperitoneally administered
[D-Lys-3]-GHRP-6 (2?(200 nmol/mouse) on cumulative food intake in
food deprived lean mice: *p<0.05, **p<0.01 compared with the
control group by Bonferroni?8s t test. (B) Effects of
intracerebroventricularly administered [D-Lys-3]-GHRP-6 (0.2?(20
nmol/mouse) on cumulative food intake in food deprived lean mice.
ACSF, artificial cerebrospinal fluid. (C) Antagonistic effects of
[D-Lys-3]-GHRP-6 administered intracerebroventricularly (20
nmol/mouse) on feeding induced by intraperitoneal injection of
ghrelin (3 nmol/mouse) in non-food deprived lean mice. (D)
Inhibitory effects of intraperitoneally administered [D-Lys-
3]-GHRP-6 (20?(200 nmol/mouse) on the gastric emptying rate one
and two hours after injection in lean mice. (E) Effects of
intraperitoneally administered [D-Arg-1, D-Phe-5, D- Trp-7, 9,
Leu-11] substance P (10?(100 nmol/mouse) on cumulative food intake
in food deprived lean mice. (F) Effects of intraperitoneally
administered [D-Lys-3]-GHRP-6 (20?(200 nmol/mouse) on cumulative
food intake in mice with diet induced obesity who received a high
fat diet for two weeks.
Acute effects of intraperitoneally administered
[D-Lys-3]-GHRP-6 (200 nmol/mouse) on cumulative food intake in food
deprived ob/ob obese mice: *p<0.05, **p<0.01 compared
with physiological saline treated controls. (B) Chronic effects of
[D-Lys- 3]-GHRP-6 administered intraperitoneally (20?(200
nmol/mouse every 12 hours for six days) on body weight gain in
non-food deprived ob/ob obese mice.
Effects of [D-Lys-3]-GHRP-6 administered
intraperitoneally (20?(200 nmol/mouse every 12 hours for six
days) on food intake, epididymal fat mass, gastrocnemius
muscle, and blood glucose, insulin, cholesterol, triglyceride,
and free fatty acid concentrations in ob/ob obese
mice
|
|
|
| Food intake (g/day) |
4.845 (0.160) |
4.527 (0.261) |
4.285 (0.298) |
| Fat pad mass (g) |
0.974 (0.066) |
0.897 (0.169) |
0.860 (0.086) |
| Skeletal muscle (g) |
0.300 (0.012) |
0.314 (0.009) |
0.326 (0.013) |
| Glucose (mmolli) |
13.01 (1.538) |
12.06 (1.549) |
7.489 (1.081)* |
| Insulin (pmol/1) |
8294 (1676) |
6242 (1628) |
5481 (1304) |
| Cholesterol (mmol/1) |
6.649 (0.346) |
5.663 (0.291) |
5.906 (0.565) |
| Triglycerides (mmol/l) |
0.518 (0.049) |
0.436 (0.040) |
0.465 (0.068) |
| Free fatty acids (meq/l) |
2.164 (0.075) |
2.036 (0.121) |
1.646 (0.078)** |
|
| *p<0.05,
**p<0.01 compared with physiological saline treated
controls. |
Mutational mapping of the binding site for the
agonist Ghrelin and the inverse agonists: [DArg1, DPhe5, DTrp7,9,
Leu11]-substance P (SP-analog) and fQwFwLL using a library of 17
mutant versions of the Ghrelin receptor with substitutions
systematically placed through the main ligand-binding crevice. The
potency (EC50) of the compounds in respect of either stimulating
(agonist) or inhibiting (invers agonist) the constitutive
stimulation of inositol phosphate accumulation was determined in
COS-7 cells transiently transfected with either the wild-type or the
mutants forms of the Ghrelin receptor. Fmut indicates the fold shift
in potency induced by the structural change in the receptor as
compared to the wild-type receptor. In the first column is shown the
constitutive activity of the mutant receptors expressed as percent
basal signaling activity compared to the maximal Ghrelin stimulated
activity.
Holst B, et al. Mol Pharmacol. 2006 Jun 23; [Epub ahead of
print]
|
|
|
%Ghrelin%
%substance P%
|
|
|
