Transcript Introduction - University of Arizona

INTRODUCTION
• a-MSH and ACTH are two classical hormones derived from
proopiomelanocortin (POMC) precursor protein. They have been
studied for many years for their biological activities at the
pigmentary melanocortin 1 receptor (MC1R) and at the adrenal
receptor (MC2R), respectively.
• Recently, three additional melanocortin receptors have been cloned
and stably transfected in cell lines: the melanocortin 3 receptor
(MC3R -- found primarily in the brain); the melanocortin 4 receptor
(MC4R -- found primarily in the brain); and the melanocortin 5
receptor (MC5R -- found throughout the body).
• The endogenous ligands for these receptors are unknown but likely
candidates include POMC peptides such as a-MSH, g-MSH, bMSH and modified versions of these peptides.
POSSIBLE BIOLOGICAL ACTIVITIES
ASSOCIATED WITH NEW MCRs
• FEEDING BEHAVIOR -- Obesity, Anorexia
• ERECTILE FUNCTION
• CARDIOVASCULAR FUNCTION -- Blood
Pressure and Heart Rate
• KIDNEY FUNCTION -- Natriuresis
• THERMOREGULATION
• LEARNING
• OTHERS
GOALS OF RESEARCH
To develop peptides and peptidomimetic derivatives
and analogues of POMC peptides that have high
affinity and selectivity for melanocortin 3, 4 and 5
receptors and that are stable to enzymatic
breakdown in vitro and in vivo. Both AGONISTS
and ANTAGONISTS are needed.
METHODS
• Computer assisted design based on conformational and
topographical considerations.
• Solid phase synthesis including cyclic (backbone backbone, side chain - side chain, and backbone - side
chain) and acyclic analogues.
• Biological
assays
including
binding
affinity
determination and second messenger measurements
using cloned receptors, and in vitro and in vivo
bioassays.
• Conformational analysis, X-ray crystallography, NMR,
and other biophysical methods for selected analogues.
IMPORTANT STRUCTURES
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2
a-MSH
H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH
g-MSH
Ac-Ser-Tyr-Ser-Nle-Gly-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2
NDP-a-MSH (Super-agonist)
Ac-Nle-Asp-His-DPhe-Arg-Trp-Lys-NH2
MT-II (Super-agonist)
Ac-Nle-Asp-His-DNal(2')-Arg-Trp-Lys-NH2
SHU-9119 (Potent Antagonist, MC3R/MC4R)
FROG SKIN BIOASSAY FOR CYCLIC
“SOMATOSTATIN-RELATED” ANALOGUES
H-DPhe-Xxx-His-DPhe-Arg-Trp-Yyy-Thr-NH2
Comound
No.
GH-4d
Xxx
Hcy
Yyy
Cys
EC50
(nM)
3.0
Ring
Size
21
GH-11
Cys
Cys
100
20
GH-14
Cys
DCys
0.10
20
GH-15
Cys
Pen
1,000
20
MT-II *
Asp
Lys
0.10
23
*: Ac-Nle as N-terminus, while CONH2 as C-terminus.
AFFINITIES OF CYCLIC “SOMATOSTATINRELATED” ANALOGUES
H-DPhe-Xxx-His-DPhe-Arg-Trp-Yyy-Thr-NH2
Compound
No.
GH-40
GH-1
GH-14
GH-15
MT-II*
Xxx Yyy
Hcy Cys
Cys Cys
Cys DCys
Cys Pen
Asp Lys
IC50 (nM) (EC50, nM)
hMC3R hMC4R hMC5R
2.0(4.0) 0.60(0.20) 9.0(27)
<10-5
810(1350)
<10-5
21(2.6)
2.4(0.1) 166(560)
1330(Ant.) 29(16) <10-5(Ant.)
11(40) 0.60(0.30) 6.0(5.0)
*Ac-Nle as N-terminus while CONH2 as C-terminus.
ANTAGONIST ACTIVITIES OF
CYCLIC LACTAM ANALOGUES
EC50 (nM)
Compound
mMC3R
mMC4R
mMC5R
Ac-Nle-c[Asp-His-DNal(2')-Arg-Nal(2')-Lys]-NH2
pA2 8.9
pA2 10.3
4.5
Ac-Nle-c[Asp-His-DNal(2')-Arg-DNal(2')-Lys]-NH2
pA2 9.5
pA2 10.3
2.3
Ac-Nle-c[Asp-Trp-DNal(2')-Arg-Trp-Lys]-NH2
pA2 ~9
pA2 9.2
pA2 ~9
Ac-Nle-c[Asp-Trp-DNal(2')-Arg-Nal(2')-Lys]-NH2
pA2 ~9
pA2 ~10
PA2 ~7
Ac-Nle-c[Asp-His(1-Me)-DNal(2')-Arg-Trp-Lys]-NH2
pA2 ~9
pA2 ~10
pA2 ~7
Ac-Nle-c[Asp-His(1-Me)-DNal(2')-Arg-Nal(2')-Lys]-NH2
pA2 ~9
pA2 ~10
PA2 ~8
BINDING AFFINITIES OF CYCLIC
MELANOTROPIN PEPTIDES
IC50 (nM)
Peptide
hMC3R hMC4R hMC5R
a-MSH
50.4
39
560
NDP-a-MSH
1.2
1.2
0.86
MT-II
8.6
0.72
44
Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Ala-Lys]-NH2
25
0.35
26
Ac-c[Asp-His-DPhe-Arg-Trp-Ala-Lys]-NH2
2.0
1.9
100
Ac-Nle-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2
2400
78
>1000
Ac-Nle-c[Asp-His-DPhe-Arg-DTrp-Ala-Lys]-NH2 137
72
140
Ac-Nle-c[DAsp-His-DPhe-Arg-Trp-Ala-Lys]-NH2 260
91
>1000
CONCLUSIONS I
• Effective ligands for the MC1R based on Somatostatin
have been successfully designed, synthesized and bioassayed.
• All the synthesized peptides are being tested at the
MC3-5 receptors.
• It was demonstrated that one class of compounds can
be converted into another class via rational design
based on our understanding of conformationally and
topographically constrained ligands and their receptor
interactions.
• New applications of these compounds and of this
novel concept for designing effective bioactive
compounds are being applied to other areas.
CONCLUSIONS II
• CYCLIC LACTAM ANALOGUES of MT-II
substituted in positions 6, 7 and 9 with various
modified, constrained aromatic residues can lead to
potent antagonists.
• SELECTIVITY for MC4R vs MC3R and MC5R
most readily obtained.
• POTENCY and SELECTIVITY of cyclic
Somatostatin-derived melanotropin analogues at
MCRs are highly dependent on ring conformation.
• MC3, MC4 and MC5 receptors have different
structure-activity relationships with melanotropins.
SUMMARY OF CURRENT STATUS OF SELECTIVE
AGONIST AND ANTAGONIST LIGANDS FOR MC3-5Rs
• MC3R
– Have Both Agonists and Antagonists
– Selectivity
• Good vs MC5R (>300)
• Modest vs MC4R (10-50)
• MC4R
– Have Both Agonists and Antagonists
– Selectivity
• Good vs MC3R (>100)
• Excellent vs MC5R (>500)
• MC5R
– Have Agonists and Recently Discovered Antagonists
– Selectivity
• A Few Equil. vs MC3R
• A Few Equil. vs MC4R