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FIG. 1. (Upper) The synthetic peptide combinatorial library used to
screen for and study MSH receptor antagonists was composed of 96
sublibraries. Z96 is used to represent the unique amino terminus
position 1 molecule that comprises the 96 sublibraries. Position 1 is
composed of 1 of the 48 molecules listed for positions 2 and 3, or 1 of
48 acetylated equivalents. AAl to AA37 at positions 2 and 3 are the
20 standard L-amino acids (not prefixed) minus cysteine plus 18
equivalent dextrorotatory (D) isoforms; 11 additional molecules, as
listed, bring the total to 48. Abu, 2-aminobutyric acid; a-Abu, 4-aminobutyric
acid; E-Ahx, 6-aminohexanoic acid; Aib, 2-aminoisobutyric
acid; 3-Ala, 3-aminopropionic acid; Hyp, trans-hydroxyproline; Nle,
norleucine; Nva, norvaline; Orn, ornithine. (Lower) Video image of
a-MSH antagonist-like responses produced by beads from the combinatorial
sublibrary containing D-Trp-l. (Left) Image showing a 6-cm
culture dish of agarose-covered Xenopus melanophores (pretreated
with 1 nM melatonin for 30 min and 15 nM a-MSH for 2 min) just after
the application of "600 beads from the D-Trp-l sublibrary. Melanophore
cells aggregate intracellular pigment granules in response to
melatonin and disperse pigment once a-MSH is added. (Right) After
60 min, white circular patterns appeared in response to release from
nearby beads; patterns are consistent with blockade of the MSH
receptor. Circle diameters increase over time as active molecules
diffuse from their bead of origin through the gel matrix, allowing more
cells to aggregate their pigment.
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FIG. 2. Peptides that reverse ca-MSH-induced pigment darkening in diffusion assays are tested in vitro. (Left) Candidate antagonists are tested
for their ability to independently inhibit a-MSH-induced pigment dispersion. The graph shows an example of inhibition curves for three peptides
Al, A4, and A6 (see Table 1) identified from diffusion assays. Two other molecules not found in the library screens, A9 and All, are included
for comparison. Each point represents the mean and sample SD of four independent measurements taken 2 hr after addition of 15 nM a-MSH
plus the indicated concentration of test antagonist. Results are expressed as a percentage relative to treatment with a-MSH alone. (Center)
Competitive inhibition of a-MSH by D-Trp-Arg-Leu-NH2 (dWRL) is demonstrated by using Schild regression analysis (28). The negative logarithm
of the equilibrium Kd is 7.2 ± 0.1, and the slope of the regression is 1.06 ± 0.03. Broken lines indicate 99% confidence level. dr, dose ratio. (Right)
Dose ratios (dr) for Center were obtained from concentration-response curves for a-MSH taken in the absence (0) and presence (*) of 1 ,uM,
10 ,uM (A), and 100 ,LM (0) dWRL. ECso for a-MSH alone is 2.5 ± 0.3 nM and for a-MSH + dWRL is 4.8 ± 0.6 ,uM. Each point represents
the mean and sample SD of four independent transmittance measurements. Ti = initial transmittance (2 min); Tf = final transmittance (60 min).
dWRL causes no change in EC5o values for either vasoactive intestinal peptide or AVT (data not shown).
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FIG. 3. D-Trp-Arg-Nle-NH2 (dW-R-Nle), at 40 ,uM, blocks 10 nM
a-MSH-mediated cAMP second-messenger stimulation in cultured
Xenopus melanophores but does not block cAMP stimulation evoked
by 8 nM AVT. Oxytocin antagonist GVT {[Pmp , Tyr(Me)2,
Orn8]vasotocin from Peninsula Laboratories, where Pmp = 1-(,Bmercapto-,
B,l-cyclopentamethylene)propionic acid} at 20 AM is used
as a control to block responses evoked by 8 nM AVT. Each bar
represents the mean and sample SD of four independent measurements.
*t test, P < 0.001, for all groups except those bearing asterisks.
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FIG. 4. Functional antagonism of a human MSH receptor by
D-Trp-Arg-Leu-NH2 (dWRL) is demonstrated in Xenopus fibroblasts
(26) transfected with "Vector alone" (pcDNAI/NEO; Invitrogen) or
with "hMel MSHR" (pcDNAI/NEO containing a human melanoma
MSH receptor insert; gift from Roger Cone of the Vollum Institute,
Portland, OR; see ref. 29). Control = no additional drugs. MSH = 5
nM a-MSH. dWRL concentration is 10 ,uM. Forskolin = 100 ,uM
7(3-deacetyl-713-(y-N-methylpiperazino)butyrylforskolin from Calbiochem).
Test drugs were added with 3-isobutyl-1-methylxanthine
present for 45 min, and intracellular cAMP was extracted with 1 ml of
60% ethanol per well. Each bar represents the mean and SSD of three
independent measurements, except for the control-HMel MSHR group,
where n = 6. *By the t test, P < 0.006 for all groups except other groups
bearing a single asterisk. **By the t test, P < 0.006 for all other groups.
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FIG. 5. Structure-function comparisons at the third position for
the six most potent L amino acids identified by random screening from
subpools containing D-Trp at position 1 and Arg at position 2 (see Fig.
1 Upper). Three additional substitutions at position 3 not found in the
random screen are included for comparison. Antagonist activity
correlates with hydrophobicity and charge characteristics of the R
group found at position 3. Chain length and steric hindrance by
3-carbon substitution are also important.
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FIG. 6. MSH antagonists isolated from diffusion assays are active in vivo. (Left) D-Trp-Arg-Leu-NH2 (dWRL; 40 ,umols/kg) induces pallor when
injected into Xenopus (white animals), while injection of a control peptide (D-Trp-Abu-Arg-NH2) at the same concentration causes no response
(dark animals). (Right) Topical application of the tripeptide dWRL (1 mM in H20) to the skin of Xenopus laevis causes a local lightening in
pigmentation.
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